CA2176259A1 - Chimeric oligonucleoside compounds - Google Patents

Abstract

Chimeric oligonucleoside compounds and methods of preparing and formulating the same are disclosed. The compounds and compositions are useful in activating RNaseH-mediated cleavage of target ribonucleic acid sequences and in treating disease conditions relating to such sequences.

Description

wo gsll3834 ~ 1 7 6 2 ~ 9 PCI7US94/13387 ., 1, DESCRIPTION
Chi~exic Oliqrn~ eo~ C ""~
Field of the Invention The present invention relates to antisense oligonu-cleoside compounds cnnt~;n;n~i modified internucleoside linkages, and optionally other structural modifications.
The compounds are capable of hybridizing to target nucleic 5 acid sequences and activating RNaseE~-mediated cleavage of the target.
Related AD~liCati This int~rn~tinn~l application is a rnnt;nll~tion-in-part of commonly-assigned U. S . Patent Application Serial No. 08/238,177, filed May 4, 1994, which is a rnnt;n~
tion-in-part of commonly-assigned U.S. Patent Application Serial No. 08/233,778, filed April 26, 1994, which is a cnnt;nl~tion-in-part of commonly-assigned U.S. Patent Application Serial Nos. 08/154, 013 and 08/154, 014, both filed NOY ^r 16, 1993. The entire disclosures of all of these applications are incorporated herein by reference.
B~ckaround o~ the Invention SnnF~ rable attention hag been directed in recent years to the design of antisense nucleic acid oligomers 20 or use in studying, treating and ~ nnc;n~ conditions attributable to ~n~ln~nnus or f oreign nucleic acid se -guences in living organisms. For example, it is now well known that a nucleic acid oligomer having suitable anti-sense complementarity to a target mRNA can hybridize to 25 the target mRNA and, in some cases, disrupt translation of the mRNA. The antisense approach presents great promise or the eventual therapeutic treatment of disease condi-tions caused by foreign te.g., viral) genetic material, or by misfunctioning or altered eldoyell~us genetic material 30 (e.g., cancer and genêtic disêase conditions).

wo 95/13834 PCr/l~S94113387 However, despite the great promise of the antisense approach, a number of challenge3 still remain. First, antisense compounds are generally subject to degradation in the cellular milieu due to endogenous endo- and exonu-cleases . While a number o~ ~odif ied antisen3e ~tructures have been described having improved resistance to nuclease degradation, further i ~ uv~ q are desirable in order to increase the potency and half-life of the, ~ u-lds.
Second, it is generally required that an antisense com-pound have a high 3pecificity toward the intended target nucleic acid so aE to avoid disruption of activity of unintended native sequences. Although a number of re-searchers have described approaches designed to increase the binding af f inity of an antisense compo-.md to a target sequence, very few results have been reported with respect to structural r~f; n ts which avoid disruption of the activity of unintended genetic sequences while still rf-~ ~; n; ng maximum ef f icacy against the target sequence .
One approach toward disrupting the expression of undesired target mRNAs involves forming a duplex hybrid between the target mRNA and an antisense strand, followed by cleavage of the target mRNA by an ~ldu~:uuus RNaseH.
See Dash, P., et al, Proc. Natl. Acad. Sci. U.S.A.
84 :7896-7990 (1987) . Xowever, because the mode of action of RNaseH is fairly speciflc, this approach is subject to a number of constraints. First, }~NaseH enzymes act in nature to cleave the oligoribonucleic acid strand of an oligodeoxyribonucleotide-oligoribonucleotide duplex, but do not cleave DNA-DNA or RNA-RNA duplexes. This ha6 been attributed, at least in part, to the polar nature of DNA-RNA hybrids which, in contrast to DNA-DNA and RNA-RNA
hybrids, have 2'-O~I groups on one (but only one) strand.
Crouch, R.J. & Dirksen, M. -L., "Ribonucleases H, ~ in Nucleases (Linn ~ Roberts, eds . ), Cold Spring Harbor Laboratory (1982), at 212. As a result, one putative requirement of the antisense RNaseH cleavage approach is that at least some of the nucleosides of the antisense ~W0951l3834 ~B2~9 PCT/US94113387 nucleic acid strand must have characteriatics in common with deoxyribonucleotides (a5 opposed to ribonucleotides~, particularly, the absence of a polar yroup on the 2 ~ -position of the antisense nucleoside sugars. Perhaps 5 related to this is the additional requirement that at leagt some of the sugar groups in the Ant1 ~n~e compound must be in a 2'-endo (~) conformation as found in deoxy-ribonucleosides, as opposed to the 3'-endo (~) conforma-tion found in ribonucleosides. Cook P.D., PCT Publication No. W0 93/13121 (1993), at 18-19.
It has further been reported that various 2~-position substituents (e.g., 2~-0-alkyl and 2~-fluoro) will render the substituted portion of an antisense strand non-acti-vating to RNaseH, even though binding affinity toward the target nucleic acid is increased . Inoue , H ., et al ., FEBS
Letters 215 ~2~ :327-330 (1987); Monia, B.P., et al., J.
Biol. Chem. 268 (19) :14514-14522 (1993) . Likewise, the Monia, et al. report indicates that a minimum of five consecutive 2 ~ -deoxy residues is required in order to achieve efficient activation of ~ n (HeLa) RNAaeH, and that this 2'-deoxy segment (if ~cl~ ;ed by 2'-substituted residues in the same antisense compound) must be centered in the oligomer sequence in order to achieve efficient RNaseH activation in vitro or expression inhibi-tion in cells.
Another reported requirement of ~the antisense F~NaseH
cleavage approach is that, in order to achieve RNaseH
activation, at least one portion of the ;ntprn~ ]~side "backbone" of the antisense, ~ olln~ must include charged (anionic) rhnsph~rus-c-nt~;n;n~ linkage groups. Cook, P.D., PCT Publication No. W0 93/13121 (1993), at 18. In studies of chimeric antisense compounds including both methylphosphonate (uncharged) and rhnsrhr,r~; ester or phosphorothioate (charged) linkages, Agrawal, et al.
reported that the minimum number of consecutive charged h~rl~h~n~- linkages required for efficient activation of r -l;~n RNa5eH in vitro is five. ph~sph~ ;ester linkag-_ _ _ _ _ _ _ _ Wo 95113834 PCrlUS94/13387 21~2~ `
es positioned in either the t~rm;n~l or center portion of the oligomers were reportedly more ef f icient than phos-phorothioate linkages in activating RNase~I, whereas oligomers rnnt~;n;ng only methylrhnsphnn~te, phn~phnro-N-morpholidate or phosphoro-N-butylamidate linkages were inactive . Agrawal , S ., et al ., Proc . Natl . Acad . Sci .
U.S.A. 87:1401-1405 (1990) .
While phosphodiester linkages, being charged, are suitable to allow activation of RNaseX, they suffer from the disadvantage of being subject to degradation by naturally-occurring endo- and/or exonucleases. A variety of alternative linkage groups, some of which are nuclease-resistant, have been developed or proposed for use with antisense compounds. Among these are charged linkage groups such as phosphorothioate, phosphorodithioate, phosphoroselenate and phosphorodiselenate linkers. In general, deoxyribonucleoside antisense oligomers contain-ing these non-natural linkage groups tend to have lower binding af f inity toward complementary RNA target strands than the corresponding phosphodiester-linked antisense oligomers, although higher af f inity may be achieved where the antisense strand comprises r; hnnl~rl en~ides or 2 ' -substituted ribonucleosides (rather than deoxyribonucleo-sides). See Metelev, V. & Agrawal, S., PCT Publication No. W0 94/02498 (1994), at 9. Among the uncharged phos-phorus-containing linkage groups that have been reported are the alkylrhn~rhnn~te (e.g., methylrhnsphrn~te), aryl rhn~Fhnn~te, alkyl and aryl rhnsrhnramidate, alkyl and aryl phosphotriester, 1IydLu~ phosphonate, boranophos-phate, alkyl and aryl phosphonothioate, phosphoromor-pholidate, and rhnsrhnropiperazidate linkers. See Cook, P.D., PCT Publ;r~tjcm No. W0 93/13121 (1993), at 7;
Pederson, T ., et al ., U. S . Patent Nos . 5 ,149, 797 and 5,220,007; Padmapriya, A. & Agrawal S., PCT Publication No. WO 94/02499 (1994). Non-phosphorus-based linkage groups have also been reported, ; nrll~li n~ peptide, mor-pholino, ethy~ene glycol, amide, and other linkers. See Wo g~/13834 ~ 2 1 7 6 2 ~ g `` PCTtllS94/13387 Reynolds, M.A., et al., PCT Publication No. WO 92/02532 (1992); Cook, P.D., PCT Publication No. WO 93/13121 (1993), at 7. As with the charged phosphorug--~nnt~;n;n~
linkers noted above, many of these other non-natural 5 linkage groups may exhibit lower binding affinity (com-pared to phosphodiester linkages) toward complementary RNA
target strands, at least in the case of linked 2 ' -unsub-stituted ~ntiqPnqe nucleotides, and particularly in the presence of salt ions.
Various workers have attempted to identify combina-tions of linkage groups and/or structural modifications for antisense oligome~s that might lead to improved RNaseH
activation, binding affinity, nuclease resistance and/or target specif icity . Thus, Cohen, et al . have reported improved half-life for antisense and non-ilnt; q~nqe oligo-deoYyribonucleotides ~-Qn~;n;n~ at least one phosphoro-thioate linkage located, for example, at either terminus of the c ~ In~l, or throughout the compound. Oligomers cnnt~;n;n~ all phosphorothioate linkages were shown to have anti-viral (anti-HIV) activity, wherea:
phosphodiester- and methylrhnsrhnn~ tP- linked compounds were reportedly inactive . Cohen, J . S ., et al ., U . S .
Patent No . 5, 264, 423 . Walder et al . have proposed the use of a 3'-terminal non-phosphodiester linkage, optionally combined with a 5 ' -terminal non-phosphodiester linkage or a 5'-tPrm;n~l "cap" group, to avoid 3'-initiated (and optionally 5 ' -initiated) exonuclease degradation of oligodeoxyribonucleotides. RNaseH cleavage activation reportedly required retention of at least four, and preferably at least seven, contiguous phflsrhr~; ester linkages in the antisense oligomer. The preferred com-pounds contained at least lO, and preferably at least 15, nucleotides, the majority of which were phosphodiester-linked. Walder, ~.A., et al., PCT ~Publication No. WO
89/05358 (1989). Padmapraya & Agrawal have reported that the incorporation of nonionic alkyl or aryl rhn,~phnn~-thioate liDka~e~, preferably at one or both termini of the _ _ _ _ . . . , ,, , ... _ ... _ .. _ . ,, . , .. . . _ . , WO 95/13834 ' PC rlUS94113387 oligomer, res~lted in; uv~d nuclease resiætance, albeit with a reduction in Tm of 1-2C/rhnsrhnnnthioate linkage.
PCT Publication No. WO 94/02499 (1994).
Pederson, et al. have reported the use of "mixed 5 phosphate h~rkhnn~" oligomers cnnt~inln,r both a phospho-diester- or phosphorothioate-linked segment for RNaseH
activation, and one or more non-RNaseH-activating, un-charged linkage group segments. It was found that a segment of five or 8iX consecutive phosphodiester linkages 10 was efficient, in a 15-mer compound, to effect RNaseH
cleavage of a target RNA strand, whereas similar compounds with fewer phosphodiester linkages, or with up to six consecutive phosphorothioate linkages in place of the rhnRrhn~liester linkages, had low activity. Pederson, T., et al., U.S. Patent Nos. 5,149,797 and 5,220,007.
Giles & Tidd have reported that the target specifici-ty of an ~nt; ~n~e oligomer can be improved by the use of a chimeric structure comprising t~rmin~l methylrhnsrhnnn-diester sections separated by a central RNaseX-activating 20 phosphodiester region having a high A+T to G+C ratio. The observed reductions in non-specific cleavage were attrib-uted to the lower Tm caused by the methylphosphonate segments, the reduced hybridization strength of the small, A/T-rich phosphodiester region, and the reduced prospects 25 for partially-complementary hybridization at the shortened RNaseH activation site. Giles, R.V. & Tidd, D.M., Nucl.
Acids Res. 20 (4) :763-770 ~1992) .
Ohtsuka, et al. have described the use of partially

2~-substituted (e.g., 2'-lower alkoxy substituted) 30 oligomers for, site-specific RNaseH cleavage of RNA targets with or without secondary structure. RNaseH cleavage was reportedly localized to a site (or sites) on the target corresponding to the non-substituted (i.e., deoxyribonu-cleotide) portion of the antisense compound. Single-site 35 cleavage was reportedly optimized by use of a tetradeoxy-r; hnnllrl Potide segment located centrally in the compound between two 2'-substituted terminal segments. Inoue, H., ~76259 Wo 95t13834 Pcr/uS94113387 et al, FEB Letters ~:327-330 (1987); .qh;hAh~ra, S., et al., Nucl. Acids Res. ~ :4403-4415 ~1987); Ohtsuka, E., et al., U.S. Patent No. 5,013,830. The use o~ par-tially 2'-substituted oligomers additionally rf~ntil;n;ng 5 one or more non-phosphodiester linkages has also been reported. See .~h;h~hA~a, S., et al., European Patent Application Publication No. 0 339 842 A2 (1989) (reporting

3'-5' or 2'-5' linked oligomers having phosphorothioate or other linkages); Cook, P.D., PCT Publication No. WO
93/13121 (1993) (reporting increased binding affinity attributable to 2'-substitutions, and nuclease resistance attributable to , e . g ., ph~srhnrothioate and phosphoro-dithioate linkages); Monia, B . P ., et al ., J . Biol . Chem.
2Ç8(19) :14514-14522 (1993) (reporting effects of 2~-15 substitutions in phosphorothioate-linked oligomers);
Metelev, V. & Agrawall S., PCT Publication ~o. Wo 94/02498 (1994) (reporting use of 2 ' -substitutions in phosphoro-thioate- or ~hf~srhnrodithioate-linked oligomers); McGee, D.P., et al., PCT Publication No. WO 94/02501 (1994) 20 (describing preparation of various 2'-substituted nucleo-sides and ~h~sph~amidites)~
- of tbe Invention The present invention relates to improved RNa6eH-activating Ant;qPnqe oligonucleoside, , ~lq rnntA;n;n~
25 selectively modified ;n~rnllr~eoside linkages, and option-ally other structural modifications. The compounds exhibit improved target specificity and potency ~_...~a~ ~d to other RNaseH-activating antisense ~ . They are useful both in vivo and in vitro in reducing or ~1 ;m;ni:lt-30 ing the translation of target mRNA sequences, most prefer-ably sequences related to disease conditions.
In one aspect, the present, ~ lq incorporate one or more polynucleoside segments having chirally-pure or chirally-enriched modified (non-~h~-sph~ ;ester) inter-35 nucleoside linkages. The chirally-selected linkage 8egments are preferably 8elected to include linkages ,, , , , _ , . . , , _ . .... ,,, . . , _ . _ . .. .

Wo 95113834 2 1 ~ ~ 2 5 9 PCrlUS94113387 ;
having R chirality at the asymmetric phoaphorus atom of one or more of the linkage structures ( "Rp chirality" ) .
Preferably, at least about 40% of the linlcages in a given chirally-selected segment will be Rp-chiral. Also included 5 are segments selectively including one or more Sp-chiral linkages. In one preferred embodiment, chirally-selected segments are situated at the tor~;nAl (3' and 5' ) portions of the compound, surrounding (fl~nk;ng) a central RNaseH-activating re~ion . The f lanking chirally- selected seg-10 ments preferably are subst;~nt;;~lly non-RNaseH-activating.
The RNaseH-activating region, if linked with asymmetric (chiral) linkage groups, may alternatively or additionally be chirally selected. In a related embodiment, the RNaseH-activating region is situated at or near one 15 terminus of the compound, and all or a portion of the ~, ;n~r of the compound is chirally selected and prefer-ably is non-RNaseH-activating.
The chirally-selected Rp-~nr; rh~l segments of the invention serve to increase the binding af f inity of the 20 compound as compared to racemic ~ 1uu--ds. In addition, because the chirally-selected modified linkage structures are more resistant to degradation by endo- and/or exonu-cleases than are non-modified phosphodiester linkages, the chirally-selected segments will tend to protect the 25 compound from degradation in the in vivo environment.
In another aspect, the present ~ __ 'q incorporate one or more 1 polynucleoside segments comprising mixed modified (non-phosphodiester) ;nt~rn1l~1eoside linkages.
Two or more ~ifferent ;nt~rnllcl~nR;de linkage structures 30 are ;n~ in the mixed linkage segment, and one or more of these may be a modified linkage structure. One or more of the linkage structures in the sequence may be chirally selected. Preferably, the mixed linkage segment includes multiple linkage sequence blocks (synthons) each contain-35 ing two or more different ;ntPrn11clenc;r~ linkage struc-tures, or a single such synthon that is repeated two or more times n the mixed linkage segment. Where the _ _ _ Wo 95/13834 PCT/US94113387 ~176259 compound r~ntA;nc more than one mixed linkage segment, the linkage sequence blocks may be the same or dif f erent in the respective segments. In one preferred embodiment, mixed linkage segments are situated at the terminal (flanking) portions of the compound, surrounding a central RNaseH-activating region. The RNaseH-activating region may alternatively or additionally comprise a mixed linkage segment. The fl~nk;ng mixed linkage segment5 are prefera-bly non-RNaseH-activating. In a related embodiment, the RNaseH-activating region is situated at one terminal portion of the compound, and all or a portion of the ,~ ;n~l~r of the compound contains a mixed linkage segment and preferably is non-RNaseH-activating.
The mixed linkage segments of the invention may be racemic or chirally selected; in either case the identity of the int~rn~ ncide structures and/or the linked nucleoside substituents can be selected to afford greater binding affinity to the compound while ~~~;ntc;n;n~ target specificity and nuclease resistance and increasing poten-cy. Because the mixed linkage segments of the compound include one or more modified ;nt~rn~1- leoside linkage structures that are resistant to degradation by endo-and/or exonucleases, the ~ ~uul~ds will have higher potency in the in vivo environment.
In another aspect, the present invention ; n~ Pc uved RNaseH-activating segments comprising linked n-1~le~Pides having mixed int~rnllolpr~siclp linkages. In one preferred : ~ 1; t, the R~aseH-activating segment ;nA1~ c at least five consecutive 2~-unsubstituted li.e.
DNA) n~ residues linked by two or more differe~t charged (anionic) ;nt~rnll- leoside linkage structures in an alternating sequence. Preferably, the RNaseH-acti~ating segment includes at least four such charged ;nt~rnl~rleo-side linkage structure5. One or more of the internucleo-side linkage structures in the RNaseH-activating segment may be chirally selected if an asymmetric phosphorus atom is present in the linkage 8tructure.
_ _ _ _ _ _ _ _ . .. .. . . ,, _, , .. . _, .... ...

wo 9~/13834 ~ PCTNS94/13387 In another a3pect, the pre3ent invention provide3 chimeric structures for anti3en3e oligonucleoside com-pounds that maximize activity while r-int~;n;nrJ the ability to effect 3elective RNaseH-mediated cleavage of 5 the intended target strand These goals are achieved by structure3 which provide, on the one hand, controlled binding affinity and, on the other hand, controlled RNaseH-activation char~rtf~r; Rt; c3 .
Thus, in one ~mhn~;r t, binding a~finity is con-lO trolled (3electively increa3ed) through the u3e ofchirally-3elected Rp-chiral internucleo3ide linkage3 in one or more portion3 of the ~ ~CJU~1~. Alternatively or additionally, one or more Sp linkage3 may be used to 3electively decrea3e binding af f inity In a related 15 embodiment, binding affinity i3 controlled (3electively increa3ed) through the use of multiple or repeated linkage seriuence blocks (3ynthon3) in one or more mixed linkage segments of the compound; the linkage structure3 may be racemic or chirally-3elected. In another related embodi-20 ment, binding affinity is controlled (selectively in-crea3ed) through the u3e of 2'-3ub3tituent3 on one or more nucleo3ide sugars in the compound, preferably in conjunc-tion with altPrn~t;n, linkage segments and/or chirally-selected int~rnllrl ~n~ l ink~ , RNaseH-activating 25 characteristics can simultaneously be controlled (substan-tially eliminated, or selectively increased) in these segments of the ~ _ ~1 by the use of 2 ' -substituted or unsubstituted nucleo3ide sugars and/or by the selection of uncharged or charged linkage structures for a given 30 segment of the co~n~o11n-1.
Likewise, RNaseH-activation characteristics are controlled (selectively increased or decreased) by the selection of mixed or uniform charged ; nt -~nllrl eoside linkages in the RNaseH-activating region of the compound 35 RNase~I-activating characteristics can be selectively decreased, particularly in the RNaseH-activating region of the compound, by the use of linkage structures such as W0 95/13834 217 6 2 S 9 PcrNS94/13387 ~hc~grhnrothioate or especially phosphorodithioate struc^
tures that are poorer substrates f or RNaseH . RNaseH-activating characteristics are also controlled by the inclusion of non-R~aseH-activating portions in the com-r 5 pound such that only a portion of the compound is effec^
tive in activating cleavage of the target genetic se-quence, f or example by d,U~l u~ ~ iate selection of linkage structures, 2'-substituents and other features as de-scribed herein.
Among the highly preferred compounds of the invention are those having subst~nt;~lly non-RNaseH-activating, chirally-selected, mixed linkage segments at the two terminal (flanking) portions of the ~ . uul.d, and an RNaseH-activating region positioned therebetween. Also preferred are, ~ A.c: having subst~nt;~lly non-RNaseH-activating, racemic mixed linkage segments at the two terminal (flanking) portions of the compound wherein one or more of the linked nucleosides in the mixed linkage segments is 2'-substituted, and an RNaseH-activating region is positioned in the compound between the mixed linkage segments . Especially pref erred compounds include those chosen from the following structures:
'-T~r"l~.~l R~ Acti~.r~tlng 3~-T-nnin~l Portio~ R-~io~ Portio 25 YP ~R) /DE DE IIIP !R) /DE
2 ' OMeMP 1 R ) / 2 ' OMeDE PS 2 2 ' OMeMP ~ R ) / 2 ' OMeDE
YP ~ ~) t 2 ~ OMeMP P S MP ~ R ) / 2 ' OMeMP
llP ~R) eslriched P82/DE MP ~R) enriched 2 ' OMeNP ~R) enriched P8 /DE 2 ' OMeMP ~ R) enriched 3 0 XP ~R) /P8 PS/P82 MP ~R) /PS
2 ' OMeMP (R) /2 ' OMeP8 2 ' OMeMP ~R) /2 ~ OMePS
IIP ~R) /P52 ~P ~R) /P82 2 ' OMeMP ~ R ) / 2 ' OMePS 2 2 ' OMeMP ~ R ) / 2 ~ OMeP8 2 2 ' OMeMP / 2 ' OMeDE 2 ' OMeMP /2 ' OMeDE
3 5 MP/2 ' OMeDI~ MP/2 ' OMeDE
MP (R) /PA~n ISP ~R) /PAIIL
2 ' OMeMP ~R) /2 ' OMePAm 2 ' OMeMP ~R) /2 ' OMePA~
2 ' OMeYP /2 ~ OMePAm 2 ' OMeMP / 2 ' OMePAm llP/2 ' OMe~Am MP/2 ' OMe~A~

,., . ~.

MP (R) /TE MP !R) /TE
2 ' OMeMP (R) /2 ' OMeTE 2 ' OMeNP (R) /2 ~ OMeTB
2 ' OMeMP/2 ' OMeTE 2 ~ OMeMP/2 ' OMeTE
MP / 2 ' OMeTI~ MP / 2 ' OMeTE
5 MP (R) /MPS . llP (R) /MP8 2 ' OMeMP (R) /2 ' OMeMPS 2 ' OMeMP (R) /2 ~ OMeMPS
2 ' OMeMP/2 ' OMeMPS 2 ~ OMeMP/2 ' OMeMPS
MP/2 ' OMeMPS MP/2 ' OMeMPS
MP (R) /PF MP (R) /PF
2 ' OMeMP (R) /2 ' OMePF 2 ' OMeMP (R) /2 ' OMePF
2 ' OMeMP/2 ' OMePF 2 ' OMeMP/2 ' OMePF
~P/2 ~ OMePF MP/2 ' OMePF
MP (R) /PB~3 MP (R) /PBB, 2 ' OMeMP (R) /2 ~ OMePBE, 2 ' OMeMP (R) /2 ~ OMePB}I, 2 ' OMeMP/2 ' OMePBM~ 2 ' OMeMP/2 ~ OMePBI}3 MP/2 ' OMePBII, MP/2 ' OMePBX, MP (R) /Rsi MP (R) /Rsi 2 ' OMeMP (R) /2 ' OMeRSi 2 ' OMeMP (R) /2 ~ OMeRSi 2 ' OMeMP / 2 ' OMeRSi 2 ' OMeMP/2 ' OMeR8 i 2 0 MP/2 ' OMeRSi NP/2 ' OMeRSi MP (R) /C}I~ MP (R) /C}I, 2 ~ OMeMP (R) /2 ' OMeCHl 2 ~ OMeMP (R) /2 ' OMeOE
2 ~ OMeMP / 2 ' OMe CJIl 2 ' OMeMP / 2 ' OMe CH
MP/2 ' OMeCEIl MP/2 ~ OMeCHl ~y: MP . racemic methylr~ - linkage (between linked n~lrl~ .e); MP(R) ~ chirally-selected Rp-methylrhnerh~nA~e lincage; DE - rh~erhn~ Rter linkage; PS = ~ a ~ L~Lh_oate lin~age; PS2 = PI~JA~I~VL~ h; ~A'-~ li~kage PAm = 1 ~1A to lincage; TE ~ ~I.oa~1wLLieater linkage; MPS ~ alkyl (particu_arly metlyl) ~11oa~ ,L~,Lhioate linkage; PF = ~ A~ JLI~1UOr date lin~age; PB}I~ linkage; Rsi = silyl (espec_ally alkyl-disuhstituted silyl) linkage, C}~i = f~rr^--e~Al linkage 2'0Me ~ 2'-methoxy-s--hA~ (or cther lower alkoxy, allyloxy or halo 8~hA~;t~ ) n~rlf.~ o reaidue, linked using the listed liLkage structurei "enriched`' refers to a segment of li~kages preferahly ~ n~A;n;ng at least ahout 40~ (and up to 100'6) Rp-aelected linkages among the linkages in the segment and thus includes a mixed se~uence of racemic and chirally-selected R
;nr~rn~ rlr~r~A;Ar~ linkage aLLu~.LuL~_; linkage DLLu~ LUL_D grouped with slashes denote a mixed linkage segment including the listed linkage DLLuuLULC:s~ optionally in a serie.s of multiple or repeated mixed linkage seguence blocks.
In another aspect, the present invention includes improved antisense oligonucleoside compositions useful in treating or diagnosing diseases or other conditions in living or~anisms attr;h~l~Ahle to the expression of endoge-nous or f oreic~n genetic inf ormation . The compounds and -Wo 95/13834 PCINS94/13387 ~176259 compositions are also useful in studying such conditions in vitro or otherwise. In another aspect, the invention provides methods for treating, diagnosing or studying such conditions .
Other aspects and objects of the invention will be apparent from the following detailed description.
Briel~ De~criDtion of the Draw; n~
FIG~RES 1 and 2 are graphs showing nuclease stability of various compounds and segments of the present inven-tion, compared to other mixed linkage compounds, over time .
FIG~ S 3 and 4 are bar graphs showing dose-response activity of a chirally-selected compound of the present invention, versus a non-chirally-selected ,- ~, in inhibiting target (Fig. 3) and non-target (Fig. 4) protein synthesis .
FIG~JRE 5 is a graph showing RNaseX activity of a chirally-selected compound of the present invention, versus a non-chirally-selected compound, over time.
FIGllRES 6-lO depict sythong and ;n~l 3;;ltes useful in constructing ~ ,uullds of the present invention.
FIG~RE ll is a graph showing kinetic data relating to RNA cleavage by various 2'-sugar-substituted and unsubsti-tuted ~ ou~ds of the invention.
Detailed De2~cri~tion A full appreciation of the present invention requires an understanding of the competing parameters underlying the present RNaseX cleavage technique. There are a number of parameters of primary concern, including oligonucleo-side-target binding affinity, RNaseX cleavage rate, specificity/mismatch effects, oligonycleoside displacement by processing ribosomes, and nuclease stability. As will be seen from the following discussion, a proper balance of these competing parameters requires that the oligonucleo-~ide compound have a binding affinity (as quartitated for .. ... ,,, _, , , _ _ _ WO 95113834 ~ 1 ~ 6 2 ~ ~ PCr/US94/13387 t, ~

example by the af f inity constant KA) that is not too large relative to the RNaseH cleavage rate. The present inven-tion provides structures that satisfy this requirement as well as other re~ i ~ outlined below .
The present technique of RNaseH cleavage of a target genetic sequence requires that the oligonucleoside com-pound hybridize with the target sequence, and that the oligonucleoside have a hybridization occupancy time that i8 sufficiently long to effect cleavage of the target sequence by the RNaseX enzyme. The initial step of oligonucleoside-target hybridization is governed, from a first-order kinetic standpoint, by the forward and reverse rate constants (kl and k l) that define KA' where KA = k1/k 1-The rate of cleavage of the target (which is essentially irreversible) is then governed by the rate constant k2, as ~ollows:
Oligomer + Target Strand kl I ~ k l Hybridized Oligomer/Target Strand k, ~ [RNaseH]
Released Oligomer + Target Cleavage Fragments Other considerations aside, it would appear that target cleavage would be optimized by ~-~r;mi7.;n~ both KA
and k2. However, this does not take into account the problem of non-specific binding (i.e. mismatches) between the oligonucleoside and lln;nt~on~d nucleic acid sequences that exist in the cleavage (e . g . cellular) medium which could result in undesired cleavage of the lln;n~n~
sequences. Nor does this simple approach take into account the fact that an oligonucleoside with high binding affinity will typically be displaced from its hybridized state, and thus will be unable to activate RNaseH-mediated cleavage, each time the host ribosome processes along the target ,mRNA sequence.

Wo 95/13834 2 1 7 6 2 5 9 PCr/US94/13387 Consider first the rhilllPnr,e of achieving high target speci~icity with an antisense cleavage compound. Mammali-an cells typically contain an RNA population comprising about 3 x 10' ribonucleotides. By assuming a statistically 5 random distribution of the four naturally-occurring nucleotides within this pop~ t; nn, the total number of "match" ser~uences in the population having exact base-by-base compl~ -t~rity, and the number of "mismatch'~ se-quences having one or more base mismatches, can be approx-lO imated for a target sequence of any given length. (O~course, the actual distribution of ri hnnl~r~ eotides in a given 1 i~n cell population will not be truly random, but nevertheless such statistical analyses can shed light on the probabilities of a mismatch sequence occurring. ) 15 The following table lists the number of targets that would exist in such a population as a function of number of mismatches (zero to five) and target ser~uence length (12, 15 or 18).
M~ ~ trh~ encrth ~rget~
0/12 1 . 8 0/15 2 . 8 x 10-2 1/15 1 . 24 0/18 4 . 4 x 10-~
1/18 2 . 4 x 1o-2 2/18 0 . 62 3/18 9 . 6

4/18 109 It will be seen that an appreciable number of pote~tial mismatch seg,uences may exist even for target sequences as long as 12 nucleosides, particularly as the number of single-base mismatches increases. If the K,~ for a given 35 mismatch duplex is sufficiently high as to allow apprecia-ble hybridization of an ~nt;cPn~e oligomer to a mismatched Wo 9S/I3834 ~ ~ PcrluS94/13387 2~7~2~
-target, then unintended and undesirable cleavage of the mismatched target can result.
Take, for example, the case of a one-base mismatch between a 12-to-18 nllc~ en~ anti3ense oligomer and an unintended mismatch R~A sequence. The present inventors have a8certained that the KA for the correct "match"
hybridization typically does not exceed the KA for the incorrect "mismatch" hybri-l;7at;nn by more than a factor of one hundred. Furthermore, the forward rate constant of hybridization (k1) will be approximately the same for both the match and the mismatch, because the forward hybridiza-tion i9 typically governed in large part by the physics of solution-phase intermolecular exposure which tend to obscure the effect of the single-base mismatch. In this case, the hybr;~l;7At;nn ~'off rate" (k l) can be no more than 100 times greater for the mismatch than for the correct match. It will now be seen that, if the cleavage rate constant k2 is not subst~nt;~lly smaller than the reverse rate constant k 1 for the mismatch, then unintended mismatched nucleic acid sequences will be cleaved (along with the properly matched target sequence). It will also be seen that specif icity f or the intended target se~auence will be optimized if k2 has a value on the order of k l (match), but much less than k l (mismatch):
k l (match) -- k2 c~ k l (m; F~m-tnll) In addition, the present invention takes into account the ribosomal displ ~c t of hybridized oligonucleosides that typically occurs in the coding region of a target mRNA during the process of R~A translation. The ribosomal pron~qsi nn~l rate varies somewhat from RNA to R~A but in general is calculated to pass any single point on an mR~A
every 10-15 seconds . If the KA (match) for a given oligo-nucleoside is 101 M~l and the KA(mismatch) is lO~ M~1, then the half-life hybri~;7~t;nn occupancy times (tl/2) will be about 28 minutes and 17 seconds, respectively, for the match and the mismatch. But because the r;hosn--~l proces-sional rate i~ 80 fast, the correctly-matched oligonu-Wo 95/l3834 2 17 ~ 2 ~ 9 PCr/US94113387 cleoside will be displaced from the target sequenee just about as frec~uently as the m; I trh~ oligomer, and the effective oceupancy times will be approximately the same.
The result in this case is that, from a specificity standpoint, the high affinity constant for the correctly matched hybridization goes for naught, and nonspecific cleavage will occur at lea8t as frec~uently as the intended sequenee-specific cleavage. In fact, nonspecific cleavage may occur even more frequently if more than one mismatch sequence exists in the "target" RNA population.
Given considerations such as these, the present inventors have discoYered that it is beneficial to limit the binding affinity constant of the subject RNa8eE~-activating oligonucleoside compounds to values that are typically no greater than 10l M~l for targets in the coding region of a target mRNA. Preferred K,~ values for the present compounds are in the range 107-10l M~l. In such a ease, beeause the ~off rate" will be relatively high eompared to compounds with higher binding affinities, it is possible and desirable to utilize compounds having a relatively high cleavage rate. Thus, the inventors have discovered that it is benef icial to control the cleavage rate constant of the subject compounds to values in the range of 1 to 10-5 sec~l, preferably 10~1 to 10-~ sec~i, and most preferably 10-' to 10-3 sec 1 The cleavage rate is preferably selected to give at least a 3 :1 cleavage rate of a perfect "match" relative to a 2-mismatch target.
In eontrast, in the non-coding region of a target mRl~A site (e.g., the 5~-cap region, the 5'-untranslated region, the initiation codon region, the 3 ' -untranslated region, splice acceptor or donor sites, intron branch sites, and polyadenylation site8), inhibition of protein prorl1lrt; ~n can be achieved prior to the translation process by suitable hybridization of an antisense oligonu-eleoside, and r;hc~ pl~ of the hybridized oligomer generally does not occur. As a result, oligonu-eleosides having higher binding affinities ~and higher _ _ _ _ _, _ _ . .. . . ... .. . .. _ . . . _ _ _ _ WO 9S/13834 ~ ~ . PC rlUS94/133~7 half-life occupancy times) can be utilized in the non-coding region without the 1088 of 6pecif icity described above with respect to the coding region. In this case, an upper limit on binding affinity will be imposed by the lif etime of messages in the mRNA pool relative to the lifetime of mismatch hybrids. Thus, the lifetime of a typical mRNA molecular species ~taking into account repl~n; ~' -nt of the mRNA pool via transcription) is on the order of five hours. If the hybrid lifetime of mismatch sequence approaches an hour or more, then the translation of the mismatched message will be p~LLuLl-t:d by steric blocking effects apart from any RNaseH cleavage r -h~n;~T As a result, KA(match) should generally be in the range 107-1013 M-1. Furthermore, a relatively low concentration of oligonucleoside is preferably used in this case so that the total level of mismatch occupancy over time (in addition to the miamatch hybrid lifetime of a single m; o~--t.h~d oligonucleoside) is low. (Of course, the rate of RNaseH-mediated cleavage, k" should still be much lower than k l(mismatch) for targets in the non-coding region, just as it is for coding region targets, in order to avoid non-specific mismatch cleavage. ) Values for KAI k1, k 1 and k, can be ascertained using methods known in the art. The ~t~orm;n~t;~ of RAI the equilibrium binding constant, requires the measurement of the c~n~ n~rations (~h~ol~l~e or relative) of single and multimeric species, as well as enough time to ensure complete equilibration. The equilibrium hybridization of oligomers can be studied by direct methods which physical-ly separate the single and multi-meric species, such as gel shift (Lima et al., Biochemistry 31, 1205~-61 (1992) ), strand cleavage (Young, S., Wagner, R.W., Nucleic Acid Research 19, 2463-70 (1991) ), filter binding (McGraw, R.A.
et al., BioTechniques 8, 674-678), or equilibrium dialysis (Bevilacqua, P.C. & Turner, D.E., Biochemistry 30, 10632-40 (1991) ) . Indirect methods rely on physico-chemical properties of the multimeric and single-stranded states, Wo 95~13834 Z 1 ~ ~i 2 5 9 PCrlUS94/13387 -. .

and include method9 6uch a9 optical melting (Albergo, D.D.
et al., Biochemistry 20, 1409-13 (1981)), and differential scanning calorimetry (Albergo, D.D. et al., op. cit . ) .
These publications are incorporated by reference herein.
t 5 Kinetic meatiuL q of on-rates (kl) and of ~-rates (k l) uge many of the same detection methods as equilibrium binding constant determinations, but rely on accurate correlations of species formation or disappearance with time. Off-rates can be studied by the direct methods described above, as well as indirectly by optical methods, and nuclear magnetic resonance of ~f~ut~rl 1~- exchange of protons (I,eroy et al, Journal of Molecular Biology 200, 223-38 (1988) ) . On-rates can be determined ~rom Ka and k "
using the e~uation kl = KA x k 1. Mea~uL -nt of oligomer kl can be measured by specialized kinetic techniques such as temperature jump k;n~t;--c (Williams, A.P. et al., R;c~hf~miEltry 28, 483-4291 (1989), and Turner, D.H. in Investiqati~nE3 of Rates An~ MF~-~h~n; E ~ o~ Reac~ nE3 6, 141-189) . The foregoing publications are also incorporat-2 0 ed by ref erence herein .
It will be recognized, in light of the present disclosure, that the above preferred values ~or binding and kinetic constants will vary depending on the biologi-cal system in which the present oligonucleosides are being used . The values given above represent pref erred values based on hybridization of the oligonucleoside to a single-stranded target sequence that does not have substantial gecondary structure. Where the target sequence is located in a region of the mR~A molecule that has substantial 8econdary structure, the binding affinity of the oligonu-cleoside with respect to the secondary-structured target region may be much lower than that measured with respect to a non-structured (e.g., synthetic) target sequence having the same nucleoside sequence. In some cases the ~
for the non-structured strand may be as much as 107-fold greater than that of the structured strand. If the resulting ~ with reSpeCt to the intended gE~ ry-.. .. _ ,, . ,, _ _ _ Wo 95113834 PCrlllS94/13387 217~2S~

structured target is too low relative to, for example, a non-structured mismatch sequence, problems of specificity may result.
One preferred approach t~o thi9 situation is to target

5 a region in the target mRNA f or RNaseH-mediated cleavage that does not have sufficient secondary structure to adversely af f ect the binding af f inity of the sub; ect oligonucleoside. The secondary structure of nucleic acids can be determined directly by the use of nucleases, base l0 modification chemicals, or sugar-phosphate backbone modifying reagents, as recently reviewed by Jaeger et al., Annual Reviews in R;rl~hPmt~try 62, 255-287 (1993).
Another approach is to utilize two or more antisense compounds in tandem, at least one of which is a chimeric 15 oligonucleoside of the invention, which antisense com-pounds have nucleoside base sequences selected to hybrid-ize to adjacent regions in a secondary-structured mRNA
target region . It is known that adj acently-hybridizing antisense compounds may be used to disrupt secondary 20 structure of RNA molecules and thus to enhance the effec-tive KA~ 8 of the respective ~ rt~. By using this approach, cleavage of target mRNA regions having secondary structure may be achieved with specificity using oligonu-cleoside compounds having controlled binding af f inity as 25 taught herein.
As ~ s-~1Rced above in the background section of this disclosure, a number of workers in the ;Int; C~nCe field have reporte~l various and disparate efforts to increase binding af f inity of antisense oligonucleosides, to opti-30 mize RNaseH activation, to improve nuclease resistance,and to improve target specif icity . It will be seen in light of the ~ preceding detailed description that many of these approa~hes involve competing or conflicting consid-erations. For example, as just discussed, increased 35 binding affinity is not always desirable in view of the problems it can create for target specificity. Certain structures that provide increased binding affinity, such WO95/13834 _ ~t 762$9 PCTNS94/l3387 as 2~-methoxy substitutions, or increased nuclease re8is-tance, such as methylphosphonate ;n~rn~ eoside linkages, are seemingly incapable of activating RNaseH cleavage.
Conversely, certain 6tructures that provide high RNaseH
5 activation, such as phosphodiester linkages, are nuclease-unstable while others, such as phosphorothioate linkages (and also phosphodiester linkage6), may result in cleavage rates (k2) that approach or exceed the mismatch "Off rate"
(k 1), particularly in longer linkage sequences. The 10 present invention provides improved oligonucleoside structures that address these competing considerations and meet other goals as described herein.
The oligonucleoside compounds Of the invention com-prise linked nucleosides having a base sequence that is 15 complementary to a target region Of the target ribonucleic acid sequence, and include an RNaseH-activating region and at least one non-RNaseH-activating region. When used in conjunction with l;;ln RNaseH (e.g., in l;;ln cellular systems), the RNaseH-activating region comprises, 2 0 in the pref erred embodiment, a segment of between 5 and about 9 consecutive 2'-unsubstituted nucleosides linked by 4 to about 8 charged (anionic) internucleoside linkage structures. When used in conjunction with bacterial RNaseH (e.g., in bacterial c~ r systems or in antibac-25 terial therapy in mammals), the R~aseH-activating region comprises, in the preferred ~ ; , between 3 and about 7 consecutive 2'-unsubstituted nucleosides linked by 2 to about 6 charged ; nt~rnllrl ~c~ide linkage structures .
The non-RNaseH-activating region comprises, in one 30 preferred ~mho~;- t, a single segment of at least 3 linked nucleosides, and more preferably at least about 5 linked nucleosides, cc~n~;n;n~ one or more chirally-selected Rp- linkages . In a related second pref erred embodiment, the non-RNaseH-activating region comprises two 35 separate flanking segments, each segment ront~;n;n~ at least about 2 linked nucleosides, and more preferably at lea8t about 4 linked nucleosides (or a total of at least _ . . . ,,, _ WO 9~113834 PCr/US94/13387 217~259 , ~

about 8 linked nucleosides in the two separate segments), wherein one or more of the l;nk~ is a chirally-selected Rp-linkage. The RNase~-activating region is preferably f lanked in the compound by two such separate non-RNaseH-5 activating regions. In a third related preferred embodi-ment, the non-RNaseH-activating region comprises an alt~rn~t;n~ se~uence of racemic (non-chirally-selected) internu~l~n~ linkages comprising ~l) a racemic methyl-(or lower alkyl- ) phosphonate (NP), methyl- (or lower lO alkyl-) phosphonothioate (MPS), aminoalkylphosphonate (AAP) or ~m;nr~lkylphosphonothioate ~AAPS) linkage, alternating with (2) a negatively-charged phosphate, phosphorothioate or phosphorodithioate (e.g., DE, PS, or PS2 ) linkage . In any of the above embodiments, one or 15 more of the nucleosides in the non-RNaseH-activating region may be 2 ' -substituted, particularly to increase binding affinity and nuclease resistance while controlling (selectively decreasing or eliminating) RNaseH-activation characteristics. It is particularly preferred that one or 20 more, or all, phosphodiester linkages, if present in the non-RNase~-activity region, be 2 ' -substituted, although further 2 ' -substitutions may also usefully be employed in the non-RNaseH-activity region.
As an example, the phosphonate irLternucleosidyl 25 linkages used in oligomers of the present invention may contain a lower alkyl group replacing one of the two non-bonding (or non-bridging) oxygens on the phosphorus of a rh~gph~tl;egter ;nt~rn~ 1eosidyl linkage, wherein the other non-bonding oxygen remains or is alternatively replaced by 30 sulfur. The replacement of oxygen by lower alkyl creates a chiral environment around the ph~)srh~rus which can be designated as either Rp or Sp, depending on which of the non-bonding oxygens has been replaced with lower alkyl.
The Rp and Sp configurations can be dep~cted as follows:

WO 95/13834 2 1 7 6 2 5 g PCr/uss4~l3387 Il 11 o_~3 oi~ ~
wherein X is oxygen or sulfur and R is lower alkyl.
Applicants have discovered that the binding affinity of the present R~aseH-activating oligonucle~side compounds can usefully be controlled by selectively incorporating into the compounds polynucleoside segments crntA;nin~
chirally-selected internucleoside linkage structures.
Such chirally-selected Rp-rich segments afford greater binding affinity than the corresponding racemic sequences.
Applicants have also discovered that selectively-increased binding af f inity and improved nuclease resistance can be acl1ieved in a practical fashion, with or without chiral enrichment, using multiple or repeated blocks or synthons comprising both charged (;nrl~ inr phosphodiester) and uncharged (particularly racemic or chirally-selected methylphosphonate) internucleoside linkage structures.
Such synthons preferably do not have more than one consec-utive charged linkage structure in their sequence, partic-ularly if the charged (anionic) linkage structure is a phosphodiester bond.
These controllable binding affinity polynucleoside segments oi the invention provide the benef its of in-creased nuclease resistance, controllable RNaseH-activa-tion characteristics and ease of synthesis. Thus, for example, the linkage structures can be chosen to include one or more uncharged modified (non-rhn~rl~n~ qter) linkage structures which will be substAnti~lly non-acti-vating to RNaseH and al80 nllrl~A~e-resistant~ Use of 2'-substituents as described herein also leads to increased nuclea~e re6istan~e of ~ _ t~ including charged linkage _ _ _ _ _ _ . ,, .. . _ .. .. . . ..

Wo 95113834 ~ ~ 7 ~ 2 5 ~ PCrlUSg4/13387 structures, particularly phosphodiester linkages. Fur-thermore, individual synthon6 can be prelim;n=rily assem-bled as synthetic blocks which are then readily combined to provide a controllable binding af f inity segment con-5 taining two or more dif f erent block structures, or asingle repeated block structure.
While the described technic~ue of chiral selection can usefully be employed in both the RNaseH-activating and non-RNaseH-activating regions of the present compounds, it lO is most advantageously used in the latter region. In addition, chiral selection is preferably achieved with multiple or repeated mixed linkage structure blocks as described h~r~; n= f ter .
A chirally- selected polyn~ segment of the 15 present invention includes a sequence of ;nt~rnllcleoside linkage structures that is enriched or pure with respect to Rp chiral linkages. Such a sec~uence is considered chirally-enriched if at least about 75% of the chiral (asymmetric) linkage structures in the segment, or alter-=
20 natively at least about 40% of the total linkage struc-tures in the segment, have Rp chirality A8 shown below, chiral enrichment of at least about 75% can be achieved gynth~t;~lly by coupling a series of dimer nucleoside blocks (synthons) wherein the structure linking the two 25 r)t1~ 1 er-sides of each synthon is a modif ied (non-phospho-diester) Rp-chiral linking structure, and wherein the linking strtlcture between the respective synthons is asymmetric. The coupling reaction between synthons in the series will, in the simplest case, be carried out racemi-30 cally, which means that about half of the inter-synthon linkages will be Rp-chiral and about 75% of all of the internucleoside linkages in the resulting mixed chiral/racemic segment will be Rp-chiral. (It should be noted that the ~racemic~ reaction may be driven more 35 toward one diastereomer in particular cases; for example, investigations related to the present invention have shown that coupling of 2~ -O-methyl-substituted methylphosphonate WO 95/13834 . PCT/US94113387 2-~ 76259 ~nn~ ~: leads preferentially to Sp-chiral internucleoside linkages . ) It will be seen that chiral enrichment in excess of 75~ of the asymmetric linkage5 can be achieved by, for example, conjugating trimer nucleo5ide synthons wherein both internucleoside linkages within the block are Rp-chiral and the respective trimer synthons are conjugated racemically (or ~rh;r~lly). Synthetic schemes are shown below f or the preparation of such trimer synthons .
Alternatively, conjugation between individual nucleosides or between synthon5 can be carried out stereospecif ically using asymmetric linkage structures, in which case all the linkages in the segment will be Rp-chiral. While it is not considered nf~ c~ry to the preferred practice of the present invention to obtain segments having chiral enrich-ment in exce8s of about 75~ of the asymmetric linkages (or about 40% of the total linkages), such highly-enriched segments will generally exhibit higher binding af f inity characteristics .
AS seen above, a mixed chirally-selected segment of the invention may include within it one or more achiral (non-asymmetric) linkage structures. Thus, in one pre-ferred structure of~ the invention, a mixed chirally-selected segment is composed of a1t~rn~t;n~ phosphodiester (achiral) and Rp-methylphnsrhnnRt~ (or other chiral) linkage structure5. Such a repeated alternating linkage sequence segment can be prepared using dimer nucleoside blocks wherein the structure linking the two nucleosides of the block is an Rp-chiral methylphosphonate linkage structure, and where the blocks are conjugated achirally using a phosphodiester (or other achiral) linkage struc-ture. It will be seen that a polynucleoside segment prepared in this manner will be chirally pure ;nz~ h as all of the chiral linkages in the segment are of the Rp conformation, whereas subst~nti~l ly 50g~ of the total linkages will be Rp-chiral.
_ _ , . .. _ _ . . . . . .

Wo 95/13834 PCrlUS94/13387 ,21~;25 The inventors have a3certained in investigations relating to the invention that enrichment of methylphos-phonate ~p linkages gives an increase in melting tempera-ture (Tm) of about 0.9 to 1.5 C per internucleosidyl linkage that is in the Rp conformation as compared to a random racemic conformation. This translates into an increase in binding affinity (KA) by a factor of about 1.8 for each additional selected Rp linkage (or a factor of about 2 . 6 in the case of 2 ' -0-methyl-substituted resi-dues). It will now be appre~ciated that, by the judicious use of chirally-selected linkage structure segments in the present compounds, binding affinity can be controlled in a manner consistent with the objectives set forth above in the detailed description. The examples below demonstrate that increased potency can be achieved with such chirally-Eielected compounds, as compared to racemic compounds, while maintaining specif icity against the intended target sequence .
As P~r~;nP~ above, another objective of the inven-tion is to provide oligonucleoside structures having controlled R~aseH activation characteristics. This objective is obtained in the present invention by provid-ing in the compound a non-RNaseH-activating polyn~lrlPnc;de region, or regions, having reduced RNaseH-activation ~r~h; l; ties, along with an RNaseH-activating region having sufficient RNaseX-activation r~r~hil;ty to effect RNaseH-mediated cleavage of the target nucleic acid strand. Preferably, both of these segments of the com-pound are constructed to be nuclease resistant.
As is also explained above, one putative requirement of mammalian RNaseH activation is that the antisense compound must have a sequence of at least f our or f ive crncerllt~ve charged (anionic) internucleoside linkage structures (or at least two such linkages in the case of bacterial RNaseH), wherein the linked nucleosides are 2 ~ -unsubstituted. Conversely, in the practice of the present invention, the non-RNaseH-activating segment can usefully Wo 95/13834 2 1 7 ~ 2 ~ 9 PCr/US94113387 include uncharged linkage structures and/or 2 ' -substi-tuents. By making use in the non-R~aseX-activating region of modified (non-ph~-srhn~l; PCter) uncharged linkage struc-tures such as those described herein, the present com-pounds achieve increased nuclease resistance. Moreover, the use of 2 ' -substituents as described herein leads to selectively controllable increa8es in binding af f inity .
Thus, the inventors have ascertained in investigations relating to the present invention that the use of 2'-0-methyl nucleosides in methylph~srhnn~te-linked oligomers results in additional increases in T= o~ about 1C per substitution of 2'-deoxy with 2'-0-methyl nucleosides.
Furthermore, the inventors have ascertained that the use of 2~ -substituents on nucleosides linked by phosphodieater bonds also leads to increased nuclease resistance.
Consistent with these obj ectives, pre~erred 2 ~ -substituents of the invention include lower (l to about 3 carbons) alkoxy, allyloxy, and halo (preferably fluoro) substituents. A methoxy group is especially preferred.
In general, 2 ~ -substituents that are electron-withdrawing are useful in increasing the binding affinity and nuclease resistance of the present compounds, as such substituents are believed to create a 3 ' -endo conformation in the substituted sugar group.
It has further been discovered that a limited propor-tion of charged linkage structures, ; n~l u~; ng phospho-diester linkages, may usefully be incorporated into the non-RNaseH-activating segment, particularly in a linkage setauence c~r~;n;n~ multiple or repeated blocks of charged 3 0 and uncharged linkage structures . Such segments lead to controllable increases in binding affinity, nuclease resi8tance, and controlled RNaseH activation characteris-tics, and result in compounds having r~nh~nr Pd speci~icity for the; nl-r~n~r~r~ target nucleic acid se~auence .
Preferred linkage structures and 2~-substituents for the non-RNaseH-activating se_ c of the invention include the f ollowing:
. _ _ _ _ _ _, . . , _ . ,,, .. . , _ _ WO95/1383~ PCrlUS94/13387 MP (R) /DE
2 ' OMeMP (R) /2 ' OMeDE
NP (R) /2 ' OMeNP
NP (R) enriched 2 ' OMeNP ~R) enriched NP ~R) /PS
2 ' OMeNP ~R) /2 ' OMePS
NP ~R) /PS2 2 ' OMeNP (R) /2 ' OMePS2 2 ' OMeMP/2 ' OMeDE
NP/2 ' OMeDE
MP (R) /PAm 2 ' OMeMP (R) /2 ' OMePAm 2 ' OMeNP / 2 ' OMePAm NP/2 ' OMePA
MP (R) /TE
2 ' OMeNP (R) /2 ' OMeTE
2 ' OMeNP/2 ' OMeTE
MP/2 ' OMeTE
MP (R) /MPS
2 ' OMeNP (R) /2 ' OMeNPS
2 ' OMeNP/2 ' OMeNPS
NP/2 ' OMeMPS
NP (R) /PF
2 ' OMeNP (R) /2 ' OMePF
2 ' OMeNP/2 ' OMePF
NP/2 ' OMePF
NP ~R) /PB~J
2 ' OMeNP ~R) /2 ' OMePBi}3 3 0 2 ' OMeNP/2 ' OMePBH3 NP/2 ' OMePB~3 NP ~R) /RSi 2 ' OMeMP ~R) /2 ' OMeRSi 2 ' OMeMP/2 ' OMeRSi MP/2 ' OMeRSi MP (R) /CII~
2 ' OMeMP (R) /2 ' OMeC~2 wo 9~13834 ~ 1 7 ~ 2 5 9 PCT/US94113387 2 ' OMeMP/2 ' OMeCH~
MP/2 ' OMeCH, 3~ey: MP = racemic methylphosphonate linkage ~between linked nucleoside~); MP(R) = chirally-selected Rp-methylrhnsrhnn~te linkage; DE = rhnsrhn~;ester linkage, PS = phosphorothioate linkage; PS2 = phos-phorodithioate linkage; PAm = phosphoramidate link-age; T3 = phosphotrieæter linkage; NPS = alkyl (particularly methyl) phosphorothioate; PF = phos-phorofluoridate linkage; PBH3 = boranophosphate linkage; RSi = silyl (especially alkyl-disubstituted silyl) linkage; CH, = formacetal linkage; 2'0Me - 2'-methoxy-substituted (or other lower alkoxy, allyloxy or halo substituted) nucleoside residue, linked using the listed linkage structure; "enriched" refers to a segment of linkages preferably rQnt~;n;nr at least about 4096 (and up to 100~6) Rp-selected 7;nk;~ c among the linkages in the segment, and thus includes a mixed -seQIuence of racemic and chirally-selected R
internucleoside linkage structures; linkage struc-tures grouped with slashes denote a mixed linkage segment ;nr~ ;nr the listed linkage structures optionally in a series of multiple or repeated mixed linkage sequence blocks.
Also preferred are compounds having a segment chosen from the above listing wherein one or more (or all) of the methylphosphonate (MP or MP (R) ) linkages are replaced with lower alkyl-, especially methyl-, phosphonothioate (MPS or MPS(R) ) linkages, or with ~m;nn~lky~rhn~rhnn~te (AAP or AaP(R)) or ~m;nn~lkylphosphonothioate (AAPS or AAPS(R)) linkages . Such compounds include 2 ~ -substituted residues cnnt~;n;nr such linkages, as well as . ~ Jul~ds "enriched"
in these Rp-chiral linkages. Examples of the latter include compounds having an alternating se~uence of MP
(racemic) and AAP (R) linkages, or an alternating sequence of MP(R) and AAP (racemic) linkages, or an alt~rn~tins sequence of ~AP (racemic) and AaP (R) linkages . Also preferred are compounds chosen from the above listing wherein one or more (or all) of the Rp-chiral methylphos-phonate (MP(R)) linkages are replaced with racemic methyl-phosphonate (MP) linkages, preferably in an alternating sequence with a second dif f erent linkage structure, and most preferably in an alternating or other mixed sequence wo gSrl3834 2 1 ~ ~ 2 5 9 PCTrUS94rl3387 , i.

with phosphodiester, phosphorothioate or phosphorodi-thioate linkages.
Each of the mixed linkage segments listed above will contain at least one of each of the linkage structures 5 listed. From a aynthetic standpoint, it may be convenient to alternate the listed linkage structures or to use a repeated sequence rnrlt:~;nlng both structuree, although this is not n-or~q~ry. Two or more of the mixed linkage segments listed above may be serially combined within a 10 given non-RNaseH-activating region of the compound. In this case, it may be convenient from a synthetic stand-point to select discrete synthons from the respective mixed linkage groups and ~combine them in the single region .
Thus, it will be seen that the pr~sent invention provides synthetic oligomers having one or more segments ;nr~ ;nr~ mixed int~rnllrl~osidyl linkages, particularly oligomers having chirally pure or enriched rhr,sphrn~l~r ; nt~rnllrleosidyl linkages interspersed with single non-20 rhnqrhrn~te internucleosidyl linkages and methods for their preparation. Such phosphonate internucleosidyl linkages include lower alkylphosphonate ; ntPrnllrl eosidyl linkages of 1 to 3 carbon atoms and lower alkyl rhr~rhr,nn-thioate (alkylth;r~ph~rhrn~te) internucleosidyl linkages 25 of 1 to 3 carbon atoms. These mixed oligomer segments preferably have phosphonate ;nt~rnl~rlP~ yl linkages interspersed between single non-rhnsrhrn~te internucleo-sidyl linkages in a ratio of from 1 to about 1 to 1 to about 4 non-phosphonate linkages to rh~l~rhr~n~te linkages.
3 0 According to a pre~erred aspect, such oligomers have alternating chirally pure phosphonate internucleosidyl l;nki~r~ which alternate with non-phosphonate ;nt~rnllrleo-sidyl linkages. Oligomers comprising such segments, particularly in one or more non-RHaseH-activating regions, 35 may be used to prevent or interfere with expression or translation of a single-stranded RNA target sequence The chimeric oligonucleosides have an overall nucleoside base -2~762$9 seriuence, inrl~ ;nrJ the RHa8eH-activating and non-RHaseH-activating regions, which is sufficiently complementary to the RNA target sequence to hybridize therewith.
Preferred chirally pure phosphonate linkages include 5 Rp lower alkylrhr~sphnr)~te linkages, and more preferred are Rp methylphrsrhnn~te ;nt~rn1~rleo~idyl linkages. Preferred non-phosphonate linkages include phosphodiester, phos-phorothioate and phosphorodithioate, while phosphorami-date, phosphorofluoridate, boranophosphate, formacetal and 10 silyl int.orn11rlPosidyl linkages may also be used. Accord-ing to an especially preferred aspect, Ry-enriched oligomers are provided having chirally pure Rp-methyl phrsrhnn~te linkages which alternate with phosphodiester linkages in the non-RHaseH-activating regir~n of the 15 compound . These alternating oligomers have been f ound to exhibit ~nh~nrf~rl binding affinity for an R~A target ser~uence and also increased nuclease resistance and specif icity .
The present invention likewise ; nrl ~ chimeric 20 antisense oligomers having ~nhAnrod potency as antisense inhibitors of gene expression comprising one or more segments with methylphosphonate ;ntl~rn~1rl~osidyl linkages F~nh:~nr~d for the E~p configuration which are interspersed between non-rhr.srhrn~te ;nt~rn11rleositlyl linkages, prefer-25 ably phosphodiester or alternatively rhn8rhnrothioate orrhr~rhnrodithioate linkages. We have found that chirally enriched oligomers hybridize more tightly to RNA target sequences and should show Pnh~nr~tl potency inhibiting translation of RNA targets as compared with oligomers 30 having racemic MP ;nt~rn11rleosidyl linkages mixed with the same non-phosphonate internucleosidyl linkages.
As explained above, the RNaseH-activating region of the present invention can have varying minimum and optimum lengths ci~r~nrlin3 on the species (~ n or bacterial) 35 of the RNaseH enzyme that is utilized for cleavage. In either case, the RNaseH-activating region preferably compri8es a sequence of Consecutive 2 ' -unsubstituted _ _ _ _ _ _ _ _ _ _ . . . .

21~62S~
32 ~
nucleosides linked by charged 1 nt.orn11cleoside linkage structures. Preferred linkage structures and mixed linkage structures for the RNaseH-activating region are selected from among the following:
DE

PS

PS /DE

One especially preferred linkage structure is the phos-phorothioate (PS) linkage.
In a related embodiment, two oligonucleosides of the invention having t~rmin~lly-positioned RNaseH-activating 15 regions may be used in tandem to effect cleavage of a target mRNA site. The nucleoside base sequences of the reepective compounds are selected to be complementary to adj acent regions in the target mRNA strand. The RNaseH-activating regions may be used in tandem to effect cleav-20 age of a target mRNA site. The RNaseH-activating regions are situated at the 5'- t~ n~ and the 3'-terminus of the respective ~ de such that, upon co-hybridization to the adj acent regions in the target, the two RNaseH-activating regions abut one arother and are hybridized to 25 adjacent target subregions in the overall target region of the mRNA strand. The two R~aseH-activating regions act to complement one another with respect to RNaseH-mediated cleavage of the target region. Shorter RNaseH-activating regions may be used in the two compounds than might 30 otherwise be required, and specificity should be increased to the extent that dual hybridization is required to ef f ect cleavage .
Chimeric oligomers of the invention, or segments thereof, hav~ng a predetermined base sequence of nucleo-35 sidyl units and having chirally pure rhnsph(~n~t~ inter-n~1r~ idyl linkageg mixed with non-phosphonate 1; nk~
wherein the phosphonate linkages are interspersed between Wo 95/13834 2 1 ~ 6 2 5 9 Pc~r/uss4ll33~7 single non-phosphonate linkages may be prepared by cou-pling to one another individual nucleoside dimers, trimers or tetramers of preselected nucleoside base sequence having chirally pure or race~ic phosphonate or other 5 int~rn~ idyl linkages.
In this regard, chirally pure or racemic synthons of the formula:
O Z
X I o ~0 j -- --n \
O Z
\Cp lO may be utilized wherein X is oxygen or sulfur, R is lower alkyl of l to 3 carbon atoms, Bl is a removable blocking group, Z is hydrogen, alkoxy o~ l to lO carbon atoms, halogen or alkenyloxy of 3 to 6 carbon atoms; ~3 is an optionally protected purine or pyrimidine base; n is l, 2 15 or 3 and Cp is a coupling group. The coupling group Cp is conveniently selected 80 as to give the desired non-n~te ' nt~ntl~l ensidyl linkage when coupled toanother synthon.
According to one preferred chirally-selective syn-20 thetic method, nucleoside dimers having a ~hnsrhnn~telinkage connecting the two ~ucleosidyl units of the dimer are prepared and separated into their Rp and Sj, isomers.
The resulting dimers which have a defined chirality at the _ _ , .. . . . _ . . . . .. _ _ _ . . . .

2~g wo 95113834 PcrluS94113387 ~ ., , . .
!-, 34 phosphonate linkage, a~e then derivatized so that they may be coupled together using an automated DNA synthesizer.
The dimers may have coupling groups which result in any one of a variety of internucleosidyl linkage6 between 5 dimers. From a stock of 16 dimers, oligomer segments of any nucleoside base sequence may be synthesized by linking together the a~Lu~Liate dimers. Dimers are added to the growing oligomer chain until an oligomer segment having the desired number of nucleosides is obtained. The 10 resulting oligomer segment has a defined chirality at every other internucleosidyl linkage (i.e., those linkages originally derived from the coupled dimeric units). The L~ ;n;n~ ;ntF~rn~ leo8idyl linkages comprise non-phos-phonate internl1n1~nR;rlyl linkages, such as phosphodiester, 15 phosphorothioate, phosphorodithioate, morpholino, phos-phoramidite, phosphorof luoridate, boranophosphate, f orma -cetal, silyl or other non-rhnRrhnn~te internucleosidyl linkages .
Alternatively, larger blocks of nucleosides such as 20 trimers and tetramers may be coupled to give a chirally enriched oligomer. Trimers having two chirally pure internucleosidyl linkages may be conveniently prepared by coupling the c-~L-~l~Liate chirally pure dimer synthon to another nuclec)side and, for example, if Rp chirality is to 25 be selected, then separating the resulting Rp-Rp and Rp-Sp trimers. The resulting trimer has defined chirality (i.e., is chirally pure) at both inter-nucleosidyl linkages. The trimers are then derivatized to give trimer synthons 80 that they may be coupled together using an 30 automated D~A synthesizer. The trimer synthons have coupling groups which allow them to be coupled together to give a chirally enriched rhnsrhnn~t~ oligomer segment.
From a stock of 64 trimers, oligomers of any base se~uence may be synthesized by linking together the c-~L.,~Liate 35 trimers. Trimers may be seqll~nt;~11y added to the growing oligomer chain or alternatively coupled with nucleoside ~, dimers and/or tetramers until an oligomer Wo 95113834 PcrluS94113387 2~62~9 segment having the desired number of nucleosides is obtained. The reæulting chimeric oligomer has a defined chirality at those ;ntPrn~ Pnsidyl linkages in the chirally-seleCted 6egment derived from the internucleo-5 sidyl linkages of the coupled chirally-selected dimers, trimers or tetramers. Thus, use of these trimers will result in an oligomer segment having phosphonate linkages of def ined chirality at about two out of every three internucleosidyl linkages. By following analogous tech-10 ni~ues, tetramers having three chirally pure internucleo-sidyl linkages may be prepared and coupled to each other or to other synthons (including monomers) to give other chirally-selected segments or portions thereof. Alterna-tively, dimers, trimers and other short oligomers having 15 ;ntGrn1lnl~o~;~lyl linkages of defined chirality (such as pure Rp) may be coupled together or. to other synthons in d~L~L~Liate se~uence to give an oligomer segment or portion thereof of a particular desired se~uence and length. Such a chirally-selected segment cdn be coupled 20 with additional nucleosides forming a separate segment of the compound, particularly a segment of consecutive 2'-unsubstituted nucleosides linked by charged linkage structures forming an RHaseH-activating region.
According to an alternative synthetic method, cou-25 pling conditions for nucleoside synthons (or dimers) areused which direct coupling to give an PnhAn~P~ yield of the desired chiral-configuration. This method may be used to couple individual n~ nsi~P synthons or alternatively the chirally pure dimers and, thus, obtained are oligomer 30 segments, particularly non-RHase~I-activating segments, enriched for the desired chiral configuration at each of the ~hnsphnn~te ;ntPrn-l~ leosidyl linkages.
The chirally-selected methylE~hn~phnn~te and other ~ - ~, dimers, trimers and the like taught in the 35 examples and Detailed Description herein can be coupled together by a variety of different methods leading to the following, non-exclusive, types of ;n~Prnll-~leosidyl ~ ~ = == _ _ _ _ _ _ . . , . . . . _ .. _ .. . _ , .. . .. , _ WO 95/13834 ~ 1 7 6 2 5 9 PCr/US94113387 :. , ~.,, .. .

linkages: phosphodiester, phosphotriester phosphoro-thioate, phosphorodithioate, phosphoramidate, rh~l~rhf~ro_ fluoridates, boranophosphates, formacetal, and silyl.
Internucl eosidyl phosphodiester linkages can be obtained by converting the 3'-OH of a chirally-selected or racemic synthetic unit ~monomer, dimer, trimer, poly-nucleoside, etc. ) to either a phosphotriester synthon (Reese, C.B. (1978) Tetrahedron 34, 3142-3179), phosphora-midite synthon (Beaucage, S.L. and Lyer, R.P. (1992) Tetrahedron 48, 2223-2311), H-phosphonate synthon (Froehler, B.C. in Agrawal, S., ed. Protocols for Oligonu-cleotides and Analogs, Synthesis and Properties, Methods in Molecular Biology Vol. 20, Humana Press, Totowa, NJ, 1993, pp. 63-80), or phosphoromonochlo~idite reagent (Hogrefe, R.I. (1987) dissertation, Northwestern Universi-ty, Evanston, IL).
Tn~rnllrleosidyl phosphorothioate 1 ;nk~rJ-~F can be obtained by converting the 3 ' -OH of a synthetic unit to either a phosphotriester synthon (Stec, W.J., et al.
(1991) Nucl. Acids Res. 19, 5883-5888) ), phosphoramidite synthon (Lyer, R.P., et al. (1990) JACS 112, 1254-1255), H-phosphonate synthon (Seela, F. and Kretschmer U. (1991) J. Org. Chem. 56, 3861-3869), or rh~Rrhnromonochloridite reagent (Hogrefe, R.I. (1987) Dissertation, Northwestern University, Evanston, I~. ) .
Internucleosidyl rh~rh~rodithioate linkages can be prepared as by the disclosures herein and by U . S . Patent No. 5, 213, 088 to Gorenstein et al . Internucleosidyl phosphotriester linkages can be obtained by converting the 3 ' -OH of a synthetic unit to either a phosphotriester synthon (ReeGe, C B. (1978) Tetrahedron 34, 3143-3179), phosphoram.idite synthon (Beaucage, S.~. and Lyer, R.P.
(1992) Tetrahedron 48, 2223-2311), H-phosphonate synthon (Froehler, B.C. in Agrawal, S., ed. Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Methods in MrlPrlll~r Biology Vol. 20, Humana Press, Totowa, NJ, 1993 , pp . 63-80), rh~ h~romonochloridite Wo 95/13834 2 1 7 6 2 5 g PCTIUS94/13387 reagent (Hogrefe, R. I . (1987) Di8sertation, Northwestern University, Evan5ton, IL.), or post synth~t;r~lly (see U.S. Patent No. 5,023,243 to Tullis.
Int~rn~lrlensidyl phosphoramidate, phosphorofluor-5 idate, boranophosphate, formacetal, and silyl linkages canbe obtained by converting the 3'-OH of a 8ynthetic unit to the ~L-~Liate synthons. (See Agrawal, S., ed. Protocols f or Oligonucleotides and Analogs, Synthesis and Proper-ties, Methods in Molecular Bioloqy Vol. 20, Humana Press, 10 Totowa, NJ, 1993, for synthetic protocols to obtain synthons for each of the above. ) Chemical structures for synthons and reactive inter-mediates useful in the present invention are depicted in FIGS. 6-10, and are discussed in further detail in U.S.
Patent Application Serial Nos. 08/154,013 and 08/154,014.
The following example8 demonstrate various signifi-cant aspects of the present invention, but are examples only, and should not be considered as limiting the scope of the present invention.
r 1,,~
r le 1 Pre~aration of MP(R~) /DE and ME ~l?s) /MP Dimer Svnthr~nc A. Preparation o~ a ~CT) Dimer Havinq a ~h;~all~ pl~re Methvl~hosl~honate Internudeosidvl T-; nk~qe Usinq Solution Pha8e Chemistrv Into a 2 L roto-evaporator flask was placed 10 . 0 g (28 mM) of 3 ' -tert-butyldimethylsilyl thymidine and 26 .1 g ( 3 5 mM ) of 5 ~ - dime thoxytri tyl - NÇ - i 8 obutyryl - 3 ~ - me thyl -N, N- di i sopropyl ~m; noFhr~sphoramidite - 2 ~ - deoxycytidi~e . The solids were dissolved in 500 ml of acetonitrile and evaporated to dryness under vacuum. This process was repeated with another 500 ml of acetonitrile and then the flask was released under argon and stoppered with a rubber ~epta .
... . . . . . . .

W095/13834 21 762S9 PCr/Uss4ll3387 .

This dry solid foam was then dissolved in 500 ml of acetonitrile ( "ACN" ), and with manual stirring, treated all at once with 404 ml tetrazole (180 mM, 0.45 M
tetrazole in THF) . Manual stirring is ~ nt;n~ l for 30 seconds and then the flask is allowed to stand for another 2.5 minutes, after which time the reaction mix is treated all at once with 275 ml of an oxidizer solution (I2/H20/lutidine/THF; 25 g/2.5 ml/100 ml/900 ml). The solution was stirred manually and allowed to stand at room temperature for 15 minutes. The resulting dark amber solution was then treated with bisulfite (2 g/25 ml H20), which upon addition, turned the solution light amber as it reacted with the excess iodide. The reaction mix was then concentrated to a thick oil and taken up in ethyl acetate ~"EtOAc") (500 ml) and washed with saturated sodium bicarbonate (2 X 250 ml) and H20 (2 x 250 ml). The organic phase was dried over MgSOi, filtered and ~ nrf~ntrated to a light colored solid foam, which upon further drying yielded 35 grams of crude dimer.
The crude dimer was run on HPLC (reverse phase, Waters C18 b~n~r~k) with a program (ACNMETH) starting with 5096 acetonitrile and O.1 M triethylammonium acetate (TEA~, pH ~ 7.0) which increased to 1009~i acetonitrile over 20 minutes with a linear gradient. Two major peaks were resolved, one at 4.5 minutes, which is residual lutidine and the other at 14 . 5 minutes which is the mixture of Rp and Sp diastereomers. The ratio of Rp and Sp was determined tauantitatively by taking a 5 mg ali~[uot of the crude product and dissolving it in 1. 5 ml of acetonitrile along with 0.5 ml of tetrabutyli 11-m fluoride (TBAF, 1 M
solution in THF) . After standing at room temperature for 10 minutes the sample was run on HPLC Two new peaks were observed at 6.5 and 7.1 minutes and the later eluting peak was gone. The first new peak, which is believed to be the Sp diastereomer, represented 66~6 (2/1) of the normalized value for the two peaks. The crude product was also analyzed by the (normal phase silica plate) in 75/25 -~ 2~76~
WO 95113834 - . PCrlUS94113387 EtOAc/CH2Cl2 ("75/25") with 5~ methanol added. The TLC
6howed two spots with Rf's of 0.45 and 0.64, respectively;
the faster running product (believed to be the Rp form~ was less intenge than the slower moving one.
The Rp diastereomer was separated on normal phase silica using a methanol step gradient in 75/25 EtOAc/CH2Cl2 . A 7 . 5 cm by 60 cm column, was loaded with 700 g of silica (first slurried in 2.5 L of neat 75/25 EtOAc/CH2Cl2) . The crude dimer was then dissolved in 75 ml of 75/25 EtOAc/CH,Cl2 and loaded onto the column. The column was started with 1~ methanol and increased to 29~
and finally 3~ where the Rp dimer began to elute. The Rp dimer eluted cleanly over several bed volumes while m~;nt~;n;n~ 3~ methanol in the eluent. The Sp dimer was eluted later with 30~ methanol. The Rp dimer yield was 11. O grams, while the Sp yield was 17 . 8 grams . HPLC
analysis (ACNMETH) was performed on the Rp dimer and one peak was observed at 14.5 minutes. The TLC (75/25 EtOAc/CH2Cl2, 5~ methanol) o~ this product, revealed a single spot product with an Rf of 0.55 which, upon treat-ment with 10~ sulfuric acid in ethanol and heat, was both trityl and 6ugar positive.
The newly resolved Rp dimer, 11. 0 g (O . 011 M) was dissolved in 110 ml of ACN and treated all at once at room temperature with 22 ml of TBAF (O . 022 M, 1 M in THF) . The reaction mixture was allowed to stand overnight at ambient temperature. The next morning the reaction was determined to be complete by TLC (75/25, EtOAc/CH2Cl2 with 10~
methanol); no starting material was detected but a small amount of 5'-DMT-dT was observed, which runs considerably faster on normal phase silica than the 3'-OH of the dimer.
The reaction mixture was .-nncl~ntrated on a rotary evapora-tor to a thick oi~ which was then dissolved in CH2Cl2 (200 ml) and washed with saturated sodium bicarbonate (2 x 100 ml) and H,O (2 x 100 ml) . The organic phase was dried over MgSO~, filtered, and c~lnr~ontrated to a light yellow solid foam, which was puri~ied on 100 grams of silica ~75/25, _, . . ... . . .... _ _ _ .. _ . .

Wo 9~13834 , ~' PCT/US94/13387 X1~ 9 EtOAc/CH2Cl2 with 596 methanol). The 5'-DMT-dT was removed but an impurity at 13 . 5 minutes (HPLC, ACNMETH) was detected which was first believed to be unreacted starting material (t-BDMS on) but after ~;t;nni:ll treatment with TBAF this was found not to be the case. A second column, using 100 g of silica and the same eluent was run and smaller fractions were taken; the column was able to successfully separate the two spots. The pure CT-Rp dimer fractions were pooled and cnn~ntrated to yield 5.5 grams of a nearly white solid foam.
B. Pre~aration of a ChirallY Pure ~CT) MP(R~) /DE Dimer SYnthon Into a 100 ml round bottom flask was placed 0.5 g (0.55 mMol) CT-3'-OH dimer (product of Example lA) which was rendered anhydrous by 3 x 20 ml co-evaporations with pyridine. The flask was released from the vacuum system under argon gas and stoppered with a rubber septa. The compound was redissolved in 10 ml acetonitrile and 200 /Ll ( 1. 4 mMol, 2 . 5 eq) TEA were added . To the resulting mixture at room temperature and with manual stirring, was added in one portion 200 ~l (0.90 mmol, 1.6 eq.) 2'-cyanoethyl-N,N-diisopropylchlorophosphoramidite. The reaction mixture was allowed to sit at room temperature bef ore being analyzed by reverse phase HP~C . The HP~C
(Beckman System Gold, C18 bnn~r~lk, ACN method; Solution A was 50/50 ACN/0.1 M TEAA in water, pH 7 and Solution B
was ACN; a gradient of 0 to 1009~ Solution B was run at a rate of 1 ml/minute over 25 minutes) showed complete conversion of starting material and a crude purity of greater than 90 percent. The diastereomers of the phos-phoramidite were not resolved. The reaction mixture was n~Pntrated under vacuum to a light yell solid foam. The foam was purified immediately by L:I1LI tngraphy on 20 g of normal flash grade silica equilibrated with 5/1/5 ethyl acetate/ acetonitrile/methylene chloride with 2~6 TEA to give 0.5 g (82~ yield) of the above-identified product as ~76259 WO 95/13834 PCr/US94/13387 an off-which solid foam having a purity of 99.39~ as determined by HPLC.
C. Preparation of a ChirallY Pure (CT~ MP (RF) ~MP Dimer SYnthon The CT-3 ' -OH dimer, 5 . 5 g (6 mM), prepared as de-scribed in part A above, was rendered anhydrous with two co-evaporations with pyridine. The resulting solid foam was released f rom the rotary evaporator with argon and stoppered with a rubber septa . The solid f oam was dis -solved in 100 ml of 9/1, ACN/CH2Cl" then treated with 1.7 ml triethylamine (TEA, 12 mM). lqith magnetic stirring, the reaction mix was treated dropwise at room temperature with 1.5 ml chloromethyl-N,N-diisopropylamino phosphine (Cl-MAP, 8 mM) . The reaction was monitored on HPLC
(ACNMETH) and a~ter 1. 5 hours was complete, showing two main products, one at 3.5 minutes which was pyridine and a second at 14 . 3 minutes which was the desired amidite .
The reaction mixture was cnn~n~rated on a rotarY
evaporator using a partial vacuum; the flask which con-tained the resulting light amber sludge was released under argon and capped. The crude product was immediately passed through a flash column ~nn~1nin~ 60 grams of silica (first equilibrated in 1/1/1 ACN/EtOAc/CH2Cl2 with 3~ TEA). The product was eluted quickly with this eluent and all U.V. positive fraction5 were pooled and concen-trated. The resulting 501id foam wa5 co-evaporated with ACN to remove any residual TEA, then dried overnight under full vacuum. The fi~al product, an off white solid foam, weight 5. 0 grams.
3 0 E~cam~le 2 Preparation of (CU) 2'-0-MethYl MP(R~)/2'-0-MethY1 DE and 2' -Q-MethYl MP (R~ /2~ -O-MethYl MP Dimer SYnthons A. Pre~aration of 2 ' -0-MethYl C Monomer A 5.0 g (8 mmol) portion of 2'-0 methyl cytidine was re~ldered anhydrous with pyridine co-evaporations (3 X 25 ml~ and then dissolved in 50 ml acetonitrile. The solu-WO 95113834 . PCTNS94/13387 ~1762~9 tion was treated with 1. 65 ml triethylamine ( "TEA" ) (12 mmol, 1. 5 eq. ) and cooled in an ice bath. The solution was then treated with dropwise addition of 1.65 ml chloro-methyl-N,N-diisopropylamino phosphine ( "Cl-M~p" ) over two 5 minutes. The ice bath was removed and the reaction mixture stirred for two hours. The reaction mixture (reaction was determined to be complete by HPLC) was concentrated to dryness. The residue was dissolved in 20 ml ethyl acetate/heptane (1:1) with 4% TEA, then loaded 10 onto 40 g silica gel equilibrated with the same solvent system. All W absorbing eluent from the column was collected and pooled, then concentrated to give 5 . 5 g of the above-identified product (yield about 90%).
B . Pre~aration of Silyl-Protected 2 ' -0-Methvl Uridine Into a 250 ml round bottom flask was placed 5 . 0 g (9.0 mmol) 5'-DMT, 2'0-methyl uridine which was rendered anhydrous with dimethylformamide (DMF) co-evaporations (3 X 25 ml). The resulting dry foam was taken up in 50 ml DMF, then treated all at once with 2.4 g (35 mmol, 3.9 eq. ) imidazole, followed by dropwise addition of 3 . 0 ml (12 mmol, 1.3 eq. ) t-butyldiphenylsilyl chloride. The reaction mixture was stirred at room temperature over-night .
The progress of the reaction was checked by HPLC (ACN
method (Solution A was 50/50 ACN/0.1 M TEAA in water, pH
7 and Solution B was ACN; a gradient of 0 to 100% Solution B was run at a rate of 1 ml/minute over 25 minutes) and thin layer chromatography ("T~C") using 5% methanol in methylene chloride, and determined to be complete (no starting material was evident). The reaction mixture was then poured into ice water. and taken up in methylene chloride, then washed several times with aqueous sodium bi~rhrn~te and water. The organic phase was dried over magnesium sulfate, filtered and then ~ n~ ntrated to give 7 . 2 g of a solid foam which gave a single spot on TLC.
The solid foam was then dissolved in 70 ml methylene WO 95113834 - PCr/US94/13387 ~76259 chloride and treated (with rapid magnetic stirring) all at once with 70 ml benzene sulfonic acid, 2~ by weight in 2:1 methylene chloride/methanol. After stirring for 15 minutes at room temperature, the reaction mixture was quenched with 10 ml TEA. The resulting detritlylated compound was stripped down to a thick amber oil which was then loaded onto 150 g. 8ilica gel equilibrated in heat methylene chloride . The product was eluted f rom the column using 296 methanol (in methylene chloride). After drying, 3.51 g of the above i~Ph~;~;.od product were obtained (yield about 80~).
C . Pre~aration of (CU~ 2 ~ -O-MethYl 25P (P~ ) Dimer The silyl-protected 2 ' -0-methyl uridine monomer (product of Example 2B) (3 . 0 g, 6 mmol) was taken up in 30 ml anhydrous ACN . The 2 ' -0 methyl cytidine amidite monomer (product of Example 2A) (5.5g, 7 mmol, 1.2 eq.) separately, was taken up in 55 ml ACN. Both solutions were allowed to stand over 3 A molecular sieves overnight at room temperature.
The two solutions were carefully decanted into a single flask and treated with 94 ml tetrazole (0.45 M in ACN, 42 mmol, 7 e~). The resulting mixture was stirred for 4 minutes and then oxidized by addition of 1.5 ml (1.2 eq. ) cumene hydroperoxide. The reaction mixture was r~ln~~~ntrated to dryness, then taken up in methylene chloride and washed with aqueous sodium bicarbonate and water. The organic phase was dried over magnesium sul-fate, filtered and concentrated to give 7.5 g. of a solid f oam . The diastereomeric ratio as determined by HP~C by comparison of areas under peaks was 57/43 Sp to Rp.
The Rp diastereomer was isolated by column chromatog-raphy using two silica columns (100:1, silica to crude product, equilibrated in 3:1 ethylacetate/methyl chloride with an increasing methanol gradient irom 1 to 5~). A
total of 1. 07 g oi pure Rp dimer was isolated.
, _ _ _ _, _ . , . . . . . . . -Wo95113834 2i7625la PCr/US94/1338 D . DeDrotection of (CH~ 2 ' -0-Methvl Dimer A 1.07 g (0.90 mmol) portion of the 2'-0 methyl CU
dimer (product of Example 2C) was dis501ved in 10 ml THF
and treated all at once with 1.5 ml (1 m in THF, 1.5 eq.) tetrabutylammonium fluoride ("TBAF"). The reaction mixture wae stirred at room temperature of r 3 0 minutes after which time HP3-C revealed complete deprotection of the silyl group had been achieved. The reaction mixture was concentrated and the ,-"n,Pntrate purified on 10 g silica gel, eluting with 3 :1 ethyl acetate/methylene chloride with 596 th;ln~ll. The clean fractions were rnnr-Pntrated to give 550 mg of the above-identified pure 5 ' -OH aimer .
E. Pre~aration of a ChirallY Pure (CU) 2'-0-Methvl (NP/DE) Dimer Svnthon A 230 mg portion of 2'-0-methyl CU 3'-0~ dimer (prod-uct of ExampIe 2D) was rendered anhydrous by 2 X ~ ml co-evaporations in ACN. The resulting dry solid foam was dissolved in 2.5 ml ACN and then 73 ~1 (2.5 eq.) triethyl-amine ("TEA") and 94 ~Ll (2.0 eq.) 2'-cyanoethyl-N,N-diisopropyl chlorophosphoramidite (,~CNE) were added. The reaction mixture was stirred at room temperature for 2 hours at which time EIP~C analysis determined the reaction to be complete. The reaction mixture was fl; RR~l VP~ in eluent (3/1/1 ethylacetate/acetonitrile/methylene chloride with 4~6 TEA) and loaded onto 2 g silica gel equilibrated with 3/1/1 ethylacetate/acetonitrile/methylene chloride with 496 TEA. The column was run using 3/1/1 ethylacetate/acetonitrile/methylene chloride with 196 TEA.
The clean fractions, 3 to 25, were concentrated, redis-solved in acetonitrile and concentrated again to a solid foam. The foam was dried overnight under full vacuum to give 200 mg of the above-irlpnt;r;ed product.

W0 95/l3834 2 ~ ~ ~ 2 S 9 PCINS94/13387 -F. Pre~arPtion of Ch;rally Pure (C~J) 2'-0-~ethyl ID?~R;) /NP Dimer Svnthon Into a 100 ml round bottom flask was placed 400 mg (0.372 mmole) of 2'-0 methyl CU dimer (product of Example 2D); it was rendered anhydrous by 1 X 5 ml co-evaporation with acetonitrile. The dry foam was then released from the vacuum system under argon gas, dissolved in 4 ml ACN
and stoppered with a rubber septa. The solution was treated with 2 equivalents TE~A (103 ~Ll, 0.744 mmol), followed by 1.75 equivalents chloro-methyl-N,N-diisopropyl rh~Srh;n~ ("Cl-MAP") (118 ILl, 0.651 mmol). The reaction mixture was stirred for 1 hour at room temperature, after which time HPLC showed about 50/50 starting material/product. An additional 50 ~l TEA and 70 ~l Cl-M~P were then added and the mixture stirred for an hour.
When HPLC showed only 80~ conversion, an additional 30 ~l TEA and 3 0 1ll Cl-MAP were added and the re8ulting mixture stirred another hour. At this time HPLC revealed 6~
starting material. The reaction mixture was concentrated to dryness. The residue was dissolved in 500 ml 3/1/3 e~hylacetate/acetonitrile/methylene chloride with 496 TEA
and loaded onto 5 g silica equilibrated in the same solvent system. Fractions were collected. The early fractions were c~- nt~m;n~tPd with a yellow impurity and, thus, were pooled and concentrated separately. The product from those fractions was then repurified by chromatography using the same conditions and pooled with the clean product isolated from the first column. The ~ .' in~d products were co-evaporated with ACN (3 X 5 ml) and dried overnight under full vacuum to give 350 mg (77~
yield) of the above i ~l.ont; f i ed product which HPLC Rhowed to be 95.59~ pure.

Wo95113834 ~ ~. PCr/Uss4/l3387 æ~6~5~

rnnle 3 -Pre~aration of 2 ' -O-MethYl MpS (1~ ) / 2 ' -O-MethYl-DE and 2 ' -Q-Methvl ~SPS (R~) / 2' -O-Methvl-MP Dimer SYnthons These dimer sYnthons are prepared ky f ollowing the procedures described in Example 2, except that in Para-graph C, an equivalent amount of 3H-1,2-benzodithiole-3-one, l, 1-dioxide (Beaucage reagent) is substituted for cumene l~y~L~ ide~ The L~ .,ceduL ::8 of Paragraphs 2E and 2F, respectively, lead to the phosphodiester and methyl-phosphothioate linkage combinations.
r le ~ ~
Pre~aration of MPS (R~) /DE Dimer SYnthons These dlmer synthons are prepared by following the procedures of Example 1, except in Paragraph A, an e~uiva-lent amount 3-E-l~2-benzodithiole-3-one~ 1,1-dioxide (Beaucage reagent) is substituted for the oxidizer solu-tion (I~/H2O/lutidine/THF) .
.nr)le 5 Pre~aration of MP (~ ) /PS2 Dimer Svnthons The MP(Rp)/PS2 dimer synthons are prepared as follows.
Isometrically pure Rp dinucleosides having a free 3'-OH are prepared according to the methods described in Example lA.
The dinucleoside is converted to the corrPspr~n~l;n~
th1 nph~sph~.ramidite using procedures such as those of Plotto et al. (Plotto et al, Tetrahedron 47:2449-61 (1991)) or Gorenstein et al., U.S. Patent No. 5,218,088.
The dinucleoside is co-evaporated three times with anhy-drous pyridine, followed by three co-evaporations with toluene. A portion of dinucleoside (10 mmoles) is dis-solved in 200 ml anhydrous dichloromethane, then three equivalents of anhydrous diisopropylethylamine followed ~y 1.5 equivalents of ~ chloro-N,N-diisopropylamino-th; ~ th~yphosphine are added at 0C with stirring. The reaction is monitored by TLC until ~pt~orm;npd to be complete.

WO 95/13834 2 1 7 ~ ~ ~ 9 PCI/US94/13387 The product i8 worked up and purif ied using the procedures of Example lB for isolation of the MP (Rp) /DE
phosphoramidite .
Examl~le ~i PreParation of ~SPS (R~) /PS2 Dimer SYnthons The MPS (Rp) /PS2 dimer synthon6 are prepared as follows. The isometrically pure Rp dinucleoside with a free 3'-OH is prepared according to the methods of Example 4. Using the dinucleoside, the dimer synthon is prepared by the methods of Example 5.
rnle 7 - =
Pre~aration of ~tPS(R~)/2'-0 Me~hY1 DE D;r-r ~Ynthon~
The MPS (Rp) /2 ~ -O-methyl DE dimer sYnthons are prepared using procedure6 analogous to those of Examples 1 and 3 but using the c.~L~,pLiate protected 2'-deoxynucleoside and protected 2 ' -O-methyl nucleosidea .
Exam~le 8 Prel:1aration of a PQlv-CT Oliqomer Xav;n~r Alternatinq MP (R~) /DE InternucleosidYl J,; nkAqes An oligomer having the sequence 5~ - (C T) - (C'T) - (C-T) -(C-T) - (C-T) - (C-T) - (C T) -A-3' was prepared using a C-T
MP (P~) /DE dimer synthon prepared according to Example 1.
The grouped dinucleosides indicate where the stereochemis-try is fixed as the fast eluting isomer on 8ilica gel (putative Rp) and the asterisks indicate the chirally pure linkages .
Manual couplings were used to synthesize the oligomer to conserve reagent, although the process can be done on an automated DNA synthesizer. The sequence was synthe-sized from the 3'-term;nl~q starting with methacrylate support bound deoxyar~-~nn~;n,~.
The protected dinucleoside methylphosFhnn~m;~;te (22 mg each per required co~rl;n~) freshly co-evaporated with pyridine and toluene to ensure dryness were placed into _ _ _ _ _ ~ ., . . , . . _ .. . . _ .. ... . .. . .

~ 217~2Sg WO 95/13834 ~ ~' PCT/US94/13387 dried l ml glass autosampler vials and dissolved in anhydrous acetonitrile to a cnn~ ntration of O.l M (200 ~l per coupling). The vessels were purged with argon and tightly sealed with screw caps with tef lon septa .
A l ~mole scale DNA synthesis column (Milligen) was f illed with 1 ILmole of methacrylate support bound deo~;y-Arlf~nns;nf-. The column was attached to a ring stand in a vertical orientation . A male-male luer ~f itting was attached to the bottom along with an 18 gauge needle to control the effluent. The column was washed with lO ml acetonitrile using a syringe. The support bound nucleo-side was detritylated by passing 3 ml of 29~ dichloroacetic acid in dichloromethane through the column over l. 5 minutes. The orange, dimethoxytrityl cation bearing solution was reserved. The column was washed twice with ml each of anhydrous acetonitrile.
The $irst collr1;n~ was accomplished as follows: lO
ml more anhydrous acetonitrile was passed through the column. Then, 200 ~Ll of the CT methylrh~srhnnAm;~ite was drawn into a 1 ml syringe. Next, 200 Ill of 0.45 M tetra-zole in anhydrous acetonitrile was likewise drawn into the syringe c~nt~;nlng the methylphosrh~nAm;~;te. The re-agents were rapidly mixed in the syringe, then slowly passed through the column dropwise over three minutes, being sure to lightly draw the plunger up and down to ensure adequate mixing with the support. A$ter 3 minutes, 1 ml of the rn~;~l;7;n~ reagent (O.l M I, in 73~ tetrahydro-furan, 259~ 2, 6-lutidine and 29~ water) was passed through the column over one minute. The column was washed with 20 ml acetonitrile and then treated with 600 ILl of a solution ~ntA;n;n~ 205~ (v/v) acetic anhydride, 30~6 (v/v) acetoni-trile, 509~ (v/v) pyridine and 0.312~ (w/v) dimethylamino-pyridine. The column was then washed with 20 ml acetoni-trile .
The above-described synthetic cycle was repeated until the synthesis was completed. The overall coupling Wo 9S/13834 ~ 1 7 ~ 2 S 9 PCr/lJS94/13387 efficiency based on dimethoxytrityl absorbance was 95.7~, for an average of 99.3~ per coupling.
The oligomer was then cleaved from the support and 'deprotected. The support bound oligomer was removed from the synthesis cartridge and placed in a glass 1 dram vial with a screw top. The support was treated for 30 minutes at room temperature with 1 ml of a solution of acetoni-trile/ethanol/NH~OH ~9/9/1). Then, 1 ml of ethyl-~nf~1Ar-;n~
was added to the reaction vessel and the reaction allowed to sit for 6 hours at ambient temperature in order to go to completion. The sUp~rnAt~nt cnn~A;n;n~ the oligomer was then removed f rom the support and the support was rinsed twice with 2 ml of 1/1 acetonitrile/water; the washings were combined with the supernatant. The combined solution was diluted to 30 ml total volume with water and neutralized with apprn~;r-~t~ly 4 ml of 6 N HCL. The neutralized solution was desalted using a W~ters C-18 Sep-Pak cartridge which was pre-equilibrated with 10 ml acetonitrile, 10 ml of SO~ acetonitrile/100 mM triethyl-ammonium birArh~nAte~ and 10 ml of 25 mM triethylammonium birArhnnAte, seql~n~;A11y. After the reaction solution was passed through the column, it was washed with 30 ml of water. The product was then eluted with 5 ml of 1/1 acetonitrile/water .
The oligomer was purified on HPLC using a Beckman Ultrasphere-reverse pha5e 4 . 5 X 250 mm column with an increasing gradient of acetonitrile in O . 5 M triethyl -ammonium acetate (0~ to 403~ over 40 minutes). The isolat-ed yield was 41 OD,60 units (35~). The compound was characteri~ed by electron spray mass ~e- LL, ~ (calc .
4391/found 4391).
Alternatively, the above-identi~ied oligomer can be synthesized on an automated DNA synthesizer. In this case the c.~Lu~liate dimer synthons (as used above in the manual synthesis) are dissolved in acetonitrile to a rnnrC~n~ration of O.1 M as described above. The amidite so~ utions are placed in conical vessels on a Millipore Wo 9~/13834 PCrlUS94/13387 7~2~ ~o Expedite DNA Synthesizer. All other reagentæ (oxidizer, deblock, capping reagents and activator) are prepared as described above for the manual synthesis, and applied to the d~ ,L,Liate positions on the instrument as instructed in the manual. P'U~L 'nq parameters for one synthesis cycle are as given in Table I in U. S . Patent Application Serial No. 08/158, 014. The deprotection and purification of the oligomer is carried out as described above f or the manually synthesized oligomer.
Exam~le 9 PrenAration of a Polv-CU Qliqomer Havinq Alternatinq 2'-O-MethYl NP(~) /2' -O-Methvl DE and 2~ -O-Methvl NP(R,~ /2' -O-Methvl NP Internucleosidvl T,; nk;~qe5 An oligomer having the sequence= 5' (C~U) - (C`U) - (C-U) -( C'U) - ( C'U ) - ( C'U ) - ( C U) - A- 3 ' was prepared us ing 2 ' - O - me thyl NP(Rp)/2'-O-methyl DE dimer synthons prepared according to Example 2 hereinabove.
The d~ Liate dimer synthons were dissolved in acetonitrile to a ron~-~ontration of 0.1 M. All other reagents used were as described in Example 8.
A 1 llmole scale DNA synthesis column (Millipore) was f illed with 1 ~Lmole of methacrylate support bound deoxy-adenosine. The dimer synthons were coupled geq~ nt;Ally from the 3'-terminu~ as described in Example 8 except that the co-lrl; n~ time was ~t~n~ to two minutes . The overall ~ o~rl;n~ efficier,cy based on dimethoxytrityl ~hp~rhAn~e was 50%, for an average of ~919~ per coupling.
The dimethoxytrityl group was removed from the oligomer at the end of the synthesis.
3 o The deprotection was carried out as described in Example 8. The crude yield was 103 OD26~ units. The oligomer was purif ied on XPI C with a Beckman Ultrasphere-Rp using an increasing gradient of acetonitrile in 0 . S M
triethylammonium acetate (10~ to 30~6 over 30 minutes).
35 The isolated yield was 39 OD26~ units (3896). The compound WO 95l~3834 PCTIUS94/13387 2~7l~2~ig was characterized by electron spray mass spectrometry (calc. 4713/found 4712) This oligomer can also be synthesized on an automated DNA synthesizer as follows. The d~L~Liate dimer 8ynthons (as used above in the manual synthesis are dissolved in acetonitrile as described in Example 8. The amidite solutions are placed in conical vessels on the Millipore Expedite DNA synthP~i7Pr. All other reagents (oxidizer, deblock, capping reagents and activator) are prepared as described in Example 8, and are applied to the a~lJL~L iate positions on the in8trument as instructed by the manual. The same coupling program a8 described in Example 8 is used except that the coupling time is extend-ed to 2 minutes.
The deprotection is carried out as described in Example 8. The oligomer can be purified on HPLC using as described above for the manual synthesis.
Using similar procedures as described in detail in Example 8 of U. S . Patent Application Serial No .
08/154, 013, the oligomer 5' - (C*U) - (C*U) - (C*U) - (C*U) - (C~U) -(C*U)-(C*U)-A-3' having 2~-0-methyl MP(R~)/2'-0-methyl MP
(racemic) mixed linkages was prepared. The product was also characterized by electron spray mass spectroscopy (calc . 4699 . 5/found 4701) . Automated synthesis may also be employed as explained above.
le lO
Pre~aration of 5' - (T A) - (G C~ - (T T) - (C C) - (T T) - (A ::) - (C'T) -(C'C) - (T'~) -C-3' }Iavinq Re~eated MP(R~) ~MP ~,1nkAqe Struc-tures The grouped dinucleosides indicate coupled dimers and the asterisk indicates where the ster~nrhPm; ~try is fixed (chirally de~ined or chirally pure) as the fast eluting isomer on silica gel (identified as Rp).
An oligomer having this se~uence was synthesized using the d~L~Liate protected dinucleotide methylphos-ph-~n~m; tii te~ prepared using methods such as those de-wo 95/13834 ; ! ~ I Pcr/Uss4ll3387 ~ I
~1~62~9 52 scribed in Examples lA and lC above. Manual couplings were used to synthesize the oligomer to conserve reagent, although the process can be done on an automated DNA
synthesizer from the 3 ' terminus starting with support-5 bound cytidine.
Each of the desired protected 7; n~ ntide methyl-rhnsrhnn;7m; dites ~22 mg each per required coupling), T'A, G'C, T T (2x), C C (2x), A G, C T, and T G, freshly co-evaporated with pyridine and toluene to ensure dryness, 10 was placed into a dried 1 ml glass ;7llt-~ ler vial and dissolved with anhydrous acetonitrile to give a concentra-tion of 0.1 M (200 ~Ll were used per coupling). The vials were purged with argon and tightly sealed with screw caps with tef lon septa .
A 1 ~mole scale Milligen DNA synthesis column was filled with 1 ~mole of support bound cytidine. The column was attached to a ring stand irL a vertical orientation.
A male-male leur fitting was attached to the bottom along with an 18 gauge needle to control the f~l 17~nt . The column was washed with 10 ml of ACN using a syringe. The support bound nucleoside was then detritylated by passing 3 ml of 29~ dichloroacetic acid in dichloromethane through the column over 1. 5 minutes . The orange, dimethoxytrityl cation bearing solution was reserved. The column was washed twice with 10 ml each of ACN ~anhydrous).
The first coupling was accomplished by passing 10 ml more ACN (anhydrous) through the column. Then, 200 1~l of the TG methylphosphonamidite was drawn into a 1 ml sy-ringe. Next, 200 ~ of 0.45 M tetrazole in anhydrous ACN
was likewise drawn into the syringe cnnt;7;n;n~ the methyl-rhn9phnn;7m; rl 7 te . The reagents were rapidly mixed in the syringe, then slowly passed through the column dropwise over 3 minutes, being sure to lightly draw the plunger up and down to ensure adequate mixing with the support.
After 3 minutes, 1 ml of the n~r;r7.;7;ng reagent (0.1 M I2 in 74.259~ THF, 259~ 2,6-lutidine, and 0.2596 water) as passed through the column over 1 minute. The column was then WO95/13834 ~17B2~9 PCrlUS94/13387 washed with 20 ml of ACN. The column was then treated for 1 minute with 600 ~il of a solution rnntA;n;n~ 20~ (v/v) acetic anhydride, 3096 (v/v) ACN, 50~ (v/v) pyridine, and O . 312~ (w/v) dimethyaminopyridine. The column was washed with 2 0 ml of ACN .
The synthetic cycle was then repeated with each dinucleotide methylpho9~hnn~m;~l;te until t~,e synthesis was completed . The order of addition of dimers af ter the initial T G cmlrl;nr, was C C, C T, A G, T T, C C, T T, G C, and T'A.
The dimethoxytrityl group was removed from the oligo-mer at the end of the synthesis.
The oligomer was the~ cleaved from the support and deprotected The support bound oligomer was removed f rom the synthesis cartridge and placed in a glass 1 dram vial with a screw top. The support was treated for 30 minutes at room temperature with 1 ml of a solution of acetoni-trile/ethanol/NH~OH (9/9/1) . Then, 1 ml of ethylPnP-l;Am;nP
was added to the reaction vessel and the reaction mixture allowed to sit for 6 hours at ambient temperature in order to go to completion. The ~llr~rn~t~nt cnntA;n;~r~ the oligomer was then removed from the support and the support was rinsed twice with 1 ml of 1/1 acetonitrile/water; the w-~h;n~c were ' inP~l with the supPrnAtAnt The combined solution was diluted to 50 ml total volume with water and neutralized with approximately 1 7 ml of glacial acetic acid. The neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-e~uilibrated with 5 ml acetonitrile, 5 ml of 50~ acetonitrile/water, and 5 ml of water, sequPnt;Ally After the reaction solution was passed through the column, it was washed with 50 ml of water. The product was then eluted with 2 ml of 1/1 acetonitrile/water.
The oligomer was purified by HPLC on a reverse phase column (Poros II R/H 4 . 6 x 100 mm) using a gradient of acetonitrile in water.

wo 95113834 ~17 ~ 9 PCrNS94/l3387 .

Coupling efficlencies are set forth in the table below .
Coupling Efficiencie~ of Dinucleotide Methylrh~ph~nnm~ ~ teEI
Dinucleotide Coupling Efficiency T G 99.7 C'C 90.2 C T 91. 8~6 A G 85.5 T T 97 . 8 C'C 83 . 696 T'T 1009~
G'C 86 . 296 T'A 92 . 496 , 5~.Y~mnle 11 Pre~aration of 5' - (G T) - (C T) - (T C) - (C A) - (T G) - ~C A) - (T'G) -(T'T)-(G'T)-C-3' Havinq RePeated MP(R~)/MP T.;nk~e Struc-tures The grouped dinucleotides indicate coupled dimers and 20 the asterisk indicates where the stereochemistry is fixed.
This sec~uence was synthesized using the appropriate protected Rp dinucleotide methylrh~cnh~n~m;~l;tes prepared and isolated using procedures such as those described in Examples lA and lC above. Manual couplings were used to 25 synthesize the oligomer in order to conserve reagent.
However, if desired, the process can be done on an auto-mated DNA synthesizer from the 3 ~ terminus starting with methacrylate jsupport bound 2~-deoxycytidine.
Each of the desired protected dinucleotiae methyl-30 rh~c~h-~n~m;dites (100 mg), G T, T T, T'G, C'A, T'G, C'A, T'C, C-T, and G~T was placed into a dried 3 ml glass conical vial and dissolved with anhydrous acetonitrile to a c~nrPntration of 0 ~ M. Molecular sieves (3 A) (0 . 5 ml Wo 9sll3834 2 1 ~ 6 2 ~ 9 PCrlU594113387 volume) were added to each vessel, the vessels purged with argon, and tightly sealed with screw caps with teflon 6epta. The reagent9 were allowed to stand overnight prior to use.
A 1 ~lmole scale 1~; l l ;3-~n DNA synthesis column was f illed with 1 ~Lmole of methacrylate support bound 2 ' -deoxycytidine. The column was attached to a ring 8tand in a vertical orientation. A male-male luer fitting was attached to the bottom along with an 18 gauge needle to control the effluent. The column was washed with lO ml of ACN using a syringe. The support bound nucleoside was then detritylated by passing 3 ml of 2 . 5~ dichloroacetic acid in dichloromethane through the column over 3 . 0 minutes. The orange, 1; hnl~ytrityl cation bearing solution was reserved. The column was washed twice with lO ml each of ACN ~anhydrous).
The first coupling was accomplished by passing lO ml more ACN (anhydrous) through the column. Then 200 ~l of the G~T methylphosphoramidite was drawn into a l ml syringe. Next, 200 1ll of 0.4~ M tetrazole in anhydrous ACN was likewise drawn into the syringe rnntA;n;nrJ the methyl~hnsphnn~m;dite. The reagents were rapidly mixed in the syringe, then slowly passed through the column drop-wise over l minute, being sure to lightly draw the plunger up and down to ensure ade~uate mixing with the support.
Af ter 3 minutes, 1 ml of the n~ ; ng reagent ( 0 .1 M I2 in 74.25~ THF, 25~ 2,6-lutidine, and 0.25~ water) was passed through the column over 1 minute. The column was then washed with 20 ml of ACN. The column was then treated for 1 minute with 600 ~l of a solution rnn~A;n;n~ 209~ (v/v) acetic anhydride, 30~ (v/v) ACN, 50% (v/v) pyridine, and 0.31296 (w/v) dimethyaminopyridine. The column was washed with 2 0 ml of ACN .
The synthetic cycle was then repeated with each tl;nllr1f~otide methylrhn~phon~m;dite until the synthesis was completed . The order of addition of dimers af ter the WO 95/13834 ?. i~ 5 9 PCT/US94/13387 initial G T coupling was T T, T G, C A, T G, C A, T C, C T and G T.
The dimethoxytrityl group was removed from the oligo-mer at the end of the synthesis.
The oligcmer was then cleaved from the support and deprotected. The support bound oligomer was removed from the synthesis cartridge and placed in a glass l dram vial with a screw top . The support was treated f or 3 0 minutes at room temperature with l ml of a solution of acetoni-trile/ethanol/NH~OH (9/9/l) . Then, l m~ of ethyl PnPr~ mi nP
was added to the reaction vessel and the reaction allowed

6 hours to go to completion. The supernatant ~ nnt~;n;ng the oligomer was then removed from the support and the support was rinsed twice with l ml of l/l acetonitrile/water; the w~ch;n~C were combined with the supernatant. The ,- ;nPd solution was diluted to 30 ml total volume with water and neutralized with approximately l . 7 ml of glacial acetic acid. The neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with 5 ml acetonitrile, 5 ml of 50~6 acetonitrile/water, and 5 ml of water, sPql1Pnt;~lly.
Af ter the reaction solution was passed through the column it was washed with 5 ml of water. The product was then eluted with 2 ml of l/l acetonitrile/water.
The oligomer was purif ied by HPLC on a reverse phase column (Poros II R/H 4 . 6 x l00 mm) using a gradient of acetonitrile in water.
r ~le 12 Pre~a~atiQrl of 5' - (G A) - (G G) - (A G) - (G A) - (G G) - (A'G) - (G'A) -(A~G) -G-3 ' Havinq RePeated MP (R~) /MP Linka3e Structures The grouped dinucleosides indicate the coupled dimers and the asterisks indicates where the stereochemistry is fixed (chirally defined or chirally pure) as the fast eluting dimer isomer on silica gel (;~lpnt;~ied as E~p).

9~/13834 ,! 1 ~ ~i 2 5 9 PCr/US94/13387 Thia oligomer was prepared using automated synthesis coupling G A, G G and A G MP (Rp) /MP dimer synthons prepared according to the procedures of Examples lA and lC.
An amount of G A, G G and A G dimer 6ynthon6 was dissolved in acetonitrile to give a concentration of 0.1 M and stored over 3 A molecular sieves (Millipore, Milford, MA) overnight.
The di6solved dimers, with molecular sieves, were placed in conical vessels on a Millipore ~xpedite DNA
Syn~h~R; 7Pr which as equipped with end-line filters to remove particulates. All other reagents ~oxidizer, deblock, capping reagents and activator) were prepared and applied to the c.~L.,~Liate positions on the instrument as instructed in the manual. The coupling program was modified to place the oxidizing step immediately subse-quent to the ~ r~; ng step in order to reduce h~ khnn~
cleavage prior to oxidation. (See Hogrefe, R. I , et al .
"An Improved Method for the 5ynthesis and Deprotection of Methyl rh~Rrh~n~te Oligonucleotides " in ~ethods in Molecu-lar Biolo~v, vol. 20: Protscol5 for Oliqonucleotides and Analoqs (ed. Agrawal, S.) pages 143-164, Humana Press, Totowa N.Y. ~1983). The ~L~yL n~ parameter6 for one synthesis cycle ~"Syn4all-1 /lmol~) are set forth in Table II of U.S. Patent Application Serial No. 08/154, 013 .
A 1 ~mole scale DNA 6ynthesi6 column ~Millipore) was filled with 1 ~Lmol o~ methacrylate support-bound deoxy-gll~n~Rinf~ and was placed on the DNA synthe6izer. The dimers were coupled 6eqll~n~i~1ly from the 3' t~rm;nl1R.
The dimethoxytrityl protecting group was removed from the oligomer at the end of the synthe6is.
The oligomer was then cleaved from the support and deprotected. The support bound oligomer wa~ removed from the synthesis cartridge and placed in a gla6s 1 dram vial with a screw top . The support was treated ~or 3 0 minutes at room temperature with 1 ml of a solution of acetoni-trile/ethanol/NH40H ~9/9/1) . Then, 1 ml of ethyl~n~A;~m;n-~
wa6 added to the reaction vessel and the reaction allowed Wo95/13834 2~ 2S;9 PCrlUS94/13387 6 hours to go to completion. The S~l~Prn~tAnt ~-nnti~in;n~
the oligomer was then removed from the support and the support rinsed twice with 1 ml of 1/1 acetonitrile/water, when ~'nml~; nPtl with the supPrn~t~n~ . The ~ '; nPd solution was diluted to 50 ml total volume with water and neutral-ized with approximately 1. 7 ml of glacial acetic acid.
The neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-es~uilibrated with 5 ml acetonitrile, 5 ml of 50~ acetonitrile/water, and 5 ml of water, se~uentially. After the reaction solution was passed through the column, it was washed with 5 ml of water. The product was then eluted with 1. 8 ml of 1/1 acetonitrile/water .
The crude yield was 87 OD260 units. The Oligomers was purified on ~IPLC using a ~-cyclobond standard phase 4.5 X
250 mm column (Azetec, Inc. Whippany, NJ) ~.vith a decreas-ing gradient (80% to 4096) of acetonitrile in 0 . 05 M
triethylammonium acetate (pH 7 ) . The isolated yield was 22 OD26~ units (25~). The product was characteri2ed by electron spray mass spectrometry (calc. 5407/found 5401).
F le 13 Pre~aration of an Oliqomer Havinq Alternatinq MP ~R~) /PS
InternucleosidYl Linkaqes An oligomer having altPrn~t; n~ MP (R~) /PS; nt~rnllcl eo-25 sidyl linkages is prepared using dimer synthons. All theparameters of the synthesis, deprotection and purification are as described in Example 8, except that the oxidizing reagent is replaced by a 0 .1 M solution of 3H-1, 2-benzo-dithiole-3-one, 1,1-dioxide or a 0.1 M solution of sulfur 30 in 1/1 carbon disulfide/diisopropylethylamine.
F le 14 Preparation of an Oliqomer Havinq AltPrnPtln~ MPS(R~)/DE
TnternucleosidYl Linkaqes An oligomer having alternating MPS (R~) /DE internucleo-35 sidyl linkages is prepared using the dimer synthons of wo 95/13834 2 i 7 6 2 S g PCT/US94/13387 Example 4. All other parameters of synthesis, deprotection and purification are as described in Example 8.
Exam~le 15 5 Pre~aration of an Oliqomer Havi n~ ~l t~rn~t; nq MPS (RF) /PS
Tnterrll-rlensidvl Linkaqes An oligomer having alt~orn~t; n~ NPS (Rp) /PS; nt~rnll~ l eo-sidyl linkages is prepared using the dimer synthons of E~cample 4. All of the parameters of synthesis, depro-10 tection and purification are as described in Example 8,except that the nl~1~1i7;n~ reagent is replaced by a 0.1 M
solution of 3_-1,2-benzodithiole-3-one, 1,1-dioxide or a 0.1 M solution of 9ul~ur in 1/1 carbon disulf ide/diisopropylethylamine .
~YAr'-le 16 Pre~aration Qf an Oliqomer Hav;n~ Alternatinq NP(R~)/PS2 Internucleosidvl Linkaqes An oligomer having alt~orn~t; n~ NP (Rp) /PS2; nt~rnllcl eo-sidyl linkages i8 prepared using the dimer synthons of 2 o Example 5 . All of the parameters of synthesis, depro-tection and purification are as described in Example 15.
F le 17 Pre~aration o~ an Oliqomer Havinq Alternatinq NPS (~) /PS2 Internucleosidvl Linkaqes An oligomer having alternating NPS (R~) /PS2 inter-nucleosidyl l; nk~PR is prepared using the dimer synthons of Example 6. All of the parameters of synthesis, depro-tection and purification are as described in Example 16.
ExamDle 17A
Prel~aration of an oliqomer Havinq Altorn~t;n~ MP (R~) /2 ~ -0-Meth~l ~E Internucleosidvl Linkaqes An oligomer having alt~rn~t; n~ NP (R9) /2 ' -0-Methyl DE
int~rnucleo_idyl linkages is prepared using dimer synthons 2sg Wo 95/13834 . ~ , PCr/US94/13387 .

similar to those of Example 7. All other parameters of synthesis, deprotection and purification are as described in Example 9.
T;~ le 18 Pre~aration o~ an Oliqomer Havinq Alternatinq MP (Rq) /MPS
Internucleosidvl :I.inkaqe8 The preparation of an oligomer having alternating MP (R7) /MPS internucleosidyl linkages is aL~ h~d using dimer synthons prepared according to Examples lA and lC
and dissolved and stored over molecular 6ieves. The oxidizing reagent is a 0 .1 M solution of 3H-1, 2-benzo-dithiole-3-one, l,l-dioxide ("Beaucage Reagent", see Iyer, R.P. et al., JACS 112:1254-1255 (1990)) or a 0.1 M solu-tion of sulfur in 1/1 carbon disulfide/ diisopropylethyl-amine, with synthesis proceeding generally as described in Example 12 .
~Y~ e 19 P~eT~aratiorl of ;~n Oliqomer Havinq 2 ' -O-Methvl Nucleosidvl Unite and Alternatinq MP(R~)/MPS Internucleosidvl rink~es This oligomer is prepared using the dimer synthons as described in Examples 2A-2D and 2F and ~ollowing the general synthetic procedures of Bxample 8 of U. S . Patent Application Serial No. 08/154,013, except that the oxidiz-ing reagent described therein is a 0 . lM aolution of 3~-1l2-benzodithiole-3-onel 1,1-dioxide or a 0.1 M solution on 1/1 carbon diaulf ide/diisopropylamine .
r - ~le 20 Pre~aration of an Oliqomer Havinq 2'-O-Methvl Nucleosidvl TTn;ta and Alternatinq MPS(F~)/MP Internucleoaidvl ~inkaqe~
This oligomer is ~~ epa, e~ using dimer synthons a~
described in Example 3 above and by f ollowing the parame -ters of synthesis, deprotection and purification of Example 19.

-WO 9~/13834 ~ I ~ 6 2 ~ 9 PCr/US94113387 F le 21 Pre~aration of an Oliqomer Havinq Alternatinq MPS (~ ) /MP
InternucleosidYl J,i nkA~re$
This oligomer i5 prepared using dimer synthons pre-pared according to Examples lA and lC, substituting Beaucage reagent for the oxidizer in Example lA, and by following the parameter8 of synthesis, deprotection and purif ication as described above in Example 12 .
F le 22 Pre~aration of an Oliqomer Xavinq Alternatinq ~PS ~R~,l /lLPS
InternurleosidYl T,inkAqeg This oligomer is prepared using dimer synthons as referred to in Example 21 and by following the parameters of synthesis, deprotection and purification as described above in Example 12, except that the oxidizing reagent used therein is replaced by a 0.1 M Rn11lt;nn of 3_-1,2-benzodithiole, l,l-dioxide or a 0.1 M solution of sulfur in 1/1 carbon disulfide/ diisopropylethylamine.
~A mn 1 e 23 Prel~aration o~ 2'-F Dimer Syntl~nnR
Dimer 8ynthons useful in the preparation of the oligomers of the present invention may be prepared using 2~-fluoronucleosides. Methods for preparation of 2~-fluoronucleosides have been reported and are known to those skilled i~ the art. lSee, e.g.: Codington, JOC
Vol. 29 (1964) ~2'-F U); Mangel, Angew. Chem. 96:557-558 (1978) and Doen, JOC 32:1462-1471 (1967) (2'-F C);
Ikehara, Chem. Pharm. Bull. 29:1034-1038 (1981) (2'-F G);
Ikehara, J. Carbohydrates, Nucleo8ides, Nucleotides 1:131-140 (1980) (2'-F A), and also Krug, A, Nucleosides &
Nucleotides 8:1473-1483 (1989).) The preparation of dimer synthons using 2'-fluoro-nllrler,R; ~1~R may be accomplishing using the procedures analogous to those de8cribed for the 2 ' -O-methyl dimer 35 sYnthons (See, e.g., ~xamples 2, 3, and 7~. The resulting

7 &2S9 W095/13834 2i PCrlUS94113387 dimer synthons may be used to prepare oligomers using methods analogous to the methods used f or the 2 ~ -O-methyl dimer synthons such as in Example 9.
~ mn 1 e 2 4 5 Pre~aration of MP(~)/MP(R,)/DE and MP(R~)/MP(R~)/MP Trimer 5yr~thons The above- identif ied trimer synthons are prepared using the MP (Rp) /MP dime~ synthons of Example lC. The dimer synthon is coupled to a 5'-hydroxy, 3'-silylated 10 nucleoside according to the methods of Example lA for the coupling of the 3' -nucleoside: to the monomer phosphorami-di te The selected 5'-hydroxy, 3'-silylated nucleoside (1 equivalent ) and isomerically pure Rp dimer methylphos -15 phonamide (1. 25 equivalents) are weighed into a roundbottom flask and dried by co-evaporation with acetoni-trile. The resulting foam is dissolved in acetonitrile and treated with a solution of O . 45 M tetrazole in aceto-nitrile (4.5 equivalents) . After 3 minutes, the reaction 20 mixture is oxidized and the reaction product i8 worked up as described in Example lA. The diastereoisomers of the 3'-silylated trimer are resolved on a silica gel column as described in Example lA f or resolution of the dimer isomers . The conf iguration of the separated diastereo-25 isomers iB determined using 2-D nmr (ROSE~) . The trimer having the desired chiral conf iguration (Rp/R;,~ of the two internucleosidyl linkages i8 converted to a trimer synthon by reaction with chloro-j~-cyanoethoxy-N,N-diisopropyl-amin~.L,h~ hn~ c~midite using methods as described in Example 30 lB. The trimer synthon is worked up and purified using methods as described in Example lB to achieve the MP t}~) /MP (Rp) /DE trimer .
Using similar procedures, an MP(Rp)/MP(Rp)/MP phos-phoramidite synthon may be obtained by using chloromethyl-35 N,N-diisopropyl~m;nnrhn~phine in the fi:nal reaction as described in Example lC for the corresponding dimer Wo 9S/13834 ~ ~ 7 6 2 ~ 9 PCr/uSg4113387 . .

synthon. Workup and purification are as described in Example lC .

F le 25 - ~
PrsPaxation Of 2 ' -O-Allyl p;r and Trimer SvnthnnR And Their Use in Oliqomer ,3vnthes; R
The dimer and trimer 9ynthons described, for example, in Examples 1 and 24 can be prepared using 2 ' -0-allyl nucleosides . The preparation of 2 ' -O-allyl nucleoside6 and their use in the preparation of oligomers has been reported (see e.g. Iribarren, et al. (19gO) Proc. Natl.
Acad. Sci. (USA~ 87:7747-51; and I,esnik et al. ~1983), Biorh~m; Rtrv 32: 7832-8), and such substituted nucleosides are commercially available. The nucleosides are used to prepare dimer and trimer fiynthons using procedures de-scribed hereinabove. The synthons are used to prepare oligomers using methods such as those described in Exam-ples 10, 11, 12, 13 and others above.
le 25 Pre~aration of an OlicrnmPr Hav; n~ MP ll~, ) /MP~DE Internllr] eo-sidvl Tl;nk~re8 The above-; ~l~on~ Ol; ~ r is prepared using the trimer synthons of Example 24, or by tho8e in Example 20 of U.S. Patent Application Serial No. 08/l54, 014, and by following the methods described in Example 8, substituting the trimer synthons for dimer synthons. All other parame-ters o~ synthesis, deprotection and pur;firA'r;nn are as described in Example 8.

Wo 95/l3834 2 1 7 6 2 5 ~ PCT/US94/13387 le 27 Pre~aration of an Oliaomer Havinq MP(~,)/Mp(R~)/MP Inter-nucleosidYl Linkaaes The above-;~lrnt;f;ed oligomer is prepared using the 5 procedures described in Example 14 of IJ.S. Patent Applica-tion Serial No. 08/154, 013 .
F le 28 Pre~aration of Olicro~ibonucleosides O1igor;h~n1~r~ tides used in the present examples may 10 be synth~ ; 7~rl using general procedures such as described below .
The appropriate 5'-D-dimethoxytrityl-2'-0-tert-butyldimethylsilyl - 3 ' -0 -N, N- diisopropyl - ,B -cyanoethylphos -phoramidite nucleosides (Millipore, E~ilford, MA) were used 15 or synthesis . Syntheses were . done on a 1 l~mole scale with a Milligen 8750 automated DNA synthesizer using standard Milligen phosphoramidite procedures with the exception that the coupling times were ~lct~n~ to 12 minutes to allow ader~uate time for the more sterically 20 hindered 2'-0-tert-butyldimethylsilyl RNA monomers to react. The syntheses were begun on control-pore glass bound 2'-0-ter~-butyldimethylsilyl r;hon-~c~eosides pur-chased from Millipore. All other oligonucleotide synthe-sis reagents were as described in Millipore' 8 standard 25 protocols.
After synthesis, the olis~n~rlr~tides were handled under sterile, RNase-free conditions. Water was steril-ized by overnight treatment with 0 . 5~ diethYlpyrocarbonate followed by autoclaving. All glassware was baked for at 30 least 4 hours at 300C.
The oligonucleotides were deprotected and cleaved f rom the support by f irst treating the support bound oligomer with 3/1 ammonium hydroxide/ethanol for 15 hours at 55C. The supernatant, which rfnt;l;nl~rl the oligonucle-35 otide, was then ~ nt~d and evaporated to dryness. Theresultant residue was then treated with 0 . 6 mL of 1 M

Wo 95113834 2 i 7 6 2 ~ 9 PCr/US94113387 tetrabutylammonium fluoride in tetrahydrofuran (which t-"ntsiin~c~ 5~ or less water) for 24 hours at room tempera-ture. The reaction was c~uenched by the addition of 0.6 mL
of aqueous 2 M triethylammonium acetate, pH 7. De6alting 5 of the reaction mixture was accomplished by passing the solution through a Bio-Rad l~DG column uæing sterile water. The desalted oligonucleotide was then dried.
Purif ication of the oligoribonucleotides waæ carried out by polyacrylamide gel electrophoresis ~PAGE) contain-ing 1596 19/l polyacrylamide/bis-acrylamide and 7 M urea using ~tandard procedures (See Maniatis, T. et al., Molecul~r l~ nin~: A Labor~torY MAnllAl, pages 184-185 (Cold Spring Harbor 1982) ) . The gels were 20 cm wide by 40 cm long and 6 mm in width. The oligoribonucleotides ~60 OD Units) were dissolved in 200 /LL of water C~ntAining 1.25~ bro--~rh~nnl blue and loaded onto the gel. The gels were 1-un overnight at 300 V. The product bands were visualized by W bA~-k~hArl~wing and excised, and the product eluted with 0 . 5 M sodium acetate overnight . The product was desalted with a Waters C18 Sep-Pak cartridge using the manu~acturer supplied protocol. The product was then 3~P l~h~llecl by kinA~;n~ and analyzed by PAGE.
r le 29 pre~ara~ion of Rac~m; c Methvl~hoqnh~n~te Olic~onucleotides Various racemic oligomers were synthesized using 5 ~ -(dimethoxytrityl) deoxynucleoside-3' - [ (N,N-diisopro-pylamino) methyl] -phosrh~nm~m; dite ~ ~ . Solid-phase synthesis was p~LLo, 1 on methacrylate polymer supports with a Biosearch Model 8750 DNA syrJth~s; ~or according to the manufacturer's r~ ~At;r)n~ except for the fol-lowing modifications: the monomers were dissolved in acetonitrile at a ~onr~ontrations of 100 mM, except dG, which was dissolved in 1/l acetonitrile/dichloromethane at 100 mM. DEBLOCK reagent = 2 . 5~ dichloroacetic acid in dichl~ thAnf~ OXIDIZER reagent = 25 g/L iodine in 0.259~ water, 259~ 2,6-lutidine, 72.596 tetrahydrofuran. CAP

Wo 95/l3834 2 1 7 6 2 ~ 9 . . PcrrUsg4rl3387 A = 10% acetic anhydride in acetonitrile . CAP B = O . 625 N,N-dimethylaminopyridine in pyridine.
The dimethoxytrityl group was removed f rom the oligonucleotide at the end of the synthesis.
The oligonucleotide was then cleaved from the support and deprotected. The support bound oligonucleotide was removed from the synthe5i5 cartridge and placed in a glass 1 dram vial with a screw top. The support was treated for 3 0 minutes at room temperature with l ml of a solution of acetonitrile/ethanol/NH,OH (9/9/1). Then, 1 ml of ethyl-l;Am;nf~ wag added to the reaction vessel and the reaction allowed 6 hours to go to completion. The super-natant cnnti~;n;nq the oligonucleotide was then removed f rom the support and the support rinsed twice with 2 ml of 1/l acetonitrile/water, when combined with the superna-tant. The combined solution was diluted to 30 ml total volume with water and neutralized with approximately 4 ml of 6 N HCl. The neutralized solution was desalted using a Waters C-18 Sep-Pak cartridge which was pre-equilibrated with lO ml acetonitrile, 10 ml of 509~i acetonitrile/lOo mM
triethylammonium ~icarbonate, and lO ml of 25 mM triethyl-ammonium bicarbonate, se~l~nt;;-lly. After the reaction solution was passed through the column it was washed with 3 0 ml of water . The product was then eluted with 5 ml of 1/l acetonitrile/water.
The oligonucleotide was purified by HPLC on a reverse phase column ~Whatman RAC II) using a gradient of acetoni-trile in 50 mM triethylammonium acetate.
F le 30 ~'h; -riC oliqonucleo~ide Assemblv From MP ~R~) /MP and ~5P (R~) /DE ~imer Synthnn~ and PhosPhoramidite and MethYl-~hosl~honamidite Monomer SYnthons MP (R~) /MP dimer synthons contained a methylphosphor-amidite coupling group at the 3 ' end. When coupled together to make an oligomer, these synthons give racemic methylphosrhnr-~t-~ linkages at every other position.

Wo95113834 '~ ;9. PCrlUS94/13387 NP (Rp) /DE dimer synthons contained a ,~-cyanoethyl phos-phoramidite coupling group at the 3 ' -end. Both types of dimer synthons were synthe9ized as described in ~xample 1.
Methylphosphonamidite monomer synthons were synthesized at 5 ~BL Scientific ~San Luis Obispo, CA). Betacyanoethyl phosphoramidite monomer synthons were purchased from Milligen/Biosearch .
All synthons were coupled using a Milligen Expedite'R
automated DNA synthesizer The coupling ~LU~L~ for each 10 8ynthon were as tabulated below. To generate a phosphoro-thioate bond during a coupling step, the program "Thioate-511M" was used with either a dimer or monomer synthon containing a ,B-cyanoethyl phosphoramidite coupling group.

WO 95/13834 2 1 ~ 6 2 ~ g - PCI/US94/13387 _ _ _ _ _ _ _ /i Function Morde Amount Time(sec) nl~ 1r~1nn / i /Argl /Arg2 5 /i ~ri Sn..hl nr-r ~ n 0; /i ;r ~ault i/ d~IT l S ~a;t"
l4 ~i ~hotometer S ~ .TAET data rn~ inn /i blk i/ ~-TLSE 1 1 0 'lblk to column"
6 /i ~blk ~ SE 20n 49 '~eblock"
/i qsh A to Cl i/ ~ ISE 8n ~ ~lush sy~temwith Wsh A"
15 l.. _ /i 'hotometer S i/ I 1 ~MTOP data rnll~r~inn~
/i r,as A to Cl i/ ? I,SE 1 0 ~r-as A to Cl waste~
-.- /t Adv~nce Frar i/ X ~ ~ O ' vent Out r~FF
/i qsh A i/ ?J~SE 20 o n sh A"
$Couplilg _ /i qsh i/ ~U.S l0 n Flush system with Wsh"
/i ~ct i/ ~r . 1 n ~Flu~h syr;tem with Act"
l /i ~ + Act i/ 'U, O IlMonomer + Act to column"
+ Act i/ YCr. l~l 61' ~Couple monomer"
/i ~ct i/ ~; . ll~ nCouple monomer"
/~ qsh i/ ~J, I 56 - "Couple monomer~
/i qsh i/ ~, so ~Flush ~yst~m with W3h~
~Capping 2 /i Wsh A i/ PUI,SE 25 O Caphs to columr"
5r;xidizing i/ PUI,SE lS0 0 'End oi rycle wash"
5 /i Ox i/ PU~SE 50 30 "Ox"
z /i Wsh A i/ PULSE 50 0 ~Flush system with Wsh A"
5 rlpping 353 /t Caps i/ pu~SE 25 0 "Caps to column~
2 /i Wsh A i/ PUI,SE 50 0 'Wsh A"
~ /~ w-h A ~/ I'lll.SE 5~ ~ EIId o~ cycle ~ ~h~
.

wo 95113834 ~ ~ 7 ~ 2 ~ 9 ~1US94/13387 T~IOATE -- 5 ,~dY.' /~ Function Mode Amount Time(sec) npnr~irtirn /~ /Argl /Arg2 5 /"
/~ ~
_ _ _ _ Snrh~
14~ .dvanc~ PrAc ~ n~vent out ON~
l0 ~ e~ault ~/ ~IT l. " Irlit~' 1'. /~ ~hotometer S ~/ A _ " TART dzta rrllPrr~;rn blk ~ ~SB l n 1blk to column"
.r /~ blk ~/ ~I~SE 20r~ 41 '-eblock~
~ /~ 'sh A to Cl i/ '~SE 8 ) " 'lush sy6temwith Wsh A~
15 l~ hotometer 5 ~/ I. r _ n~:TOP data rnll~rt~rn~
,. /t l.as A to Cl ~/ ? I,SE 1 ~ ~r,aS A to Cl war~te"
dvanc~ Frac ~ v~nt Out OFF"
sh A ~/ ? ~SE 20 ~ ~ sh A"
$roupling 20 . /t Wsh f/ '~r 5_ 10 0 n`lush system with Wsh"
/~ Lct ~/ ~U,~ lr n n ~lush 6ystem with Act"
2 /i ~ + Act ~/ ' .. r; n n onomer + Act to column"
Z /~ + Act ~ 6~ ouple monomer"
/` .. =t t/ ~ 1 nrouple monomer~
25/~ sh ~ ,r,, 55, ~ n cuple monomer~
. /i sh ~/ ~.'J 50 I n lush system with Wsh"
$rapping _3 /~ Caps ~/ PULSE 25 0 "Caps to column~
.2 /~ Wsh A i/ PUI.SE s0 o "Wsh A"
30.2 /~ Wsh A ~/ PU~SE l50 o "End of cycle wash~
1;1 mr~ r1i 7~ nrJ
.7 /~ Aux ~/ PULSE 5 0 "SOx"
.7 /i Aux ~/ PU~SE 45 60 "SOx~
:.2 /~ Wsh A ~/ PULSE 50 o "Flush system with Wr;h A"
35S~ apping _3 /t Caps ~/ PUIISE 25 0 "Caps to columnn ,2 /~ Wsh A ~/ PU3 SE s0 0 "Wsh A"
.2 /i Wsh A ~/ PUISE l50 0 "End of =ycle wash"

WO 9S113834 PCllllS94/13387 M~T . v ~ Us ~lU_._!TE - - 5 UM
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ /t Functlon Mode Amount Time~sec) nrRr~;r~
/t /Argl /Arg2 5 /t /t __ !
~n~.hlor~;ng 14. /~ ~.dvanr/e Fra~ t/ ~ I vent out ON~
10 ~ /t )e~ault t/ q,~IT ~ 'aitr ~' /t ~hotometer !9 t/ ~,'. ' ' ~ TART data ~ollection"
; /t ~blk t/ ~ LSE 1 ~ " )blk to rlolumn~
,f' /t ~blk t/ ~ ~LSE 20n 4 I ~Neblock' I /t Ish A to Cl t/ ~ LSE 8n n ~ lush systemwith Wsh A"
15 1.. : /t ~hotomet~r S t/ '' r ~ TOP data collection"
/t ;as A to Cl t/ ~ LSE 1 ' aa A to Cl waste~
.~. /t ~dvance Frac t/ ~ (I r v~nt Out OFF"
.~ /t qsh A t/ ~ LSE 2 0 r. I' sh A' 5Coupli-g 20 /t qsh t/ ~U.- 1n D 'FluRh ~ystem with Wsh"
/t ~ct t/ ~ 1 n DFlush system with Act"
1 /t ~ ~ Act t/ ~ n ~Monomer + Act to column"
19 /t A ~ Act t/ ~U. 1' 6 "Couple monomer~
, /t ~ct t/ ~ Couple monomer~
25 /~ qsh t/ ~ ' 56 ~Couple monomer"
/t ~qSh t/ ~ 50 'Flush system with Wsh ,5 /t OX t/ PULSE 50 30 'Ox"
.2 /t Wsh A t/ PUI.SE 50 0 'Flush system with Wsh A~
3 0 $ apping - 3 /t Cap6 t/ PUI,SE 25 0 'Cap6 to column"
2 /t Nsh A t/ PULSE 50 0 ~Wsh A~
.2 /t Wsh A t/ P~LSE 150 0 ~End oi cycle wash' ~76259 WO 95/13834 ~ PCT/US94113387 MP ~R,) /~P -- 5 UM
/'Function Mode Amount Time(rlec) D~rr1rttrn /~ /Argl /Arg2 /~
/~ ~
_ _ _ _ _ $n~.hl rr~ nrJ
14 /~ ~dvance Frac ~/ ~ : '''vent out ON"
10 /~ 1efault i/ ~IT l.~ ~'~ait-hotom~ter S ~/ X~ : TART data rnll~r~;r )blk ~ lIgB 1 " Iblk to column-r~ blk ~/ lnLSE 20n 4 ~ "neblock~
~ /~ 1sh A to Cl ~/ ~1~5E 8~ r ~lush systemwith Wsh A"
15 1 /~ ~hotometer S ~ n 1~ ~TOP d~ta rrl~rr~
/~ ~as A to Cl ~ ? LSE l~ .as A to Cl waste"
1-4 /~ dvance Frac ~/ ~ n r vent Out OFF"
'` /~ sh A ~/ ~ LSE 20 n 11 sh A"
$r~ouplilg 20/~ ~sh ~/ 'U_~ l0 n ~1 ~lush gystem with Wr~h /~ ~ct ~/ 'U. l n 'lush system with Act"
Act ~/ ~J- n ~1 onomer 1 Act to column"
Act ~ l. 6r R( ouple monomer"
ct~ lr) 1~1 ouple monomer"
25/~ ~sh ~/ ' .. ' 56 - "~ ou l m om 1' /f ~sh ~/ 1 .. 50 p e on er $r~71~1~7in S /~ Ox ~/ PUIISE 50 30 1'Ox1' 2 /~ Wsh A ~/ PULSE 50 0 "Flush system with Wsh A"
3 0S ~pping 3 /~ Caps i/ PULSE 2s 0 Cap& to column"
2 /~ Wsh A ~/ PULSE 50 0 ~Wsh A~
2 /~ Wsh A ~/ PULSE l50 o End of cycle wash~

WO 95113834 2 ~ ~ ~ 2 5 9 PCINS94/13387 NP ~R,,) /DE -- 5 U16 /i~ Function Mode Amount Time 1sec) Description /~ /Argl /Arg2 /~
/
n - hl nr r; T`
l4. /~ .~.dvzmce Fr~c i/ ~ 1 "Event out ON"
101 /i efault ~/ ~IT 0 l. "iait~
'hotometer S ~ TA~T data rr~ r ~ i nn n )blk i/ ~SE l 1 " )blk to column~
6 /~ blk ~/ ~ ~SE 20ri 4 ~ n ;eblock"
/~ sh A to Cl ~/ '~SE 8 ~ ) n ~lUGh system with Wsh A"
15 l~ hotometr~r 5 ~ TOP dat~ collection~
.as A to Cl ~/ ~SE ln "~-as A to Cl waste l~c /~ . dvance Frac ~ n ~ vent Out OFF"
_ /~ sh A i/ nsE 20 n ~ sh A"
5Coupli Ig 20. /~ ~sh ~/ ~Tr.5 ln O RFlush system with W3h"
/~ ~ct ~/ ~tr, 1 I nFlULh system with Act"
Act ~/ ' . "Monomer ~ Act to columr"
Act ~/ ', l, 6r1 nCouple monomer~
~ct ~/ '. . ln "Couple monomer"
2~sh ~/ '',_ ' 56. nCouple monomer"
Bh ~/ '' ._ sn "Flush GyGtem with Wsh"
Sn7,~ ~ r ' n~
7 /~ UX t/ PUDSE 50 30 i'Aux"
.2 /~ sh A ~/ PUnSE 50 0 "Flush system with Wsh A"
3 0$ apping .3 /~ CapG ~/ PlnSE 25 0 "Caps to column"
.2 /~ Wsh A '/ PlnSE 50 0 Wsh A"
.2 /~ Wsh A ~/ PUnSE lS0 0 "End of cycle wash"

~17625g Wo 95/13834 PCrtUS94tl3387 Applying one or more of these co-lrl 1 n~ routines with the u~r iate dimer or monomer synthons, one akilled in the art can recognize that each of the chimeric oligomers described in sub8e~lue~t examples can be synthesized.
Deprotection and purif ication of each chimeric oligomer was done essentially as described in Examples 8 through 12.
The identitie5 of certain chimeric oligomers made ac-cording to this Example, as well as other compounds, were confirmed by electrospray mass spectrometry as shown in the following table:

WO 95/13834 ~ i 7 6 2 5 9 PCT/IJS94113387 Seq. 1~ Sequence Backbone MW MW
Predictod 3:ound 2624-1 3--CTGTTG TACGT ACCTTCTG-5' Racemic MP 5725 5726 2371-1 3--CTGTTG TACGT ACCTTCTG.5' 75%MP(R~) 5725 5725 3130-3 3'-CCTGTTG TACGT ACCTTCTG-5' MP(R~)IDE 6028 6029 2366-1 3'-CCTGTTG TACGT ACCTTCTG-5' PS 6354 6357.9 2567-1 3'-CCTGTTG(TACGT)ACCTTCTG-5' [MP][DE]IMpl 6022 6018 2687-1 3'-CCTGTT(GTACG)TACCTTCTG-5' [7~ ' JLt~k~ oRpMP] 6022 6022 3169-1 3'-CCTGTTG(TACGT)ACCTTCTG-5' [MP(P~,,)lnF]lnF]r ''(Pp)/DE] 6033 6034 3214-1 3'-CCTGTTG(TACGT)ACCTTCTG-5' [MP(P~p)IDE][PSIDE][MP(P~p)IDE]6082 6081 10 3257-1 3'-CCTGTTG(TACGTAC)CTTCTG-5' [MP(R~,)IDE][PSIDE][MP(Rp)IDE]6100 6100 3256-1 3'-CCTGTTG(TACGT)ACCTTCTG-5' [MP(R,)IDE][PS][MP(R~)IDE] 6113 6114 3258-1 3'-CGTCCTCGATT(CCTTC)GATGGTAC-5' [MP(R;)IDE][PS DE][MP(Rp)IDE]7300 7299 3260-1 3'-CGTCCTCGATT(CCTTC)GATGGTAC-5' [MP(R~)IDE][PS][MP(P~")IDE] 7331 7331 3261-13'-~ ~lA(GTGAC)CTATATGG-5' [MP(P~")IDE][PSIDE][MP(R,)IDE]7313 7310 15 3262-13'-(~ lA(GTGAC)CTATATGG-5' [MP(R;)IDE][PS][MP(Pp)IDE] 7345 7346 3269-1 3'-ACGTCTGATCA(GTAAC)TAACTCAC-3' [MP(Rp)/Dy[PS/DE][MP(Rp/DE])7309 7308 3270-1 3'-ACGTCTGATCA(GTAAC)TAACTCAC-5' [MP(Rp)lDE][PS][MP(Rp)lDq 7341 7340 I . (T', ' ` - 1~1 the portion that activates RNAseH; thc linkage on the 5 '-side of the indic~ted nucleoside is charged.
.

Wo 95113834 2 1 7 6 2 5 9 PCr/US94/13387 le 31 ~ucl~qe Stabilitv Studies of Various R~kh-~n~ Mo~; fied (Non-Chimeric) Oliqomers In each of the experiments described in this example, 5 various bSrkh~ modified oligomers were evaluated having the f ollowing sequence: 5 ' - ~l Cl cl C'l'~ 'l'A- 3 ' ( f or 2 ' -deoxy sugars); or 5'-W~U~:U~:uW~u~uA-3' tfor 2'-O-methyl sugars). The all-diester (DE) h~l~kh~ oligomer was purchased from Oligos Etc. The other ba/ kh(~n~ oligomers 10 were synthesized as described in the preceding examples.
(a) Stability studie~ in the ~ of puri~ied snake venom rh~ hn~l~ e~tera~e . Snake venom phosphodies-terase I (PDE-I) from crotalus adamanteus was purchased from US Biochemicals, Inc Aliquots of each oligomer (0 . 075 A260 units) were pipetted into polypropylene microcentrifuge tubes and dried in a Speed-Vac'M vacuum centrifuge (Savant, Inc.). Next, the tubes were placed on ice and aliquots of PDE-I were added to each tube (0.1 unit/mL in 95 ~L of 10 mM Tris-HCl, pH 8-8, 2 mM MgCl2, 0.4~6 glycerol). The zero time point samples were diluted immediately with acetonitrile (3511L), frozen in a dry ice/isopropanol bath, and stored at -20C for analysis at a later time . The l~ i n; n~ samples were then placed in a water bath at 37C. Samples for each specified time point were then removed from the water bath, diluted with acetonitrile and frozen as described for the zero time point samples.
At the conclusion of the nuclease degradation exper-iment, the samples were individually thawed and analyzed ; ~ t~ly by L~vel~e~ phase HPLC using a Beckman System Gold apparatus with a Model 126 binary gradient pump module and a Model 168 Diode Array Detector. The samples were injected onto the column u6ing a manual injector with a 2000 I~L sample loop. A Vydac C4 Protein column was used for these experiments (Vydac cat. no. 901019, 4.6 mm i.d.
X 250 mm long). Elution was done with a dual solvent 8y8tem: Buffer A = 196 acetonitrile in 50 mM triêthyl _ _ _ _ = = = _ . . .. . . .. .. . . ......

wo g5,l3834 2 ~ 6 2 ~ 9 PCr/US94/13387 ' ' ' ~ ' '' 1 ' ' ammonium acetate ~TEAA, pH 7.0); Buffer B = 50% acetoni-trile in 50 mM TEAA (pE~ 7.0). Solvent flow rates were increased rom 0 . 05 to 1. 0 mL/min . over the irst minute of the run and then held at 1. 0 mL/min. for the rf~m~; n~
5 of the run. G~adient condition5 for each backbone were as follows: All-DE b~Ckhnn~o- 5-2596 Buffer B (2.5 - 9 min.), 25-45~ Buffer B (9.0 - 22.5 min.) 45-100~ Buffer B (22.5 -28.0 min.); 2'-deoxy MP(Rp)/DI!: h~kh~n~- 5-3596 Buffer B
(2.5 - 12.5 min.), 35-50% Buffer B (12.5 - 22.5 min.), 50-10096 Buffer B ~22.5 - 27.5 min.); 2'-0-methyl MP(Rp)/DE~
bac~hone- 5-50~ Buffer B (2.5 - 17.5 min.), 50-659~ Buffer B (17.5 - 27.5 min), 65-10096 Buffer B (27.5 - 31.0 min.).
Average retention times for each h~--khnn~ oligomer (undegraded) were as follows:
A11-DE: 15 . 7 min .
2 ' -deoxy MP ~Rp) /DE: 18 . 5 min .
2'-O-methyl MP(R~)/2'-O-methyl DE: 18.6 min.
Degradation was determined by the appearance of earlier eluting peaks and a decrease in area (or complete loss) of the peak corr~cpnn~;ns to the full-length oligomer.
(b) Stability studies in lleLa cell lysates. HeLa cell CytQrl~elm;c lysate was purchased from ~ndotronics, Inc. (Minneapolis, MN) . This preparation is a hypotonic dounce lysis in 5 X the packed cell volume. It was buffered to pH 6 . 0 by adding 0 .4 mL of 2- (N-morpholino) eth~n.o~l~lfonate (MES, 0.5 M solution, pH 6.0) to 3.6 mL of cell lysate on ice and mixing with mild agitation.
Alisluots of oligomer were dried and then diluted with HeLa cell lysate (95 ~LL) as described in the preceding example.
Samples were then ;ncllh~t-~ at 37C and analyzed by reversed-phase HPLC exactly as described in the preceding example .
(c) Stability studies in cell lysate ~rom African Green Monkey ~idney COS-7 cells. COS-7 cell lysate for these experiments was p~a-~d as follows. COS-7 cells were grown to 90% confluency and then harvested in the presence of 0.2596 trypsin. The cell pellets were washed -Wo 95/13834 2 1 7 ~ 2 5 9 PCrlUSs4ll3387 twice with phosphate buffered saline and then frozen overnight at -20C. Next, the pellets were resll~pon~1~d in approximately an equal volume of lysis buffer (2.5 mM
HEPES, pH 7.2, Z.0 mM MgCl" 0.19c NP-40), drawn up and down ten times through a sterile 1 mL polypropylene pipette, and then centrifuged at 10,000 x G for 5 minutes. Approx-imately 4096 of the resulting sUpernatant was then used to lyse the cell pellet in a dounce homogenizer (Type A
pestle) with twenty strokes. This suspension was then centrifuged as above and the sllr~rn~t~nt was, '~in~d with the rest of the supernatant from the first resuspension.
The resulting solution represents pr~ n~nt ly cytosolic lysate without any nuclear debris and is approximately 1-1. 5 times the volume of the original packed cell pellet .
Aliquots from the resulting cell lysate were buffered with either 25 mM Tris-acetate (final pH 7.4) or 25 mM MES
(final p~ 6.0) prior to ;n, llh~tinn with oligomer.
Ali~auots of each oligomer (0 075 A260 unit) were dried in sterile polypropylene microcentrifuge tubes and then resuspended in 10 ~L of COS-7 cell iysate on ice. Water (90 ~LL) and acetonitrile (35 ~L) were added; -';~tely to the zero time samples and they were frozen in a dry ice/ethanol bath and stored at -20C for later analysis.
The 1, ;n;n~ samples were then incubated in a water bath at 37C. At specified time points, samples were removed from the water bath, dilutea with water and acetonitrile, and frozen exactly as described for the zero time point controls. Following the incubations with cell lysate, the samples were individually thawed, diluted with water (535 IlL) and analyzed immediately by reversed phase HPLC as described above.
(d) Stability ~tudie~ cell ly~ate from Esche-richi~ coli E. coli cell lysate was prepared as follows.
Approximately 2 x 101l cells were pelleted by centrifuga-tion, r~ p~nri~d in 10 mL of Tris-HC1 (50 mM, pH 7.5) and ;nrllhatF.d at room temperature for five minutes. Next, dithiothreitol and lysozyme were added to final cnnn~on~ra-WO 95/13834 , - PCr/US94113387 2~7~2~
tions of 2 mM and l mg/mL, respectively, and the resulting suspension was incubated at 37C for 30 min. The mixture was then sonicated briefly ten times on ice and centri-fuged at 7, 000 rpm for 20 min. Based on visual inspec-tion, it was estimated that this procedure had not suffi-ciently lysed the cells, so the S~ rn~t~nt (vol. = 5 mL) was collected and stored at 4 C and the cell pellet was resuspended in in l mL of Tris-HCl (50 mM, pH 7 . 5) . The res-1~p~nt8~ cell pellet was exposed to five rounds of freeze/thaw, sonicated briefly to break up the chromosomal DNA, and then centrifuged at 8, 000 rpm for 5 min. The resulting sup~rn;~t~nt (approx. 700 ,LL) was then . ` in~
with the supernatant from the previous step (approx. 5 mL) and centrifuged at 6, 000 x G for 5 min. to pellet any residual debris . The f inal supernatant was estimated to contain approximately 50~ lysed cells in approximately 57 times the original cell pellet volume (lO0 ILL). Alir~uots of the oligomers (0.050 A26~ units) were dried in sterile polypropylene microcentrifuge tubes and resuspended in 95 ~LL of cell lysate on ice. Incubations at 37C, HPLC
analysis, and ~r~uantitation of oligomer degradation were done exactly as described aboYe.
(et) Stability studies in cell ly~ate from St~p~ylo-cocc~l aureuf;. S. aureu~ cell lysate was prepared as described above for E. coli except with the following modifications: (i) the lysis was conducted with a cell pellet containing approximately 4 x lO10 cells; (ii) lysostaphin was used instead of lysozyme (500 units, Sigma, Inc. ); and (iii) a total of lO freeze/thaw cycles were used instead of five. Incubation with oligomers at 37C, HPI-C analysis and det~rD~;n~t;rn of oligomer degra-dation from the chromatograms were rnn~ ct~tl exactly as described for the experiment with ~. coli in the example above .
Results. Percent degradation was determined by comparing the peak heights and peak areas f or each time point in each experiment to the 2ero time point controls.

wo 95/13834 ~ 1 7 ~ 2 ~ 9 PCT/US94113387 The half-lives for each oligomer in the presence of PDE-I
were then determined by plotting log(~ full-length) versus time and finding the value corresponding to log (50~) =
l . 699 . The following table ~ummarizes the results from 5 these experiments:
Meta7Dolic Degradation Rate8 of pA~kh~ln~ Analog~ in Bio-logical Sy~tems.
Alternating ~tl~-llf~ of Normal 2~-0-Met_yl MP(R~,)/D3i: 2~-0-Mcthyl Annlos Phospho- ~NA Alterrating 7fP ~
diester 2 ' -0-methyl 10109~ Fetal Calf Serum, p~l 8 12 min. 40 min. 5 Ers. ~ 300 ~rs.
Green Monkey Kidney Cell c 10 mi~. ~ 5 }7rs. - 25 Elrs. Stable*
Lysate, pEI 6. O
15Green Monkey Kidney Cell ~ S min. ~ S E~rs. - 20 }Irs. sta Lysate, p}~ 7 . 4 E. co7~ Cell 1-3 min. _.2 7Irs. ~ 65 ~rs. Stahle~
Lysate S. Aureus Cell 13 min. ~ 20 Elrs. ~ 75 71rs. Stable*
Lysate Snake Ve~om 15 min . 2 . 5 min . 167 min . Stahle~
Phospho -die~7tt~r:la9~Aae * No ~t~ctAh~ r~ t~nn after 24 hour ~nrl~h5lt~r~n, r ~le 32 HYbri~;~Ation of 6~h;rAlly 7~nriGhed Antl Non-6~h;ra Oliqomers to RNA Tarqets Chirally enriched all-pyrimidine (C'T) 7A and all-30 puri~e ~A'G) ~T MP-oligomers were prepared using either Rp-or Sp-dimeric units. Control o1;~ s were also prepared using the individual I - ic units. The asterisks indicate the positions of def ined chirality.
Each oligomer was annealed to a complementary syn-35 thetic RNA target and then monitored by ~hqorhAn~-~ at 260 nm as a function of temperature. Sigmoidal transitions were observed corr~ro~;ng to the7-mal denaturation o~ the hybridization complexes. The Tm values were r~ta~rrn;nf~ at . , . . , . , _, _ _ WO 95/13834 - PCr/US94/13387 2~9 - i the midpoint of each sigmoidàl transition. Previously, we have shown that ~CT) B r~l; S forms a double-stranded complex with RNA at neutral pX, whereas (AG) 8 Oligomer forms a triple-stranded complex. Thus, we anticipated 5 that the data for each chirally enriched seriea would be applicable to double-stranded and triple-stranded MP/RNA
helices, respectively. The Tm data is summarized below:
Al ~ernatinq ~ CT), (A) 10 Oliaomer No. Seauence Confiquration' 2286-1 5'-c t-c t-c t-c t-c t-c t-c t-a-3' (Rp) 2288-1 5 ' ctctctctctctct-a-3 ' (R, S) 2287-1 5'-c't-c~t-c't-c't-c't-c't-c't-a-3' (Sp) (B) Oliqo Tm ~l:l.RNA) 4Tm~RNA) 2286-1 45.5C +10.4C
2288-1 35. 1C ----------2287-1 25.4C -9.7C
Alternatinq (AG)7 2 0 (A) Oligomer No . Seauence Conf iquration' 2323-1 5 ' -a'g-a g-a g-a g-a g-a g-a g-t-3 ' (Rp) 2 2 5 3 -1 5 ' - agagagagagagag - t - 3 ' ( R, S ) 2252-1 5 ' -a'g-a'g-a'g-a'g-a'g-a'g-a'g-t-3 ' (Sp) (B) Qli~o Tm ~l:l.RNA) ~Tm(RNA) 2323-1 55.2C +7.2C
2253-1 ~ 48.0C -------2252-1 40 . 0C -8 . 0C
As shown in the tables above, the Rp-enriched preparations have higher Tms with RNA targets. On the other hand, Sp-enriched preparations have lower Tms with RNA targets.
In separate experiments, we conf irmed that the 35 chirally-enriched (C T) ~A and (A G) ,T MP-oligomers form -WO 9~113834 PCTNS94113387 ~17~25~

double- and triple-stranded complexes with RNA at neutral pH, respectively.
These experiments demonstrate that chiral enrichment can dramatically effect the binding affinities of MP-5 oligomers in both a duplex and triplex motif.
F le 33 Tm Com~arisons for Methvlphos~honate Oliqomer9 ContAin;na Eithe~ R~-~n~iched or Racemic Backbones Racemic methylphosphonate oligomers and complementary 10 RNA targets were synthesized according to the methods described in Examples 28 and 29 . The MP (R~) ~MP oligomers were synthesized according to the methods described herein by coupling MP (E~,) /MP dimers . Each coupled MP (Rp) /MP dimer is indicated by parentheses in the table below, wherein 15 asterisks indicate chirally pure linkages.
z~nn~ ;nr reaction mixtures rr,nt~;n~ equimolar amounts of methylphosphonate oligomer and RNA target oligomer (2 . 4 llM total strand rrnr~ntration), 20 mM
potassium phosphate (pH 7.2), 100 mM sodium chloride, 0.1 20 mM EDTA and O . 039~ potassium sarkosylate . The reaction mixtures were heated to 80C and then slowly cooled to 4C
over apprrY~-t~y 4 to 6 hours. The annealed samples were then transferred to 1 cm quartz cuvettes and absor-bance at 260 nm as a ~unction of temperature was monitored 25 using a Varian Cary Model 3E Spectrophotometer rclnt~;n;n~
a 6 x 6 temperature controlled sample holder and which interfaced with an IBM compatible PC t~r. The temperature was varied from 5C to 80C at a ramp rate of 1C/minute. The Tm for each melt profile is defined at 30 the point corresponding to the first deri~ative (of the A260-temperature function) . The following table summarizes data obtained for a number of pairs of racemic versus Rp-enriched methylphosphonate oligomers. Based on the observed increases in Tm, Rp-enrichment u9ing the MP(Rp) /MP
35 dimer coupling method described herein leads to signifi-~6~5~ ~
Wo 95113834 - , PCrlUS94/13387 cant r~nh~3nc t in the binding energy between a methyl-phosphonate oligomer and its RNA target.
Comparison of Tm's for MP(R,j)/MP Enriched and Racemic MethYlPhos~honate Oliqomers Sequence Sequence Tnn ~Tm number 2288-1 5'-CT-CT-CT-CT-CT-CT-CT-A-3' 34.4C
2286-1 5'-(C-T)(C-T)(C'T)(C'T)(C-T)(C T)(C~)-A-3' 44.0C 9.6C
2253-1 5'-AGA-GAG-AGA-GAG-AG-T-3' 48.9C
2323-1 5'-(A G)(A G)(A G)(A G)(A G)(A G)(A G)-T-3' 56.3C 7.4C
2517-1 5'-GTG-TGT-GTG-TGT-GTG-TA-3'-3' 41.0C
2516-1 5'-(G-T)(G-T)(G~T)(G-T)(G'T)(G-T)(GT)(GT)-A-3' 48.8C 7.8C
1634-1 5'-TAG-CTT-CCT-TAG-CTC-CTG-3' 38.2C
2570-1 5'-(T-A)(G C)(T-T)(C C)(l-r)(A G)(C-T)(C C)(T G)-C-3' 46.9C 8.7C
2688-1 5'-ATG-GTGTCT-GTT-TGA-GGT-T-3' 40.0C
2662-2 5'-(A'T~(G-G)~T-G)(T-C)(T-G)(T~(T G)(A G)(G-T~-T-3' 47.5C 7.5C
2624-1 5'-GTC-TTC-CAT-GCA-TGT-TGT-C-3' 38.6C
2571-1 5'~G T)(C T)(T C)(C A)(T G)(C A)~G)(T'T)(G-T)-C-3' 46.3C 8.2C
2625-1 5'-GCT-TCC-ATC-TTC-CTC-GTC-C-3' 42.9C
2 0 2574-1 5'-(G C)(T-T)(C C)(A'T)(C-T)(T C)(C T)(C G)(T C)-C-3' 51.8C 8.9C
F le 34 R;nrlino Stabilitv of Various Backbone Modified Oliqomers Havinq a (CT) .A Model Seouence to Com~lementarv Svnthetic RNA ~arqets Racemic methylphosphonate oligomers and complementary RNA target oligomers were synthesized as described in previous applications. A series of oligomers having the same seo,uence but with different b~rkhnnr~C was prepared as described ~1 rewh~re in this application. Rp- (CT) dimers were used to make the 755~ Rp-enriched all-methylphosphonate and the 2'-deoxy MP(Rp)/2'-deoxy DE oligomers. Rp-(CU) dimers were used to make the 2~-0-methyl MP(Rp)/2'-0-methyl Wo 95/13834 ~ `~ PCrlUS~4113387 ~176259 DE oligomer. Oligomers cnntA;n;n~ ph~Erhnr-othioate linkages mixed with other 1; nkA~ were synthesized according to the general procedures described in Example 30 and other examples above. Control oligomers cnnt~;n;ng 5 either a normal phosphodiester (2 ' -deoxy all-DE) backbone or a 2'-O-methyl phosphodiester h~rkh~nnc (2'-O-methyl DE), and all-phosphorothioate oligomers, were purchased from Oligos Etc. Where 2'-deoxy or 2'-O-methyl substitutions are indicated below, these structures occur on all of the 10 residues in the alt~orn~;ng or repeated sequence.
~ nn~ l ;n~ reactions cnnt~;n~l e~uimolar amounts of b~rkhnn~-modified oligomer and RNA target oligomer (2.4 ~lM
total strand concentration), 20 mM potassium phosphate (pH
7.2), lO0 mM sodium chloride, O.1 mM EDTA and O.039~
15 potassium sarkosylate. These reactions were heated to 80C and then slowly cooled to 4C over a time period of approximately 4 - 6 hours . Next, the annealed samples were transferred to l cm quartz cuvettes and monitored by absorbance at 260 nm as a function of temperature in a 20 Varian Cary Model 3E Spectrophotometer cnn~;n;nS a temperature controlled 6 x 6 sample holder and interfaced to an IBM compatible PC computer. The temperature was varied from 5C to 80C at a ramp rate of 1C/min. The Tm is defined as the point corr~cronr~; n~ to the maximum o~
25 the first derivative of the thermal dissociation profile.
The binding constants at 37C (K"(37C) ) were lot~rm;n~od by a non-linear least s~uares fit of the thermal dissociation data assuming a two-state model for the melting process.
The f ollowing table summarizes the -esults:

.
WO 95/13834 2 1 ~ ~ 2 ~ ~ PCrlUS94/13387 Sequence = 5'-~L~lClCl~ lA-3' ~egu-nc~, nu~ r BAcl~bone typ- Tm(C) 1~(37-C) 2288-1 ~acemic all-MP 34.0 8.3 x 5 2781-1 2'-0-Methyl racemic all-MP 37.1 2.1 x 10' 2782-1 ~lt~7nAt;n~ racer~ic MP/DE 40.6 6.3 x 2286-1 75% '.~,-enriched all-MP 44.0 2.6 x 10' 3253-1 Alternating 2'-deoxy YP(Rp)/PS 47.3 1.8 x 2768-1 2~-0-Methyl 75% ~,-enriched all-15P 47.4 3.9 x 10' 10 2793-1 All-PS 50.4 4.3 x 10' 2760-1 Alternating 2~-deoxy MP(Rp)/DE 53.8 7.9 x 2784-1 .7~1t~.rnAtin~ 2~-0-Methyl racemic- 59.
MP/2~-0-methyl DE 10~3 x 2795-1 2'-Deoxy all-DE 60.8 7.1 x 101l 2765-1 Z8t~rnAt;n~ 2~-0-Methyl XP(R~)/2'-0- 67.9 5.2 x methyl DE 1ol' 15 2792-1 2'-0-Methyl all-DE 75.0 5.3 x 1 ol~
According to this data, a dramatic; _ .,v- t in binding stability for an RNA target is achieved with the various backbone modif ications to the original racemic all-MP oligomer.
20 Exam~le 35 ~ ~
B.;nr~;n~r Aff;n;ties of Various Chimeric Backbone Oliqomers ~lementarv RNA Tarqets The following oli~n~ P~ tides were tested for their ability to hybridize to a complementary synthetic RNA
25 target .

WO 95/13834 2 ~ ~ 6 2 ~ 9 PCT/US94/13387 I.D. # Sequence !:~escription 2~67-1 5'-GTCTTCCA~TGCAT)GTTGTCC-3' rMP] [DE] rMP]
2681-1 5'-GTCTTCCA(TGCAT)GTTGTCC-3' [MP] [PS/P~] [MP]
2687-1 5'-GTCTTCCAT(GC~TG)TTGTCC-3' [75'~MP(R") 3 [Dl!] r75~MP(Rp)]
- 5 3169-1 5~-GTCTTCCA(TGCAT)GTTGTCC-3' [MP(7,,)~DE] [D~] [MP(R~)/DB]
3214-1 5'-GTCTTCCA(TGCAT)GTTGTCC-3' [llP(Rp)/DI!] [PS/DE] [MP(Rp)/~E]
3257-1 5'-GTCTTC(CATGCAT)GTTGTCC-3' [MP(Ep)/DE3 [PS/DE] [MP(P7)/DE]
3 2 5 6 -1 5 ' - GTCTTCCA (TGCAT ) GTTGTCC- 3 ' [MP (RD ) /DE] [ PS ] [MP ( Rl,) /DE]
The bases shown in par.onth~q~o~ contain the ba~kh~
10 modi~ication indicated in the middle set of brackets for each description, and likewise the terminal portions of the oligomers contain linkage structures as shown in the terminal sets of brackets. The PS/DE notation indicates an alternating array of bases beginning with a phos-15 phorothioate linkage . For example, if there are f ivebases in a sequence denoted as P6/DE, they include three phosphorothioate (PS) bonds and two phosphodiester (DE) bond8 .
Each oligomer was mixed with its complementary 2 0 synthetic 3~NA target in a 1:1 molar ratio in a buf f er system con6isting of 20 mM sodium ~ht~s~h~te buffer (pH
7.2), 100 mM NaCl, 0. 03~ potassium sarkosylate and 0.1 mM
EDTA; total strand concentration = 2 . 4 micromolar . The resulting solutions were heated to 70C and slowly cooled 25 to 4C over a time period of approximately 4-6 hours.
Next, the annealed oligomers were monitored at 260 nm over an increasing temperature gradient of 1C/minute using a Varian Cary Model 3E W/Visible SpeuL,u~ otometer equipped with a thermostat multicell holder, temperature controller 30 and temperature probe accessories. Data was recorded and processed using a PC computer interface. The Tm values were determined from the first deriYative of the sigmoidal melt transition. The binding constants at 37C (KA(37C) ) were determined by applying a non-linear least squares fit 35 to the data and assuming a two-state model for the dena-_ _ _ _ _ _ WO 95113834 2 1 7 6 2 ~ ~ PCTNSg4113387 turation process. These values are shown in the tablebelow:
I.D. # Tint C) ~A(370C) 2567-1 45 . 6 2 . 9 x 10~
5 2681-1 44.1 2.1 x 107 2687-1 52.8 2.6 x 109 3169-1 62 . 6 6 . 0 x 10l4 3214-1 61.0 2.3 x lO
3257-1 60.9 2.1 x lOli 3256-1 60 . 1 5 . 5 x 10l3 Studies with other chimeric b~khnnP oligomers further demonstrated that compounds cnnt;l;n;n~ Rp-chiral methylphosphonate bonds have higher net binding stabili-ties with RNA targets compared to oligomers having the same compositions but with racemic methylphosphonate bonds. Determination of Tm values was done generally as described above . Data f or a variety of sequences, some having varying sizes in their RNaseH-activating regions as well as selected 2~-sugar substitutions, were obtained as follows. ~Linkage structures separated by slashes indi-cate an alternating sequence of the listed l; nkil~c; thus, in the case of the 5-base PS/DE core of compound 2681-1, a linkage sequence -PS-DE-PS-DE-PS- appears. Uridine residues were substituted f or thymidine residues in the bracketed portions of the compounds below having 2 ~ -o-methyl substitutions. 2'-O-methyl sugar substituents were incorporated on each of the methylphosphonate- and phos-phodiester-linked nucleoside sugars of the terminal non-RNaseH-activating regions of these compounds ~numbers 3341, 3336, 3339, 3337, 3382 and 3386), except for the 3'-terminal residues that were separately bound to the solid support prior to dimer synthon addition ~cf. Example 44 below) . ) 2i7~5~
WO 95/13834 - . : . PCT/US94/13387 Sequence TYPe I
5-base core: 5' [GTCTTCCA](TGCAT)[GTTGTCC] 3' 7-base corc: 5' [GTCTTC](CATGCAT)[GTTGTCC] 3 ' Tm 5ComPound r '-L ! ' Structure ~,~
2681-1 [MP(racemic)]-(PS~DE)~-[MP(racemic)] 44.1 2567-1 [MP(racemic)]-(DE)5-[MP(racemic)] 45.6 2687-1 [75% MP(R~)]-(DE)5-[75% MP(ll~)] 52.8 3256-1 [MP(I~)/DE]-(PS)5-[MP(R")/DE] 60.1 10 3214-1 [MP(II?)/DE]-(PS/DE)5-[MP(Rp)/DE] 61.0 3169-1 [MP(Rp)/DE]-(DE)s-[MP(RF)/DE] 62.6 3257-1 [MP(RpyDE]-(PS/DE)7-[MP(R")/DE] 60.9 3341-1 [2'0Me{MP(Rp)/DE}]-(PS),-[2'0Me{MP(Elp)/DE}] 65.8 3336-1 [2'0Me{MP(Rp)~DE}]-(PS/DE)7-[2'0Me{MP(Rp)/DE}] 66.8 '.5Seauence TYDe 2 5-base core: 5' [GCTTGGCTA](TTGCT)[TCCATCTTCC] 3' 7-base core: 5' [GCTTGGCTA](TTGCTTC)[CATCTTCC] 3' Tm Compound r ~ ~ ! ' StrucPJre (C. RNA) 2 03234-2 [MPIRpyDE]-(PS/DE)5-[MP(Rp)/l)E] 62.0 3233-1 [MP(Rp)/DE]-(DE)5-[MP(R,yDE] 63.6 3330-1 [MP[Rp)/DE]-(PS/DE)7-[MP(RpyDE] 61.3 3339-1 [2'0Me{MP(R,~/DE}]-(PS/DE)7-[2'0Me{MP(RpyDE}] 68.8 3337-1 [2'0Me{MP(I~)/DE}]-(PS)7-[2'0Me{MP~pyDE}] 70.3 25Sequence TYPe 3 5-bas~ core: 5' [GGTATATC](CAGTG)[A~ U~:U l~;lUl 3' Tm ComPound E ' Lin,a~e Structure (C RNAl 3383-1 [r ~ .. iL)/2'0MeDE](PS)5[~ .. ;. )/2'0MeDE] 59.6 30 3382-1 [2'0Me{MP(rac.)/DE}](PS)5[2'0Me{MP(rac)/DE}] 64.4 3386-1 [2'0Me{MP(Rp)/DE}](PS}~[2'0Me{MP(Rp)/DE}] 64.4 The data 8h3wed that a 8ignificant ~nhAn~ ' in binding affi~ity results when racemic methy1rh~,~phnnA~e WO95/13834 ~17G25~. . PCr/US94113387 ~

linkages are replaced with R~-chiral methylphosphonate6.
This observation applie9 to nucleo9ides rnntA;n;nr 2'-deoxy ribofuranose 6ugars as well as to bases cnntA;n;ng 2'-O-methyl ribofuranose sugars. Oligomers cnntA;n;n~
5 regions of alternating MP (R~) /DE linkages have higher binding affinities than oligomers having alternating MP (R~,) /MP (racemic) linkages . A further binding ~onhAnr results when 2 ' -O-methyl ribofuranose sugars are substi-tuted f or 2 ' -deoxy sugar9 . Ba9ed on the data presented lO above, it is estimated that the Tm increases by about 0.5-0 . 6 C per substitution .
r le 36 Demonstration of the Abilitv of Various Chimeric Oliqomers to Activate RNaseH from He~a Cell Nuclear E~tract The following oligomers were tested for their ability to activate endogenous eukaryotic RNaseH derived from He~a cell nuclear extracts.
r.D~ ~ S~nc~ ~ D~scrw~rn 2498-1 5'-GTCTTCCATGCATGTTGTCC-3' AII-DE
2 o 2566-1 5'-GTCTTCCATGCATGTTGTCC-3' AII-PS
3130-1 5'-GTCTTCCATGCATGTTGTCC-3' rlPal,)/l)E Al~errlting a~on-Chimeric) 3169-1 5'-GTCTTCCA(TGCAT)GTTGTCC-3' IMP/Rr!~ R']
3214-1 5'-GTCTTCCA(TGCAT)GTTGTCC-3' IMI'(I~,)/DEI[PS/DE]~MP(Rp)/DE]
3256-1 5'-GTCTTCCA(TGCAT)GTTGTCC-3' IMP(R~,)I.,_". ,~ ' ~p)lDE]
25 Each o~ these oligomers (lO IlM) was annealed to its complementary synthetic RNA target (l ~M) in a buf fer system rnn~A;n;ns 50 mM Tris-HCl (pH 8.0), 20 mM KCl, 9 mM
MgCl2, 1 mM ,B-mercaptoethanol, 250 l~g/m~ bovine serum albumin, and 25-lO0 units/mL of RNasin (Promega, Corp., 30 Madison, WI) . RA~;nlAhPl.of~ RNA having 3~P at the 5'-terminus was prepared using [ y-32P] -ATP (New England Nuclear/DuPont, Boston, MA) and T4-polynucleotide kinase (Stratagene, Inc., San Diego, CA) according to standard procedures. Approximately 200, 000 dpms of 3'P-labeled RNA
. , , _ _ _ _ _ _ _ _ _ _ , , . . . , , , , . . . , , , . , . . , , . : , . . , . : . . . . . . . .

2~7~259 was included in each reaction as a radiotracer. These samples were ~nnP;~ by heating to 65C and slowly cooling to 4C over a period of apprn~ t.oly 4-6 hours.
Stock solutions c^nt~;n;n~ EIeLa cell nuclear extract were prepared as follows. HeLa cell nuclear extract (Promega Corp., Madison, WI, Catalog # E3521, 5 mg/mL
protein) wa5 diluted 250-fold in a buffer consisting of 20 mM HEPES (pH 8.0), 20% glycerol, 0.1 M KCl, 0.2 mM para-methylphenylsulfonyl fluoride (PM.~3F) and 0 . 5 mM dithio-threitol.
RNaseH cleavage reactions were initiated by adding diluted HeLa cell nuclear extract (5 ,I~L) to each of the annealed oligomer samples (lO~L) and then the samples were ;n~llh~t~d at 37C for either fifteen minutes or two hours.
At the end of the specified incubation time, each cleavage reaction was terminated by addition of 1.5 IlL of EDTA (125 mM, pH 8) and then quickly frozen on dry ice and stored at -20C. When all of the cleavage reactions had been termi-nated they were removed from the freezer for analysis by polyacrylamide gel electrophoresis. Aliquots (5 /lL) were withdrawn from each reaction and diluted with gel loading buffer (5 ~L, 90% formamide/lxTBE buffer/0.19~ bromphenol blue/0.1% xylene cyanole blue). The resulting samples were loaded onto a 15% polyacrylamide/7 M urea gel (20 cm X 30 cm g 0.5 mm thick) prepared in lX TBE buffer (pH

8.2). The gel was electrophoresed at 1200 volts for 1.5 hours. Bands on the gel corresponding to full length and cleaved RNA products were rlPte~-ted by phosphorimager analysis using a Bio-Rad Model GS-250 Molecular Imager 3 0 (Bio-Rad Laboratories, Hercules, CA) . The amount of cleavage that occurred in each reaction was determined by comparing the phosphorimager counts for the full length band to the total counts per lane. The results are summarized below:

Wo 95/13834 PCrlUS9411338~
~762~

Oligomer I.D. # ~Na~e}~ Cleav~ge after 2 ~Ir~.
At 37C
2498-1 24.4%
2566-1 10 . 59~
3130-1 None detected 3169-1 52 . 0~6 3214-l 38 . 096 3256-1 18 . 7~
The length of each cleavage fragment was estimated from the electrophoretic mobility of its associated radioactive 10 band. From this analysis, it was determined that cleavage occurs selectively in the middle of heteroduplexes derived f rom the chimeric oligomers . More numerous cleavage products were observed with the all-phosphodiester (DE) and all-phosphorothioate ~PS) oligomers, as expected.
15 This data shows that the replacement of PS for DE linkages results in a reduction in the rate of RNaseH-mediated cleavage. There was no cleavage observed in the sample rrn~z~;n;nr an alternating MP(Rp)/DE bArkhr,ne.
r ~le 37 20 S~AhilitV of Various Chimeric Oliqomers to Nucl~oAqe Dicrestion in the Presence of Sl-~n~nllrlease The following oligomers were tested for nuclease stability in the presence of S1-Pn-~nnl~rl ease.
LD. # Sequencc Dcscription 2567-l 5'-GTCTTCCA(TGCAT)GTTGTCC-3' ~P][DE][~]
2681-l 5'-GTCTTCCA(TGCAT)GTTGTCC-3' lMP][PSll)E [lMP]
3169-l 5'-GTCTTCCA(TGCAT)GTTGTCC-3' ~Pff~p)/DE ~E][~(Rp)/DE]
3214- l 5 '-GTCTTCCAtTGCAT)GTTGTCC-3 ' ~lP~)/DE PSIDE] [1\1P(Rp)/DE]
3256-l 5'-GTCTTCCAtTGCAT)GTTGTCC-3' ~P(Rp)/DE PS][MP(Rp)/l)E]
30 Sl-~n~r~nllr~ ] ease was purchased from Promega Corp. (Catalog # E576B, Madison, WI). Aliquots of each chimeric oligomer (0.05 - 0.075 OD260 units) were individually added to polypropylene microcentrifuge tubes cr~nt~;n;nr~ S1-endonu-clease tO.5 units/mL) in 30 mM sodium acetate (pH 5.0), 50 ~WO95113834 2 1 ~ 6 ~ 5 9 PCTIUS94113387 ., mM NaCl, 1. 0 mM zinc acetate and 5~ glycerol; total reaction volume = 10 ~LL. These tubes were incubated at 37C for specified time periods, quickly frozen in dry ice and then stored in a freezer at -20C. The samples were 5 then analyzed by reversed-phase ~PLC using a Beckman System Gold chromatography system equipped with a Model 126 Solvent Module and a Model 168 Diode Array Detector.
Column = Vydac Protein C4 (catalog #214TP54, 4.9 mm i.d.
x 250 mm long) . Buffer A = 50 mM triethylammonium acetate (pH 7) /196 acetonitrile; Buffer B = 50 mM trietbyl ;llm acetate (pH 7) /50~; acetonitrile. The elution profile was 5-35% Buffer B (2.5 - 12.5 min.); 35-509G Buffer B (12.5 -22.5 min.); 50-100~ Buffer B (22.5 - 27.5 min.); flow rate = 1.5 mL/min. The samples were diluted with water (50 ,uL) 15 and injected onto the column using a 100 ~L sample loop.
Peaks corresponding to full length oligomer and its degradation products were detected by monitoring at 260 nm. The amount of degradation occurring in each reaction was determined by measuring the r~ t;r~n in peak area for 20 the full-length oligomer (;d~nt;f;~1 by comparison to an ~rt~rn~l control and/or by coinjecting undigested oligomer as an internal control). The data is shown in tabular format below, and in graphic format in FIG. ~.
OligomerLD. # ~alf-LifeforD~h -2567-1 1.7 Hrs.
2681-1 12.2 Hrs.
3 1 69-1 0.9 Hrs.
3214-l 5.0 Hrs.
3256-1 12.5 E~s.
30 * Detorm;ned as the point where 50% full length oligomer has been digested based on a least-squares fit of the data .
This data shows that the rep~ of ~h~"3phr~rothioate (PS) bonds for phosphodiester (DE) bonds _ _ , .. _ .. . , . _ . . . _ _ _ . , _ Wo 95113834 PCrlUS94113387 ~1762~9 imparts a resistance to nuclease degradation catalyzed by Sl--~n~ nll~1 ease.
E le 38 St~hilitv of Various Chimeric Oliqomers to Nuclease 5 DiqestiQn in the Presence of lO96 Fetal Calf Serum Multiple ~ samples of each chimeric oligomer were prepared in l . 5 mL polypropylene microcentrifuge tubes on ice. Each sample ~ ntz~;n~rl oligomer (O.l OD260 unit), lO9 fetal calf 8erum (FCS, Gemini Bioproduct8, ~ lAh;lc;~, CA~, 20 mM HEPES (pH 8.0), 0.2g~ paramethylsulfonyl fluoride (PMSF), 175 mM KCl, O.l mM dithiothreitol, O.l mM EDTA, 2 mM MgCl2 and 49~ glycerol -- total volume = lO0 IlL. The samples were incubated at 37C for specified time periods and then diluted with 0.49~ NP-40/acetonitrile (35 ~
15 quickly frozen on dry ice and stored at -20C. Samples were then individually thawed, diluted with water (635 IlJJ) and analyzed immediately by reversed-phase HP~C according to the method given in the preceding example (except that a 2 mL sample 1QP was used to load the samples Qnto the 20 column). Results are shown in tabular format below, and in graphical format in FI~. 2.
Oligomcr l.D. # Half-~ if e for D.~j. ' *
2567-1 5.8 Hrs.
2681-1 8.1 Hrs.
25 3169-1 3.4 Hrs.
3214-1 4.3 Hrs.
3256-1 16.2 Hrs.
* Determined as the point where 50~ full length oligomer has been digested based on a least-squares fit of the 30 f irst three time points in each data set .
This example indicates a similar enhancement in stability to nuclease degradation when PS linkages are used in place of DE linkages.

~WO 95113834 93 PCTIUS94/13387 ~Y~m~le 3 9 ACtivitY of rMP] rDEl rMP] oliqomer 2567-1 and rMP(R~)/DEl-rDEl - rMP(RF) ~DEl oliaomer 3169-1 o~ cell-free tr~n~lation of tarqet mRNA
A target mRNA having complementarity to these oligomers at the initiation codon region was prepared by standard cloning techniques with reverse-transcription catalyzed by T7 polymerase (Promega MEGAscript kit for uncapped RNA), according to the manufacturer' 8 protocol .
Control CAT mRNA was obtained from GIBCO as a control for specif icity .
Target mRNA and control CAT mRNA were translated in a cell-free translation assay in rabbit reticulocyte lysates (Promega), in the presence of 35 [S] -Cys (NEN/DuPont) following the m-nllf~n~llrer's directions.
Oligos 2567-1 and 3169-1 were added to individual trans-lation reactions at 0, 0.2, or 1.0 M, final concentra-tions. RNAse-H (Promega Corp. ) was added to all the translation reactions at 0 . 04 units/ul . Each condition was run in triplicate. Translation reactions were incu-bated at 37 C for 1 hour. At the end of the translation reactions, proteins were denatured with ~aemmli Sample Puffer (Novex) and the amounts of target proteins synthe-sized in each case were evaluated after immunoprecipi-tation with an hyperimmune antibody serum followed by gel fractionation of the protein products (10-20% gradient SDS-PAGE~ gels, Novex) and phosphoimage analysis. The amount of control CAT protein synthesized in each case was evaluated after gel frant;nn~inn of one aliquot of the denatured translation reaction (10-20 ~ gradient SDS-PAGE
gels, Novex) and phospho-image analysis.
As shown in FIGS. 3 and 4, oligomer 3169-1 produced approximately 50% and 90% inhibition of target mRNA
translation when pre8ent at 0 . 2 or 1 ~LM, respectively.
Oligomer 2567-1 produced approximately 0% and 5096 inhi-bition of target mRNA translation when present at 0 . 2 or 1 IlM, respe~tively. Both oligo8 produced little inhibi-WO 95/13834 21 ~ 6 2 5 9 ~ PCT/US94/13387 tion of control CAT mRNA translation, indicating good specif icity .
This result indicates that replacement of racemic MP
ends by chirally-selected MP (R~) /DE linkage segments 5 significantly increases the ability of an oligomer to block cell-free translation of the target mRNA.
~m~le 4 Cleavaqe of tarqet mRNA. in the ~resence of RN~qeH, o~
r~lPl rDEl rMPl oliqomer 2567-1 and rMP(R9) /DEl - rDEl -rMP ~R~) /DEl oliqomer 3169-1 A target mRNA having complementarity to these oligomer6 at the initiation codon region was prepared by standard cloning techni~ues with transcription using a T7 polymerase cell-free assay tPromega MEGAscript kit for uncapped RNA), according to the manufacturer~ s protocol .
The resulting mRNA transcript is approximately 340 nt in length .
The ability to cleave this target mRNA, in the presence of RNAseH and either of oligomers 2567-1 ~ [MP] -2C [DE] - [MP] } and 3169-1 { [MP (Rp) /DE] - [DE] - [MP (R~) /DE] } was t,orm;n--~ as follows.
Cell-free transcribed mRNA (100 nM) was incubated at 37 C, in a cell-free translation buffer (rnnt~;n;n~ 3 5 mM MgCl~, 25 mM KCl, 70 mM NaCl and 20 mM potassium acetate), in the presence of 0 . 04 units/~Ll of RNAseH
(Promega) and either of oligomers 2S67-1 or 3169-1 at 0, 0.01, 0.1, 1, or 10 IlM. After 30 minutes, the RNA was extracted, denatured and run in a denaturing gel. After the run, the RNA was stained with ethidium bromide and its integrity was determined by visual observation of the RNA
bands present in the gel.
As shown in the table below, a good dose-response effect was obtained for both oligomers at the concentra-tions tested. Oligomer 3169-1 was more active than oligomers 2567-1 ~3169-1, at 1 ~M, cut ~98 96 of the target mRNA present in the reaction, while oligomer 2567-1, at ~wo 95/l3834 2 ~ ~ ~ 2 5 ~ PCrlUS94/13387 the 8ame concentration, cut -50~ of the target mRNA
present in the reaction). Both oligomers showed good specificity, cleaving the target mRNA in one position.
Cle~ivage of t~rget mRNA, in the presence of RNAseH, of IMP]IDE~[MPI oligomer 2567-1 and [MP(Rp)tDE]-[DE]-[MP(Rp)/DEI oligomer 3169-1 Oligomer 2~67-1 3169-1 Backbone [MP]-[DE]-[MP] [MP(I~)/DE][DEIIMP(}~,)/DE
Ol~gomerconcen- 0.01 0.1 1 iO 0.01 0.1 1 10 tr~tion (yM) /. of tsrf~et mRN~ ~ 15 50 80 5 40 9 100 clenvn~e~
) Estimated values obtained by visual inspection of the ~el Exam~le 4 1 15 ;rnh;hitiQn of Protein Svnthesis in a Cell Culture With t~hir-ric .~nt-i qense Oliaomers T~rqeted to a Non-~llk~rvotic Re~orter Gene, Chll h~ni col Tr~nqfer~qe The following example shows the ability of chimeric antisense oligomers to selectively inhihit protei~ syn-20 thesis in a eukaryotic cell culture 8ystem. COS-7 cells were transiently transfected with plasmids encoding either a target reporter gene or a control non-target reporter gene. These cells were then treated with various chimeric antisense or control oligomers and then assayed f or the 25 expression of the reporter genes.
Plasmi~q The following plasmids were used in this example.
pG1035: Splicer CAT, in8erted i~to a pRc/CMV vector pG1036: Wild-type ~AT, in8erted into a pRc/CMV vector . , . _ . . .. . . . . . . .. _ _ _ _ _ .

21 ~2~9 0 WO 95/13834 : ~ PCrNS94113387 , 96:
pGl040: UCAT, inserted into a pRc/CMV vector pGL2: Lucifera9e expressing plasmid (Promega) pSV~ galactosidase expressing plasmid (Clonetech) A description of plasmids pGl035, pGl036 and pGl040 5 follows.
l. pGl035 (SplicerCAT) and pGl036 (wild-type CAT) and the sequences of the synthetic splice sites:
A. Sequence of the wild type CAT gene used to create plasmid pGl 0 3 6:
+409 +410 GCC UAU WC CCU AW IJCC CUA AAG GGU WA WG AGA A~A ~ ~
B. Full sequence of the intron inserted within the CAT coding sequence to create SplicerCAT and plasmid l~ pGl035:
+409 l ... UAU WC CCU AW UCC CUA i~AGI quq aqu qac uaa cua ac,u cqa cuq caq acu aqu cau ua(~ ) uuq aqu qua aca aga ccg gau ~7 +410 auc uuc qaa ccu cuc ucu cuc ucu c~a GGU WA WG AGA ...
The region of the CAT gene into which the intron was inserted is shown in sequence A above. Wild type CAT DNA
(Pharmacia) was inserted into pRc/CMV (Invitrogen) to create plasmid pGl036. The sequence is shown as the mRN~.
Bases 409 and 410 are labeled for comparison to pGl035.
A synthetic intron, shown as sec~uence B above, was insert-ed into the CAT DNA to create plasmid pGl035. Mature mRNA
sequences are shown uppercase, intronic sequences are lower case. The canonical guanosine of the splice donor is labeled +409, which corresponds to base 409 of the CAT
open reading ~rame . The f irst base of the intron is labeled l. The canonical branchpoint ;~ n~;nF~ is base 39 ~Wo 95113834 ~ 1 7 6 2 ~ 9 PCrlU594/13387 and the canonical intronic splice acceptor guanosine i8 base 87 of the intron. Base 410 marks the resumption of the CAT open reading frame The sequences against which the oligomers are targeted are underlined The consensus splice site bases are given in bold face italics (Smith et al. 1989; Green 1986) .
The clDne pG1035 was created using synthetic DNA PCR
primers to create a Hind III-Spe I 5'fragment cfmtA;n;n~
the first 2/3 of the open reading frame and half of the synthetic intron and an Spe I-Not I fragment containing the second half of the intron and the last 1/3 of the open reading frame. These were ~ ' ;n~d with Hind III-Not I
cut pRc/CMV in a 3-way ligation to yield the final plas-mid. The artificial CAT gene ~nntA;n;n~ the intron is named SplicerCAT. References applicable to the foregoing include Smith CWJ/ Patton JG/ and Nadal-Ginard B/ (1989) /
"Alternative splicing in the control of gene expression, "
Annual Reviews in Genetics 23: 527-77; Green, MR (1986), "Pre-mRNA splicing, " Annual Reviews in Genetics 20: 671-708.

Wo 95/l3834 2 1~ 6 25 9 Pc~rluS94/13387 2. pG1040 (UCAT) 5' untranslated regions and amino terminus:
Wild-t~e CAT:
5' ~1 ll-t Glu Ly~ Ly~ B-r aly uuu uc~ gga gcu aag gaa gcu aaa aug gag aaa aaa ayc acu gga 3' Tyr Thr Thr uau acc acc l8G104 0 . UCAT:
5' +1 15~t Glu LyEI Ly~ S~r Gly agu qca qqa qcu aaq qaa qCu acc auq qaq aaq aaq auc acu qqa 3258-1 3 AUG 31te 3' Tyr Thr Thr uaU aCc acc The se auences of wild type and pG1040 UCAT around the AUG start co~on are shown . The target sites f or the oligomers are named and underlined, and the numbers of the chimeric oligomers against each target site are shown beneath .
UCAT was made from wild-type CAT DNA (Pharmacia) using synthetic DNA PCR primers . The resulting f ragment was cloned as a Hind III (5' end), Not I (3' end) fragment into the vector pRc/CMV (Invitrogen) . The first ;~ n~sin~
oi the open reading frame is designated +1. The amino acid changes between wild-type and pG1040 are conserva-tive .
Chimeric Oliqonucleotides were as follows.
5' AUG oliqomers (~osition -21 to +3):
3258-1, 24~er, ~MP(R~)/DE) (PS/DE) (MP~Rp)/DE):
5' cat ggt ag(c ttc c) tt agc tcc tgc 3' ~Wo 95113834 2 1 7 6 2 ~ 9 PCrlVS94113387 3260-1, 24mer, (MP(Rp)/DE) (PS) (MP(R5)/DE):
5 ' cat ggt ag (c ttc c) tt agc tcc tgc 3 ' 3 ' AYG oliqome~s (position +4 to +27):
3261-1, 24mer, (MP(Rp)/DE) (PS/DE) (MP(R~)/DE):
5 ' ggt ata tc (c agt g) at ctt ctt ctc 3 ' 3262-1, 24mer, (MP(R7)/DE) (PS) (~P(Rp)/DE):
5 ' ggt ata tc (c agt g) at ctt ctt ctc 3 ' 3636-1, 24mer, (MP(R~)/DE) (PS) (MP(Rp)/DE):
5' ggt a (ta tcc) agt gat ctt ctt ctc 3 ' 3638-1, 24mer, (MP(R~)/DE) (PS) (MP(Rp)/DE):
5 ' ggt ata tcc agt (gat ct) t ctt ctc 3 ' 3637-1, 24mer, (MP (R~) /DE) (PS) (MP (Rp) /DE):
5 ' ggt ata tcc agt gat c (tt ctt) ctc 3 ' 3640-1, 24m~r, (MP(Rp)/DE) (PS) (MP(Rp)/DE):
5 ' ggt ata tc (a agt g) at ctt ctt ctc 3 ' 3639-1, 24mer, (MP(R")/DE) (PS) (MP(Rp)/DE):
5 ' ggt ata tc (g agt g) at ctt ctt ctc 3 ' S~lice ~o~or oli~omers:
3264-1, 24mer, (MP (Rp) /DE) (PS) (MP (Rp) /DE):
5 ' cac tca cct t (ta ggg) aaa tag gcc 3 ' 3263-1, 24mer, (MP(Rp)/DE) (PS/DE) (MP(R~)/DE):
5' cac tca cct t(ta ggg) aaa tag gcc 3' XV-5, 24mer, all rhn~rhn. othioate:
5 ' cac tca cct tta ggg aaa tag gcc 3 ' Wo 95/13834 2 1 7 6 2 5 g PCrlUS94/13387 S~lice branch Point olicomers:
3269-1, 24mer, (MP(Rp)/DE) (PS/DE) (MP(Rp)/DE~:
5' cac tca at (c aat g) ac tag tct gca 3 3270-1, 24mer, (MP(R~)/DE) (PS) (MP(R~)/DE):
5 ' cac tca at (c aat g) ac tag tct gca 3 ' XV-6, 24mer, all rh~ h-- ~,thioate:
5 ' cac tca atc aat gac tag tct gca 3 ' S~lice acce~tor site oliqomers:
3265-1, 24mer, (MP(}I~)/DE) (PS/DE) (MP(Rp)/DE):
5 ' ccc tga ga (g aga g) ag aga ggt tcg 3 3266-1, 24mer, (MP(Rp)/DE) (PS) (MP(Rp)/D13):
5 ' ccc tga ga (g aga g) ag aga ggt tcg 3 ~
3387-1, 24mer, t2~oNe(Mp(Rp)/DE)] (PS) t2'0Me(MP(Rp)/DE)]:
5 ' ccc tga ga (g aga gag) aga ggt tcg 3 ' XV-7, 24mer, all rh~ -) oLhioate:
5' ccc tga gag aga gag aga ggt tcg 3 Cell Pre~aration and Treatment COS 7 cells were plated at 1. 5 x 105 cells/well in a 12 well plate format on the day before trans-fections 20 ~egan. All cultures were r-;nt~;n~d at 37C. On the next day, the transfection mixes were prepared. For each well of a 12 well plate, 1.0 IlM oligomer was c ' in~od with 1 ~Lg pGL2 or pSV,B + 1 ~g of the target CAT plasmid in 0 . 5 ml of Optimem (Gibco/BRL) and 18.75 ~Lg Tran~fectam (for chimeric 25 oligomers, Promega) or ~ipo~ectamine (for all PS
oligomers, Promega) also in 0 . 5 ml of Optimem. These quantities gave a 6.9 or 4.5 or 2.0 to 1 cationic lipid to oligomer plus DNA ratio, respectively, in one milliliter total. pGL2 and pSV,B servea as transfection and oligomer 30 specificity controls.

~Wo 95/13834 ~ ~ 7 6 2 5 9 t PCrrUS94/13387 The culture medium was aspirated of f and the cells were rinsed twice in one ml Optimem (Gibco/BRL) per well, and then one ml of tranfection mix was added to each well.
The cells were cultured in the transfection mix for 16 5 hours. The mix was removed and replaced with one ml of complete culture medium (DMEM plus 109~ fetal bovine serum and 1/100 dilution of penicillin/streptomyciri stock, all from Gibco/BRL) and the cells were incubated another 5 hours .
Cell lysates were prepared by rinsing twice in PBS
and then treated with 0.5 ml of lX Reporter Lysis Buffer (Promega). The released and lysed cells were pipetted into 1.5 ml tubes and frozen in CO2/EtOH once and thawed.
The crude lysate was then centrifuged 10 minutes to pellet cell debris, and the supernatant was recovered and assayed direct ly or f roz en at - 2 0 C .
The cell lysates were then assayed for CAT, and luciferase or ,~ ~t~se activity, and the total protein rf~nrPntration was detprm; nP~l as described below.
t'hl ~ Pn; col Acetvltr~ns~ferase (CAT) AssaY ProtocQl This assay was performed generally as follows.
First, the following reaction mixture was prepared for each 8ample:
65ml 0.25M Tris, pH8/0.57~ BSA, 4~ 4C-Chloramphenicol, 50 nCi/~ll (Dupont), and 5~L1 5 mg/ml n-Butyryl Coenzyme A (Pharmacia) A CAT standard curYe was ~re~red by serially fl;lllt;n~ CAT
stock (Promage) 1:1000, 1:10,000 and 1:90,000 in 0.25M
Tris, pH8/0.596 BSA. The original stock CAT was at 7000 Units/ml. CAT lysate was then added in a labeled tube with Tris/BSA buffer for final volume of 50 ml.
74 ml of reaction mixture was then added to each tube, which was then incubated for, typically, approxi-mately 1 hour in a 37C oven. The reaction was terminated by adding 500 ~Ll Pristane/Mixed Xylenes (2:1) (Sigma) to each tube. The tubes were then vortexed for 2 minutes and WO95/1383~ ` 21 76~g ~ '` 1 `; PCT/US94/1338~

spun for 5 minutes. 400 ml of the upper phase was trans-ferred to a srintillAtion vial with 5 ml Scintiverse (Fisher). The sample was then counted in a Packard srl nt; 11 Ation counter.
Luciferase Assav Protocol This assay was performed generally as follows~ ac-cording to standard procedures. 20 /11 of lysate was combined with lO0 /11 of luciferase assay reagent ~Promega) and counted in a srint;llAt;on counter (Packard) within 20 seconds (as r~r ~ 1 by Promega) .
~-Galactosidase Assav Protocol This assay was performed generally as follows. A ~-gal standard curve was prepared by serially diluting 1:1,000 and 1 9,000 in 0.25M Tris-HC1, plI8.0/0.59~ BSA.
Stock ~-gal was 1, 000 Units/ml (Promega) . Thus, for the 1:1,000 dilution, 1 ~l stock ~-gal enzyme was diluted in 1000 ~l Tris/BSA buffer, and for the l:9,000 dilution, 100 ,ul of the 1:1,000 dilution was further diluted in lO00 ,ul Tris/BSA buf f er .
75 ~Ll of lysate per well (untreated microtiter plate, Corning) was then added. 75 1ll 2X ,B-gal RF'~rt;nJl Buf~er (Promega) was added to each tube. TnrllhAt;nn proceeded for, typically, apprn~ t~ly 1-1.5 hours in a 37C oven.
Plates were read at A~os (405 nm) on a microplate reader (Molecular Devices).
prot~; n Aggav Protocol Samples were prepared in an untreated microtiter plate (Corning). A series of protein standards were prepared in duplicate as follows.
l. 6 ILl lX Reporter Lysis Buffer (Promega) 2. 6 ~l 75mg/ml BSA (Promega) 3. 6 ~11 lOOmg/ml BSA
4. 6 1ll 250mg/ml BSA
5. 6 ~11 400mg/ml BSA
6. 6 1ll 500mg/ml BSA
7. 6 111 looomg/ml BSA

~Wo 95/138~4 2 ~ ~ 6 2 ~ 9 PCT/US94/13387 8. 6 ~l 1500mg/ml BSA
Six ~1l of lysate per well was added, followed by 300 ~l Coomassie Protein Assay Reagent (Pierce) per well. The individual sample plates were then read at As70 on a 5 microplate reader (Molecular Devices ) . CAT activity values were normalized to the protein content of the lysate and other parameters as given.
The results of these experiments were as follows.
Anti-s~lice site oliqomers versus ~C71035 ~n~ ~Gl036 lO (splici~g inhibition by antisense oligomers):
pG1035=spiicing pG10~6 .. ,.. 5, '' _ Oligomer chemistry Donor Branch Acceptor Donor Branch Acceptor PS/DE 3263-1 3269-1 3265-1 3263-1 3269-1 3265-1center 65ill% ?2+1% 90+5% o~O oo~O o%

center 59+2% 56+7% 53+2% o% o% 0%

32+1% 23+15% 17_6% 35+1% 30+4% 20+4%
PS center, N.D. N.D. 3387-1 N.D. N.D. 3387-1 2'0Me ends 98i2% o%
20 Oligomers were transfected into COS-7 cells and lysates were made and assayed as described previ-ously, All oligomers were at l . 0 ~M f inal in the culture medium. The results are given as percent inhibition + std error N.D. = not ~i~tf~ ;n~d. All samples were perormed in triplicate. In the case of the chimeric oligomers (PS/DE center and PS center) the expression of the non-splicing pGl036 CAT was slightly higher in oligomer treated versus untreated cells, so the expression of pGl035 was normalized to pGl036 expression. All results were r~ormalized ~o total protein and luciferase counts.
The results show specif ic inhibition of CAT expres-sion when the splice site sequences are targeted using the WO 95/13834 ~ f ' PCTIUS94/13387 25~

chimeric oligomers. In the case of all phosphorothioate oligomers, pG1036 expression was inhibited approximately as well as pG1035, revealing large non-specific effects on gene expression. In addition, the incorporation of 2 ' -O-5 methyl groups in the fl~nk;n~ t~rminz~l portions of thesplice site acceptor oligomer 3387-1 and lengthening the PS center from five to seven r~nt;n~ phosphorothioate h;lrkhnm~ linkages increases the antisense activity against the splice acceptor site target significantly but does not 10 increase non-specific activity against the control target.
(~hi - ic ;oli~omerg tarqeted a~ainst the AUG of CAT
inhibit ex~ression:
5'AUG Target 3'AUG Target Control No No oligomer Target Oligomer 3258-l 3260-l 3261-l 3262-l 3269-l None Chemistry PS/DE PS PS/DE PS PS/DE No center center center center center treatment 15 % Inhibition 43+l9% 72+28% 96+7% 97+4% 4+14% 0+15%
oligomers were transfected into COS-7 cells and lysates made and assayed as described previously.
A11 oligomers were at 1. O ~LM f inal in the culture medium. Oligomer 3269-1 was a control that does not have a target site in pGl040, because the CAT gene does not contain a splice site. Results are ex-pressed as ~ inhibition + error. Each oligomer was tested in triplicate.
Chimeric oligomers targeted against the 5 ' AUG site (3258-1, 3260-1) were effective at blocking expression of the CAT mRNA (43-729~ inhibition, respectively). Chimeric oligomers targeted against the 3' AUG site (3261-1, 3262-1) were even more effective, giving 96 and 97~ inhibition, respectively. The control oligomer (3269-1) gave no inhibition, demonstrating that the inhibition observed for the chimeras that match the pG1040 mRNA was specific.
In conclusion, these results indicate the ability to down-regulate CAT activity using chimeric oligomers 5 introduced into cultured COS - 7 cells via cationic lipids .
The targets have been AUG sites ~present in both the pre-mRNA and mature mRNA) and intronic sites (present only in pre-mR~A in the nucleu8 of any cell). The chimeric oligomers with both PS/DE and PS centers have proven to be 10 more specific than all-PS oligomers and control chimeraE.
Both target-specific and r~ , -specific controls were included, demonstrating that the results are based on se~uence- specif ic antisense ef ~ects .
r le 42 15 S~ecificitv Determln~tion Singly and multiply mismatched, complementary gene targets and oligomers allow cross-over experiments to estimate oligomer discrimination of perfect match targets from imperfect non-specific targets. The present example 20 shows the preparation of C~T mRNA targets having 0- or 4-ba~3e mismatches with respect to the oligomers used in ~xample 41, as well as the effect of various mismatches on the specificity and activity of oligomers of the inven-tion .

WO 95113834 . - PCrlU594/13387 2~ i2~9 106 ~ Gl040 (UCAT) and ~Gl042 (UCAT 4mm) 5' untran81ated reqions and Z~m;nn term;n; ;In~1 oliqomers:
Wild-t~e CAT:
5~ +1 3 H t Glu Ly~ Ly- Il~ S-r Gly Tyr Thr Thr uuu uca gga gcu aag gaa gcu a~a aug gag aaa aaa auc acu gga uau acc acc ~G1040, UCAT:
5' +1 3' M~t alu Ly~ Ly~ :~1-- 8~r aly Tyr Thr Thr 10agu qCa qqa qcu a~q qa~ qcu acc auq qaq aaq aaq auc acu qqa uau acc acc 3~ (cgt cct cga ttc ctt cga tgg tac) (ctc ttc ttc tag tg~ cct ata tgg) 5' XV-l XV-2 ~G10 4 2 . UC~T 4 mi ll:mat ch:
5' ~1 3' 1 5 t ~ t M~t A p Arg Ly~ Thr Gly Tyr Thr T~r ' (cgt tCt caa cgC ctt cga tgg taUacq) ~cqtgc atqCcq attaq tauu aCq qqa uau acc acc Mismatches between pGl040 (UCAT) and pGlOg2 (UCAT) 4mm are marked with asterisks ( * ) . All other bases in the mRNAs produced by these plasmids are identical. The sequence of the wild-type CAT gene is shown for comparison. The first adenosine of the open reading frame is designated +l. The oligomer target sites are underlined.
Plasmids pGl040 and pGl042 were created using syn-thetic DNA PCR primers to amplify precisely mutated DNA
~1,, tC, The fragments were then cloned as Hind III (5' end), Not I (3 ' end) ~1 ~ R into the vector pRc/CMV
(Invitrogen) and positive clones were ;~l~nt;fied. -It will be noted that, for a given oligomer against either of these target genes, a control target is provided having a precisely def ined degree of mismatch . This allows testing of one oligomer against a perfect match and precisely-defined mismatch targets, as, ,1;fied by the f ollowing:

WO 95113834 ~ 2 5 9 PCTIUS94/13387 4 0, ~JCAT: --s~ +
Agu gca gga gcu aag gaa gCu aCc aug ga; ig ja jag al lag jaTT IATT lgg3 TaT ~aTc ~cc ~ctc ttc ttc tag tga cct ata tgg~
xv-2 042, IJCAT 4 mi~match:
Agu gca Aga guu gcg gaa gÇu aCc aug igiA~c jasjs alalg ITU lalcg gigl TlaT laTT ACC
~ctc ttc ttc tag tga cct ata tgg) xv-2 In this case, the oligomer XV-2 i8 a perfect match to pG1040, but has four mismatches to pG1042. The relative ef f ects of this one oligomer against two target mRNAs that are identical except in the four known mismatch bases can thus be determined.
In addition, mismatches in the target gene can be precisely controlled by the sec~uence of the PCR primers used in the amplification procedure, and a defined se-quence of precise mismatches can be created such as a series in the region just 5 ' of the AUG codon . This is shown in the following example:
s~ +
X-t alu Ly~ Ly~ 8-r Gly Tyr Thr Thr AgU gca gga gcu aag gaA gcu acc aug gag aag aag auc acu gga uau acc acc 3 ~ cct cga ttc ctt cga tgg tac s ~
1 mil 'ch:
Agu gca gga gcu aag gaa gcu ccc aug gag aag aag auc acu gga uau ACC acc 3~ cct cga ttc ctt cga Tgg tac 3 0 2 m; rh~c ~gu gca gga gcu ~ag gaa ACU CCC aug gag Aag aag auc acu gga uau acc ACC
3~ cct cg2 ttc ctt cga Tgg tac 3 m~ -rh ~:
Agu gca gga gcu aag ~a~ ACU CCC ~ug gag Aag aag AUC ACU gga uau acc acc 3~ cct cga ttc ctt cga Tgg tac 4 mi ' rh~5:
agU gcA gga gcu Gag IJaa ACU CCC ~ug gAg aag aag auc acu gga uaU acc ACC
3 ' cct c:ga Ttc ctt cga Tgg tac S ' S m~ rh ~
~0 agu gca gga ccu Gag ~laa ACU CCC aug gag aag aag auc ACU gga uau acc ACC
3~ cct cgA Ttc ctt Cga Tgg tac s~

Wo 95/13834 : PCTNS94113387 Here, the target sequenCe within the mRNA to be studied extends from -18 to +3. Mismatches in mutant mRNAs relative to the top sequence are shown in bold upper case.
The oligomer sequence in this example, a 21mer, is shown 5 beneath each mRNA and is invariant. Mismatches in the oligomer to each subsequent IrRNA are shown in upper case.
Using this method of increasing the number of pre-cisely known mismatches in otherwise identical targets, one can accurately determine the specif icity of various 10 oligomer chemistries (e.g. rh~3p~ rothioates versus chimeras) and modes of action (e.g. steric blockers versus RNaseH cleavers ) .
Tests were undertaken to study the effects on ac-tivity and specificity caused by variations in the loca-15 tion of the charged-h~-kh~-n~ RNasH-activating region within a chimeric oligonucleoside, and by various mis-matches incorporated into the base sequence of an oligo-nucleoside and/or in the target mRNA. The chimeric compounds listed below (see also Example 41) were assayed 20 for antisense activity against both the pG1040 (UCAT) target and the pG1042 (UCAT) 4-base mismatch control. The ol1 s r sequences were aa follows .
pG1040 (UCAT) tarqet mRNA and antisense oliqomers:
+1 +4 +27 l l l Met Glu LYR Ly~ 3--r Gly Tyr T}lr mRNA aug gag aag aag auc acu gga uau acc 3637-1 3' ete tte tte t~g tg~ eet at~ tgg S' 363b-1 3' ete tte tte ta~l tg- eet ~t~ tgg S' 3262-5 3' ete tte tte ta tClA cet t~ tgg S' 3636-1 3 ' ete tte tte t~g tg~ eet ~t-- tgg S ' 3639-1 3' ete tte tte tag t~ cet t~ tgg S' 3640-1 3' etc ttc tte t~sl t~ ~et ~ta tgg S' XV-2 3 ete ttc tte t~q tq/ eet ~tA tc~ S' wo 95113834 PCrlUS94113387 21762$9 The phosphorothioate linkages in these chimeric oligomer8 are immediately 5' of the underlined bases. It will be seen that the position of the phosphorothioate core is seq-l.on~ ly shifted in position with respect to the 5 target mRNA.
Antisense activity was assayed against both pGl041 (UCAT) and pGl042 (UCAT) using procedures as generally described irl Example 41, except that 0 . 5 ~M oligomer was used. It was demonstrated that mismatches in the phos-lO phorothioate core and the position of the core in chimericoligomers greatly affected antisense activity. The following table sets forth the percentage of gene ex-pression (t error) measured for each of the tested oligomers .
01igomer number 15 Target 3637-1 3638-1 3262-5 3636-1 3639-1 3640-1 79+5% 37i3% 35i:7% 70i3% 98iS% 103iS%
pG1 040 pG1042 89i3% 102i2% 88~4% 120i8% 93i2% 115i3%
The results show the effect of moving the RNAseH-activating rh~ h~rothioate core within the oligomer. The 2 0 position of the phosphorothioate core and/or the base composition of the phosphorothioate core has a large effect on antisense activity, as seen by comparing 3637-l, 3 6 3 8 - l, 3 2 6 2 - 5 and 3 6 3 6 - l . A more cent ral pos i t ion wi t~in the chimera is most active, but some activity is detected 25 even when the core is near the ends of the chimera.
A single base mismatch (denoted by an "x" above the sequences shown above) within the RNaseH phosphorothioate core se~uence of the chimeric oligomers e' iminates anti-sen8e activity in this eukaryotic cell culture assay, as 30 8een by comparing 3639-l and 3640-l with 3262-5. In a WO95113834 ; f: i ~' i " .. PCrf~S94/13387 2l~6259 separate experiment using the Game assay system, the all-phosphorothioate 24mer XV-2 gave 909~ inhibition of pG1040 (UCAT) expression and approximately 50g~ inhibition against pG1042 (UCAT) even though there were four mismatches in 5 the case of the latter target. This indicates that all-phosphorothioate oligomers are far less specific than chimeric oligomers rnnt~;n;n f short regions of phosphoro-thioate 1 ;nk~c, ;n~l nh as even a single mismatch between the chimeric oligomers 3639 and 3640 and the 10 pG1040 target abolished activity, whereas four mismatches in the case of XV-2 and pG1042 reduced activity by less than 5 0 9~ .
Exam~le 4 3 IncrP~ed RNaseH Cleavaqe Rate with Chimeras rnnt;l;n;nq 15 (~h; r~ 1 1Y Enriched Oliqonucleoside Methvl l~hns~hnn~te End-The present example demonstrates that chimericoligomers with ~nh;~nn~ binding affinity promote RNaseH
cleavage of RNA target strands at a f aster rate than lower 20 affinity oligomers having the same base sequence. Chime-ric oligonucleoside~ cnnt~;n;ng either racemic or chirally pure (Rp) methylE~hr~sr~hr~n~tes were P~m;n~d for their ability to activate RNaseH.
The following chimeric oligomers were used in this 25 example:
Sequence = 3 ' - [CCTGTTG] [TACGT] [ACCTTCTG] -5 ' 2681-1 [MP] [PS/DE] [MP]
3214-1 [MP (Rp) /DE] [PS/DE] [MP (R~,) /DE]
Each of these chimeric oligomers was synthesized according 30 to the method described in Example 30. A complementary synthetic RNA target was prepared according to the method given in Example 28. This oligomer has the following secfuence:

WO 9~/13834 2 1 7 6 2 ~ 9 PCrtUS94/~3387 5 ~ -G~.~ c~TTGCA~GGAAGAC-3 ' - A 32P-label was coupled to the 5'-end of this oligomer using [~-32P]-ATP and T4 polynucleotide kinase according to a procedure commonly known in the art.
RNaseH from bacterial ~. coli was purchased from Promega Corp. (Madison, WI) . Buffer A, used for the RNaseH
reactions ~nnt~;n~rl 20 mM KCl, 9 mM MgC1~, 1 mM 2-mercapto-ethanol, 250 f~g/ml of BSA ~Promega Corp. ) and 100 u/ml of RNasin (Promega Corp. ) .
A mixture of 5' -32P-labelled RNA target (approximately 80,000 dpms, 5 x 10-1 M) was mixed w~ith 1 molar equivalent of either chimeric oligomer in reaction Buffer A (total volume = 98 microliters). This mixture was incubated at 37C for 1 hour. Next, RNaseH (1.1 microliters, 30 units/mL, final concentration = 2 x 10-9 M) was added and the resulting mixture was incubated at 37C. Aliquots (15 microliters) were removed at specified time intervals, diluted with EDTA (0.5 M, 3 microliters) frozen on dry ice and then stored at -20C. The products of RNA cleavage were analyzed by gel electrophoresi6 uæing a 1590 poly-acrylamide/7 M urea gel (20 cm x 30 cm x 0.5 mm i.d.) equilibrated in 1 X TBE buffer (p~ 8.2). The gel was electrophoresed at 1200 volts for approximately three hourE. Bands on the wet gel were visualized by phosphor-imager analysis using a Bio-Rad Model GS-250 Molecular Imager (C~1 ~h~ , CA) .
Site-specific RNAse~I-r~';~t~od cleavage was observed with both chimeric oligomers. The lengths of the frag-ments were estimated according to their electrophoretic 3 0 mobility . According to thi3 analysis, it was determined that cleavage was limited to the center of the RNA target sequence. That is, cleavage was limited to the position of the RNA strand complementary to the negatively charged segment of each chimeric oligomer. A difference in the rate of RNase~l mediated cleavage was detected for the two dif f erent chimeric oligomers as 8hown in FIG . 5 .

WO95/13834 ~ 59 ,-, PCrrUS94/13387 It is seen that the rate of RNA hydrolysis in the presence of chimeric oligomer 3124 -1 (Cnnt~; n; n~ alter-nating MP(R~)/DE backbone segments at the 3'- and 5'-ends) i8 about 10 tlmes faster than that for the other chimeric 5 oligomer 2681-1 (rr~tA;n;n~ racemic MP harkhr,nP seqments) .
~,rAml21e 44 Effect of 2'-Suqar Substitution Location on Chimeric Oliqomer Cleavaqe Activitv The effect of the location of 2'-sugar substituents 10 relative to the RNaseH-activating region of the present oligomers was studied by measuring the cleavage activity of differently-substituted chimeric oligomers against a target RNA sequence. A synthetic 20mer RNA molecule, designated 3593, containing an AUG sequence near the 15 targeted cleavage site was prepared having the following se~uence:
3593 (target RNA): 5' AG AGA GAG AUG CAG AGA GAG 3' Chimeric 20mer RNaseH-activating ol;rnn~rleosides 3463, 3465 and 3466 were synthesized using appropriate dimer 20 synthon methods as generally described above. These c ,~ ds included a central RNaseH-activating region comprising five consecutive phosphorothioate-linked deoxyribonucleosides (shown in parentheses below) flanked by non-RNaseH-activating regions linked by alternating 25 ME' (Rp) /DE linkages . Selected nucleoside sugars in the ~ nk;nrj regions of chimeras 3463 and 3465 rnntA;nP~l 2'-O-methyl substitutions, indicated by the underlined capital-ized nucleoside abbreviation letters below (the target 3593 sequence is also depicted to show target complement-3 0 arity):

.
Wo 95113834 ~ 1 ~ 6 2 ~;9 PCrrUS94/13387 3593: 5~ AG AGA GA G AUG C AG AGA GAG 3~ (target RNA) 3463: 3' uc UcY cU(c tac g)Uc UcU clrc 5~
3465: 3' uc UC~ u(c tac g)uc lJC~J C~C 5' 3466: 3' uc ucu cu(c tac g)uc ucu cuc 5~
5 As with other chimeric oligomer compounds disclosed herein, the charged (here, phosphorothioate) linkages associated with the R~aseH-activating region are situated 5' to each of the nucleosides shown in parPnthPRPq~ Thus, compound8 3463, 3465 and 3466 above each i~clude a stretch of five consecutive, central phosphorothioate ( {P~} ) linkages, flanked on either side by a chirally-selected Rp-methylrh~qph~n~te ( {MP(R~) } ) linkage, as follows (shown 3 to 5~ ) :
. . . C~DII~}U{~SP (}1~) 3 (c{P5~t{P8}a{PS}c{PS~g) ~PS}u{~ ~ C{DE~U . . .
The underlined phosphorothioate linkage shown above [in the 6egmert . . .u{MP(Rp) } (c{PS}t. . .] can be incorporated into the compounds using dimer synthon methods as de-scribed, for example, in ~xample 13 above. The rr--;ninr, non-RNaseH-activating portions of the chimeric compounds include alt-~rn~t;nr, ~P(Pp)tDE linkage q~_ ~ incorporat-ed, for example, by successive addition of appropriate dimers following the support-bound 'lucr dinucleotide ser~uence at the 3'-tPrm;nllq of the compounds (see, e.g., Examples 8, 9 and 17A above). Thus, 2~-sugar substitu-tions shown above for compounds 3463 and 3465 can be achieved by successively incorporating suitable 2'0Me~{~P(R~) }c~DE} or 2'0MeU~MP(Rp) }2'0Me{DE} dimers into the respective oligomers.
To assess the RNaseH cleavage activity O$ the fore-going chimeric oligomers, 320 /~l of a mixture of 5~ 32p_ labelled RNA target c~ ~,IUUlld 3593 (160 dpm) and the selected test oligomer (1:1 molar ratio; rr~nr~ntrations 0.5 nM) was incubated in Buf$er A at 37C for one hour to achieve cOmp~ementary complex formation a~d equilibration.
,,,,, ,, . .. . . ... , ~

wo 9S/l3834 2 1 7 6 2 5 9 ~ PCrlUS94/13387 (suffer A: 20 mM KCl, 9 mM MgCl2, 1 mM 2-mercaptoethanol, 250 llg/ml BSA [Promega], 100 u/ml RNasin [Promega] . ) A 20 111 aliquot was removed as a time zero sample and 3 . 3 ~Ll of a 2 nM solution of bacterial (E. coli) RNa5eH (Promega) in 5 Buffer A was added (final concentration of enzyme in solution was 0 . 022 nM) . The reaction mixture was kept at 37C. Twenty microliter aliquots were removed from the mixture at appropriate time intervals and the reaction was stopped by adding 2 ILl of 0 . 5 M sodium EDTA solution and 10 then freezing on dry ice. The products of RNA cleavage were analyzed in 1596 PAGE (20 cm x 30 cm x O . 5 mm) con-taining 7 M urea and lx TBE buffer ~pH 8.1). Gels were run at 1200 V for 2 hours. Quantitative kinetic data were obtained by integration of the volumes of the bands by 15 means of Phosphor-image analysiE.
The kinetic curves for this example are shown in FIG.
11. A significant decrease (about 10-fold) in the overall rate of RNA cleavage was found when 2'-O-methyl nucleoside units were positioned next to the central phosphorothioate 20 RNaseH-activating region (compound 3463, triangle data points) as compared to the chimeric compound rnnt:~in;nrj all 2'-H nucleosides (~ , -JUlld 3466, circles) . The initial number of cleavage products was reduced for compound 3463 as compared to compound 3466 (2 instead of 25 3). When a 2'-H nucleoside instead of a 2'-O-methyl nucleoside was incorporated on the border of the alternat-ing methylphosphonate/phosphodiester 5 ' -end-block and pl~r,5rhnrothioate regions (compound 3465, r~ ), no sirjn; f; r~nt decrease in cleavage rate was found, and the 3 0 number of cleavage products also did not change as com-pared to that obtained with ~ , olln~ 3463 .
This example demonstrates that the presence of a non-hydroxy 2~-sugar substituent adjacent to the RNaseH
cleavage site has a significant ~l;m;nllt;ve effect on 35 RNaseH cleavage activity and that even a single 2 ~ -O-methyl substituent may be responsible for the reduction in cleavage activity. In contrast, the use of a 2'-substitu-W0 95/13834 ~ :1 rt 6 2 5 9 PCT/US94/13387 tion that i8 removed from the RNaseH-activating region by one or two nucleosides has a negligible ef f ect on RNaseH
binding and/or cleavage activation.
r ~le 45 Activitv of Chimeric Oliaonucleoside Com~o~1n~lc A~A;nct HPV
Taraets This example describes experiments using various chimeric oligonucleosides of the invention targeted against human papilloma virus (HPV) gene seauences.
A. Preparation of Plasmid E~pressing a Polyciatronic E 6 /E7 mRNA
An expression vector having an insert coding f or HPVll E6/E7 was prepared usin~ the expres6ion vector pRc/CMV ( Invitrogen) . The plaGmid pRC/CMV was linearized with Eind III. The recessed 3' ends were filled with the 5 ' -3 ' polymerase activity of T, DNA polymerase . A full length clone of HPV-11 cloned at the BamEI Site in pBR322 was digested with the restriction enzymes B6t II and Hinf I. The 873 base pair LL _ nt ~)rtAinlnS the E6 and E7 open reading frames was purified on agarose gel. The restriction ends of this fragment were modified by filling in the recessed 3 ' -ends with T, DNA polymerase .
The vector and insert were ligated with T4 DNA ligase and transformed into DH5~Y E. Coli. Recombinants were screened for c-~L-,~Liate insert and or;,~ntAti-~n as well as E6/E7 transcription and translation activity.
This plasmid (pRc/CMVII-E6/E7) was used in the cell free trAncl Atir~n system described below.
B. Preparation of Pla~mid HAving an E2 Insert An expression vector having an HPV-11 E2 insert was prepared using pRc/CMV (Invitrogen). The plasmid was linearized with Eind III, followed by treatment with calf thymus ~1 kA 1, nl~ phosphatase . To isolate the E2 open reading frame, a full length clone of HPV-11, cloned at Wo 95/13834 2 i 7 6 ~ ~ 9 PCTN594113387 the Bam HI site in pBR322, was digested with the restric-tion enzymes ~nmI and SspI. The recessed 3 ' ends were filled in with the 5'-3' polymerase activity of the Klenow fragment of DNA polymerase I. Hlnd III linkers were then 5 added. The 1309 base pair fragment containing the com-plete E2 ORF was agarose gel purif ied. The modif ied vector and B2 insert were ligated with Ts DNA ligase and transformed into DH5~ E. Coli. Recombinants were screened for appropriate insert, transcription and translation.
This plasmid (pRc/CMVII-E2) was used in the cell-free translation system described below.
C. Preparation of Plasmid Eaving Nonoci6tronic E7 Insert An expression vector having an HPV- 11 E7 insert was prepared using pcDNA-1 ( Invitrogen) . The plasmid pcDNA
was digested with Bam HI and with Xba I. A Cla~ t nt~;n;n~ the complete open reading frame of HPV-11 (from -30 through the termination codon) flanked by Bam HI and Xoa I restriction sites was prepared by PCR using standard protocols. The digested vector and fragment were ligated with T~ DNA ligase and transformed into MC 1061/P3 cells.
R~-_ 'I;nAntF: were screened for appropriate insert, tran-scription and translation.
This plasmid (pcDNA E7) was used in the cell-free translation system and in the transient expression assay described below.
D. Demon~tr~tion of Activity o~ ~nt; ~er~e Chimeric ol; ~ " Targeted to EPV-11 E7 in Cell Free Tr~ms-l~tion Extr~ct~3 Mono-cistronic (100 nM) HPV-11 E7 or polycistronic (50 nM) HPV-l1 E6/E7 RNA was co-translated with chloram-phenicol acetyl transferase (CAT) RNA (2 to 10 nM) in cell-free rabbit reticulocyte extracts (Promega). The c~nt~nts of e~ch assay system was as follows.
i Wo 9~13834 2 1 7 ~ 2 5 ~ PCr/USs4/l3387 COMPOI~ T FINAL l ~N~ ~:Nl~TlON
In vitro transcribed un- (As noted above) capped RNA
3ss-cysteine o . 8 mCi/mI, 5 Amino acids mixture, cys- 2011 each teine def icient Rabbit reticulocyte lysate 72 by volume RNAsin (Promega) 0.5 units/~LL
Oligomer . 1 to 10~LM
Cell free translation was performed at 37C for 60 minutes and was stopped by addition of SDS gel loading buffer and ;nllhation at 95 for 3 minuteG. Translation of E7 was evaluated after; ~recipitation with aE7 goat antiserum and protein A sepharose, followed by SDS-15 PAGE and phosphoimage analysis. This protocol was also used in the cell-free translations referred to below.
E . D L~ .-tion of Activity of ~nt; r -e O~; L 8 in Cell-Free RNAseH Cleavag~ A~say In vitro transcribed, llnc~rp~d mono-cistronic RNA was 20 prepared by transcribing plasmid pcDNAllE7 with RNA
polymerase (Ambion MegaScript ) . The E7 RNA was incubated at a c~ n~nt~ation of 100 nM in the presence of 0 . 04 units l~uL E. Coli. RNAseH (Promega), 3.5 mM MgCl" 25 mM KCl, 70 mM NaCl and 20 mM potassium acetate at 37C for 30 min-25 utes. Reactions were stopped by addition of formamide gel loading buffer followed by heating to 100C for 5 minutes.
Samples were analyzed by 4~6 Urea-PAGE analysis, followed by 8taining with e~h;~ m bromide. Percentages of cleavage of E7 MRNA, in the presence of RNAseH, of 30 methylphosphonate chimeric oligomers 2657-1, 316g-1, WO 95/13834 2t ~ ~ 2 5 g PCr/~'S94/13387 3214-1, 3257-1, 3241-1 and 3236-1 are shown in the table below. Good dose response effects were obtained for all the oligomers at the concentrations tested. The order of potency was 3169-1 - 3257-1 ~ 3214-1 - 2657-1 ~ 3236-l ~
5 3241-1. All oligomers showed good specificity, cleaving E7 mRNA in one position.
Oligomer (ILM) Oligomer Backbone 0.0 l 0. l 1 10 3169-l [MP(R~)/DE]-[DE]s-[MP(Rp)/DE] 7 45 85 loO
3214-l [MP(Rp)/DE]-[PS/DE]s-[MP(l~?)lDE] 2 20 50 ~0 103257-l [MP(Rp)/DE]-[PS/DE]7-[MP(R")/DE] 4 40 75 lO0 334'.-l 2'0Me[MP(R")/DE]-[PS],-2'0Me[MP~/DE] 5 40 60 60 3336-l 2'0Me[MP(Rp)/DE]-[PS/DE],-2'0Me[MP(Rp)/DE] 5 50 60 65 Results are percentage of cleavage of E7 m'~NA.
Estimated values were obtained by visual inspection of the gel.
F. Demon~tr~tion of Activity o$ Ant;n~nne 01;3 -~'D in TrAnDiently Tr~nDfected COS-7 CellD
COS-7 cells were seeded at 1 X 105 cells/well in 24 well plates and then cultured overnight in cell culture media (90% DMEM, 10% fetal bovine serum and 50 I.U./ml penicillin, 50 mg/ml streptomycin and 0.25 llg/ml ampho-tericin B). After 24 hours the cells were approximately 80 to 90% confluent. A transfection cocktail of 2.5 ~g/mL
pcDNA 1 E7, 50 ~g/mL Transfectam (Promega) and varying rnnr~ntrations of oligomer was prepared and incubated for 15 minutes at room temperature after a 2 second vortex mix .

Wo 95/l3834 ~ 2 ~ PCTIUS94/13387 Cells were washed on the plates two times, 1 ml/well with Optimem (Gibco-BRL) . Then 0.5 mL tran6fection cocktail per well was applied to duplicate wells. The plates were incubated for 4 hours in 5~ CO2 at 37C. After 5 incubation cells were washed two times, 1 mL/well with cell culture media and cultured overnight. Then cells were washed twice, 1 mL/well with cysteine rl~f;~ nt DMEM
and then ;nt'llhê~t~'~ for 309 minutes in cysteine deficient DMEM under cell culture conditions. Cells were labelled by incubation with 250 IlCi of 35S-cysteine/well in 500 ~L
cysteine def icient DMEM without serum f or 5 hours . The cells were then washed twice, 1 mL/well with 1 X rhnsrh~te buffered saline and then lysed with 100 ~ SDS sample buffer (50 mM Tris-C1 [pH 6.8], 100 nM dithiothreitol, 296 sodium dodecyl sulfate, 0.1~ bL- Lh~nnl blue, 10g~ glycer-ol). Wells were washed with 100 ~L~ RIPA buffer (10 mM
Tris-Cl [pH 7.4], 150 mM NaCl, 19~ Triton X-100, 0.1~
sodium dodecyl sulfate, 0.59~ sodium deoxycholate) and combined with sample buf f er lysate .
2 o B7 synthesis was evaluated by immunoprecipitation of E7 protein with goat anti-HPV-11 E7 serum and protein A
sepharose beads (Sigma). T nrrecipitated E7 protein was quantitated by SDS-PAGE and rhn~rhr~ir-~e analysis.
Total protein synthesis was evaluated by SDS-PAGE and phosphoimage analysis of a fraction of the transfected cell lysate bef ore immunoprecipitation .
Representative experiments were performed as follows.
E7 expression plasmia pcDNAllE7 (5~g/ml) and different amounts of antisense ol i ~r nl-rle - tide were transf ected into COS-7 cells in the presence of Transfectam' (Promega).
Cells were incubated with transfection mixture for 4 hours, allowed to recover in media plus serum overnight, and labeled with 35S-cysteine for 5 hours before harvest-ing. Cells were lysed and E7 protein synthesis was evaluated by i nprecipitation with ~E7 serum followed by SDS-PAGE gel fractionation of protein products and phosphoimage analysis. Total protein synthesis was W095ll3834 21 7~25g (` i ` ~ PCr/US94113387 analyzed by SDS-PAGE separation of an aliquot of the cell extract, autoradiography and phosphoimage quantitation of all the proteins present in each lane. The following table summarizes the IC50 and IC90 values obtained with chimeric oligomers 3169-1, 3214-l, 3256-l, 3257-l and 3336-1 .
POTENÇY OF OLIGQ~Rq TARÇETED TO E~PV-11 E7 IN A CELL
BASED ASS~Y
Cell-based assay Oligomer Backbone IC50 IC90 103169-2 [MP(R")/DE]-[DE]-~MP(I~)/DE] >2 ~LM >>10 I.M
3214-1 [MP(Rp)/DE:]-[DE/PS]-[MP(E~7)/DE] 0.2 IIM I ILM
3256-1 [MP(E~p)/DE]-[PS]-[MP(Rp)/DE] 0.12 1 ~M
yM
3257-1 [MP(Rp3/DE]-[DE/PS]-[MP(R")/DE] 0.06 <0,3 I~M
/LM
3336-1 2'0Me[MP(Rp)/DE]-[DE/PS]-2'0Me[MP(ilp)/DE] 0.4 ILM ~2 ~LM
It is clear from this example that chimeric oligo-nucleotides 3214-1, 3257-1 and 3256-1, which contain all rhngrhnrothioate ( [PS] ) or alternating phosphorothioate/
phosphodiester ( [PS/DE] ) linkage in the middle and chiral methylphorothioate/methylrhnsrhnn~t~ dimers linked by phosphodiester linkages ([MP(P~)/DE]) as end-blocks, are potent inhibitors of transient expre3~ion of HPV E7 protein in COS-7 cells.
Chimeric oligonucleotides with rhn~rhnrii ester link-ages in the middle, such as 3169-1, were not potent in the cell-based assay, although they proved to be very potent in the cell-free assay. This difference may be due to the Wo 95113834 2 1 7 6 2 5 9 PCrlUS94/13387 _ . .

intracPl 1 1ll Ar instability of the phosphodiester linkage .
Finally, oligonucleotides c~ntA;n;n~ 2'0Me modification in the sugar of the nucleosides present at the ends (see 3336-1) were les6 potent than the corrPAp~-n~; n~ chimeras 5 with [MP (Rp) /DE] ends .
G. Demonstration of Oligomer Activity by Microinjection in VERO CellR
(i) lIicro in~ection oligomers were microinjected together with E2 (pRc/CMV 11-E2~ or E7 (pcDNAE7) expression plasmids at 50 g/l~l into the cytoplasm of VERO cells according to the following procedure. On the day preceding injection, VERO
cells (approximately 2 X 105 cells/ml) were plated on coverslipæ. Plasmid DNA was diluted in PBS to a r~n~pnt~a-tion of 20 ng/~ul (E7) or 50 ng/~Ll (E2) in an Eppendorf tube. The tube~3 c~ntA;n;n~ plasmid DNA were centrifuged for 15 minutes at 1,400 rpm. The tubes were set on ice prior to microinjection. A 2 ~LL aliquot of plasmid DNA
solution was loaded onto a fem to top. The tip was set with the coverslip at 45. The pre6sure on the micro-injector was set at 80 and the injection was performed.
The coverslips were incubated at 37C overnight after insertion. At 16 hours post-injection, cells were fixed and immunostained with goat anti-E7 polyclonal antibody, as explained below.
(ii) Indirect Fluore~3cence T -- vv_y Prior to use in this assay, goat anti-HPV-11 E7 or HPV-11 E2 serum was preabsorbed with VERO cells as fol-lows. ~mfluPnt VERO cells from two T-150 fla~ks were scraped and then washed twice with P8S. 200 1ll serum was then added to the cell pellet and mixed at ~0C overnight.
The mixture was centrifuged and the 8upernatant was removed to a new tube. The preabsorbed 8erum was stored in 5 0 ~ glycerol at - 2 0 C .

WO 95/13834 ~ PCTIUS94/13387 Expression level of E2 or E7 was assessed using a fluorescent antibody assay. Coverslips were fixed in 1096 formaldehyde in PBS for 20 minutes at room temperature and then washed twice with PBS, followed by incubation with goat anti-HPV-11 E7 or l;PV-11 E2 protein serum preabsorbed as set forth above at a 1:1000 dilution in PBS for two hours at room temperature. The coverslips were then washed with PBS three times, five minutes per wash, and incubated with FITC-conjugated Donkey Anti-Goat IgGAb tJackson, T ~R~qearch, Cat #705-095-147) at 1:200 dilution in PBS. The coverslips were then washed with PBS
three times, air-dried, and mounted with 509~ glycerol on slide glass. Examination was done under W lights.
Results are presented in the following tables.

Cell-free assay Vero cells Oligomer Backbone Tm IC50 IC90 CAT
Inhibition 2687-1[75/~MP(l~j][DE]175%MPal~,)] 52.8 ~0.04 1 IIM 20%, 5 N-3+ (0.5 IIM) ,uM nM
3169-1IMP(Rp)/I'F][l~F]~[~- (R")/DE] 62.6 ~0.04 0.8 /IM 20%, 5 C-3+ (2 ~I~M) ~M ~LM
3214-1[MP(RV)/T ,, ' S]~IMP~B,,)/DE] 61.0 ~0.2 ~M 4 IIM No inh., C-3+ (I iLM) 10 IIM:
25%, 5 ~M
3257-1 [MP(Rp)/Dy[DElPS]7lMP(R7)/DE] 60.9 ~0.06 0.6 yM 50%, 2 C-3+ (0.5 ILM) uM rM
3256-1[MP(RV)' ,~ .,J~[MP(R,)/DE] 60.1 ~0.25 5 uM No inh., C-3+ (0.5 ~.M) uM 5 ~IM
3336-12'0MelMP(Rp)/DE]lPS]7- 66.8 N/D
2'0MetMP(R,)/DE]
3341-12~oMe[Mp(R7)lDE][ps]7- 65.8 2 ~M >~10 N/D
2'0MclMP~R,)/DE] IIM

WO 95113834 2 1 7 6 2 ~ 9 PCI'IUS94/13387 o +
~ ' _ r ~ o o ~_ +
~ o ~ ~r~ ~ ~r~
o O, ~2 " Z +
Z r~
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r~ r o . , _ o ,,~ r~ , r ~;1 U~ o .1 2~7~59 Wo 95113834 PCrrUS94/13387 E. Demonstr~tion of Activity of ~nt; ~n~e O
T~rgeted to E2 in Cell-Free Translation Extract~
E2 RNA was prepared by transcribing plasmid pRc/CMV-llE2 with T7 RNA polymerase using an Ambion MegaScript 5 kit, following the nn-n~lfA~t-lrer's directions.
In vitro transcribed E2 mRNA was cell-free translated in rabbit reticulocyte lysates (Promega). The final ~nn,!~ntrations of each, ~ n~nt of the assay system was as f ollows:
In vitro ~r~nerr~h-~ uncapped RNA: 50 nM
'sS-M~h i rn; n~ 1. 3 I~Ci/
Potassiu= Acetate: 20 mM
Amino ncid mixtures, m~thionine deiicient: 50 ~IM
Rabhit Reticulocyt~ Lys~te: ~ 339s vol~vol RNAsin: None or 0.5 units/lll Cell-free translation was performed at 37C for one hour and was stopped by addition of SDS gel loading buffer and incubation at 95C for 3 minutes. Translation of E2 was evaluated after separation of the translation mix by SDS-PAGE analysis, followed by r~n~rl~n;r-~e analysis. To determine the effect of oligomers targeted to the transla-tion initiation codon of E2, in vl tro transcribed E2 mRNA
was translated in the presence of 0.02 or 0.04 units/~l of RNAseH, and using oligonucleotide concentrations ranging from 0.01 to 10 I~M. CAT mRNA was co-translated, or trans-lated in ; n~or~nrl~nt trAn~lAt; nn reactions as control .
As shown in the following table, mea~uL~ ~ of E2 cell-free translation inhibition and of specificity with respect to CAT control mRNA were obtained with the oligomers 3170, 3233 and 3234. Parallel studies showed that these end-blocked chimeric ol; 3 s were more specific than all-phosphodiester oligomers.

~7~2ag POTENCY OF OLIGOMERS TARGETED TO

Cell-free assay Oligomer Target Backbone IC50 IC90 CAT-IC50 3170-1 AUG-12 [MP(Rp)/DE][DE]5[MP(R")/DE] ~0.06 ~M ~1 I-M 20% (5 I~M) 3233-1 AUG-4 [MP(R")/DE][DE]5[MP(Rp)/DE] ~0.1 ~IM ~1 /IM 20% (5 I.M) 3234-1 AUG-4 [MP(Rp)/DE][DE/PS]5[MP(I~)/DE] ~0.1 !-M ~1 ~LM 15% (10 I~M) I. Demonstration of Activity Of Anti~enEle Oligo~er~
T~rgeted to E6 in Cell-Free Tranalation Extr~ct~
Polycistronic E6/E7 mRNA was prepared by transcribing the plasmid pRc/CMV11-E6/E7 with T7 RNA polymerase using an A~nbion MegaScript kit, following the manufacturer~ s directions. Tn vitro transcribed E6/E7 mRNA ~50nM) was cell-free translated in rabbit reticulocyte lysates (Promega) as described in part D above. Cell-free trans-lation was performed at 37C for one hour and was stopped by addition of SDS gel loading buf f er and incubation at 95C for 3 minutes. Translation of E6 was evaluated after separation of the translation mix by SDS-PAGE analysis, followed by rhnpphn;r-~e analysis.
To determine the effect of oligomers targeted to the translation initiation codon of E6, in vi tro transcribed E6/E7 mRNA was translated in the presence or absence of the oligonucleotides shown below. Translations were performed in the presence of O . 02 or O . 04 units/lLl of RNA8e H, and using oligomer cnnr~nt~ations ranging from O . 01 to 10 ~M. CAT mRNa was co-translated as control . As 8hown in the table below, the be8t re8ults were obtained WO 95113834 PCT/13S94/l3387 ~17~

with oligomer 3215-1, a 20mer chimeric methylphosphonate oligomer targeted to AUG-10.
Cell-~ee assay Oligomer Target Backbone ICSO IC90 CAT
inhibi-tion 3255-l AUG-IO [MP(Rp)/DE][DEI[MP(Rp)~DE] I 5 IIM No inh ~M (lo ~IM) 5 3215-l AUG-Io [MP~)/DE][PS/DE][MP(RD)/DE] 0.3 2 ,~ M no inh.
.M (lO ~LM) ~ , uullds 3255 and 3215 are as follows:
3 2 5 5 - 1: 3 ' - CTGCTCC ( GTAAT ) ACCTTTCA- 5 ' 3 2 15 - 1: 3 ' - CTGCTCC ( GTAAT) ACCTTTCA- 5 ' Following are a set of examples relating to certain 10 chemistry useful in the synthesis of chirally pure 2'-0-Me dimers. The preparation of two dimers are discussed in Examples 46 and 47 to further illustrate the utility of the P(III) coupling chemistry through either a 5' or 3' phosphoramidite monomer. These two examples also demon-15 strate the ability to oxidize (with retention) inter-nucleoside methyl rhnRrhnramidite l; nk~P~ using either cumene hydroperoxide or camphorsulfonyl oxaziridine to yield the desired methylrhn~rhnn~tP linkage. Although either or both reagents may be used, our preference is to 20 use camphorsulfonyl oxaziridine because it does not have the hazards associated with cumene l~ydLu~eLu~ide. Example 48 describes the synthesis of a 2'-O-Me-gl:~nn1:;nP 5'-OE~, and is a general scheme applicable tû the preparation of other 5'-OH nucleosides. Example 49 describes the phos-.
Wo 9S/13834 ~ ~ 7 ~ 2 5 9: PC'r/US94113387 phitylation of a 2 -O-Me UC dimer with B-cyanoet_yl ("CE") ~hnsphnramidite .
le 46 Pre~aration oi a 2 -O-Me GG (5 O-DMT, 3 o-BcE. N2I8U) 5 NP(Rq) D~m~r Via 5'methvl~hos~hnn~m;tl;te MQnomer.
Into a 500 ml RBF was placed 30 .5 g (O . 05 M) of 2 ' -OMe, G(3'O-tBDPS,5'-OH, N2IBU) which was rendered anhydrous with 1 x 100 ml pyridine and 2 x 100 ml acetontrile (ACN) . The resulting dry foam was released from the roto-evaporator with argon and treated with 300 ml anhydrous ACN, 10.5 ml triethylamine (0.075 M, 1.5 eq.). The flask was stoppered with a rubber septa and treated (dropwise) with 10.9 ml chloro, methyl-N,N-diisopropyl;~m;nnrhnsphine (0.06 M, 1.2 eq.) . The reaction was allowed to stir overnight at room temperature.
The next morning, the reaction was founa to contain no starting material, as determined by HPLC (Beckman Gold, RP, Waters C18 bnn~Ar~k; A254 nm, 20 min. program 50/50 ACN/O .1 M TEAA to 1009~ ACN. ) . The reaction mix was rnnr.ont~ated then purified on 225 g silica in 3 :1 ethyl acetate/heptane rnnt~1n;n~ 256 TEA. Product was pooled and rnnr~ntri~ted to obtain 25 g (67~) of solid foam that was 86~ pure by HPLC. This product was taken up in ACN to give a 1096 solution of the desired amidite, which was stored over molecular sieves.
Into a 500 ml flamed dried RBF with argon balloon overhead, was transferred via an addition funnel with glass wool, 100 ml (10 g, 0.013 M, 1.25 eq.) of stock solution of 2'0-Me G(5'-amidite, 3'-tBDPS, N2IBU) along with 71.1 ml (7.1 g, O . 011 M, 1. 0 eq. ) of stock solution of 2 ' O-Me, G ( 5 ' DMT, 3 ' OH, N2IBU) . The reaction mixture was then treated all at once with 30 . 9 ml (259t by weight sol .
in ACN, 5 . O eg. ) of ethylthio-tetrazole (ETT) and stirred at room temperature for 5 minutes, after which time cumene lly~lLuyeI~-~ide (2.1 ml, tech., 80~6) was added all at once.
~he reaction was guenched 5 minutes later with 2 0 ml Wo 9s/l3834 2 1 7 ~ 2 5 9 PCrn3S94113387 saturated sodium bisulfite. The reaction mixture was analyzed by HPLC and determined to be 86~ dimer with a ratio of 1. 2/1. 0 (Sp/Rp) . The reaction mixture was then placed on a roto-evaporator and the ACN was removed. The resulting concentrate was then taken up in 150 ml dichlor-omethane (DCM), and washed using 2 x 75 ml sat. NaHCO3 and 1 x 75 ml water. The aqueous wa5hes were combined and then extracted with 1 x 75 ml DCM and combined with the original organic phase and dried over NaSO4, f iltered and concentrated to a light amber solid foam.
The solid foam, 12.6 g (0.0094 M, 1.0 eq.) of 2'-0-Me, GG15'DMT, 3 tBDPS, N2-iBU) MP(Rp/S;,) product was taken up in 120 ml of THF and treated all at once with 14 . 2 ml TBAF (1 M in THF, 0.014 M, 1.5 eq.) and allowed to stand at room temperature overnight. The next morning desily-lation was determined to be complete by HPLC with a purity of 84% (4496 Sp and 4096 Rp). A small amount of silica gel was added to the reaction mixture and after stirring for 10 min. the reaction mix was passed through a glass sintered funnel rrnt~;n;nrJ a small bed o~ silica gel. The product was eluted off the bed with 500 ml 1096 methanol in DCM. The filtrate was rr,nr~ntrated, taken up in DCM and washed using 2 x 75 ml sat. NaHCO3 and 1 x 75 ml brine.
The organic layer was dried over MgSO" f iltered and rrnr~ntrated to a thick oil, which weighed 14 g but had a strong cumene hydroperoxide odor.
The oil was taken up in ACN to give a 23~ by weight solution and purified on a 2 inch preparative HPLC column (Beckman Gold, RP, Kromasil C18, 10u, A295nm, 60 ml/min., isocratic 459~ ACN and 559~ H20). Three separate runs were made and the pure Rp fractions were pooled and concentrated to yield 3.3 g of 1009~ pure GG(3 -OH) MP(Rp) dimer.

~lO 95/l383~ ~ ~ 7 6 2 ~ 9 PCr~94/13387 Exam~le 4 7 Pre~aration of 2 -O-Me. CU(5 -DMT. 3 -OH, N4IBU) MP(R~) Dimer Via a 3 ' -methYl~horh~ n~m;~1; te Monsmer.
50 g (0 . 082 M, 1. 0 eq. ) of the 2 -O-Me, DMT protected - 5 cytidine was rendered anhydrous with 3 x 100 ml pyridine and 1 x 100 ml ACN co-evaporations. The flask was re-leased with argon and to it was added a stir bar, 500 ml ACN, 22 . 7 ml TEA (0 .163 M, 2 eq. ) and a septa with an argon ballon overhead. The solution was treated dropwise with 19.2 ml (0.11 M, 1.3 eq.) of Cl-MAP via a 20 ml plastic syringe and stirred overnight at room temperature.
The reaction was checked the next morning on EIPLC and starting material was gone. The reaction mixture was concentrated and purified on 300 g silica gel with 50/50, EtOAc/Heptane, with 29t TEA. Four liters of the eluent was passed through the column and all U.V. positive material was pooled and ~-nc~ntrated to a solid foam ~52 g, 9596 purity (HPLC), 84~ recovery). The product was taken up in ACN to give a 10~ solution by weight of the desired 3'-methylphos~hr~n~m;tl;te and to this solution was added molecular sieves.
After sitting over molecular sieves for one night, 100 ml (10 g, 0.013 M, 1.25 eq.) of this stock solution was added to a flame dried 500 ml RBF along with 51 ml of a stock solution of U, 5'0H (5.1 g, 0.01 M, 1.0 eq.). The ETT (67 ml, 10~ solution in ACN over molecular sieves, 6.7 g, 0.052 M, 5.0 eq.) was added all at once via an addition funnel and the reaction was stirred for 5 minutes at room temperature. The phosphite int, --l;Ate was then oxidized with 36 ml camphorsulfonyl oxaziridine (CSO) solution (10~6 in ACN over molecular sieves) for 5 minutes. The reaction mixture was checked by HPLC and found to contain 79~ dimer with a ratio of 1. 2/1. 0 ~Sp/Rp) . The reaction mix was c~- n~-~ntl^ated to a solid foam, taken up in 150 ml DCM and worked up as described above in Example 46. The resulting solid foam was 89~ dimer by HPLC and was desilylated (see below) without f urther purif ication .

=

2~2~9 The solid foam, 2 -O-Me, CUl5'-ODMT, 3'-OtBDPS, N~IBU), MP (Sp/Rp) dimer, was taken up in 100 ml THF then treated all at once with 12 . 3 ml tetrabutyl ammonium fluoride ~1 M in THF, 0.012 M, 1.5 eq.). The reaction was checked 1 hr. later by ~IPLC and det~rrrl; n~d to be complete by the disappearance of starting material. The reaction mix was concentrated and purified on silica gel (10 :1) with 3:1 EtOAc:DCM with 10~ methanol. The purified dimer (8 g, 1.5/1.0, Sp/Rp) was then purified by preparative HPLC, which following two separate runs produced 3.3 g of pure Rp dimer, 3'-OH.
Exam~le 4 8 Pre~aration of 2 ' -OMe, G (5 -OH, 3 -OtBDPS, N2IBU) Via the DMT Protected 3'-OH.
25 g of DMT protected 2'-0-Me guanosine was rendered anhydrous with 3 x 100 ml DMF co-evaporations. The solid foam was released from the roto-evaporator via argon and dissolved in 250 ml anhydrous DMF. The solution was then treated with 15.3 g t-butyldiphenylsilyl chloride (0.056 M, 1.5 eq.) and 10.1 g imidazole (0.15 M, 4.0 eq.), then stirred manual ly until the solution was ~ ~, ^ous and allowed to let stand overnight at room temperature. The reaction was checked by HPLC the next morning and found to contain no starting material. The reaction mix was then poured into 300 ml ice water while manually stirring. The solids were rol 1 e~-t~ in a Buchner funnel and rinsed with cold water and then dissolved in 250 ml DCM and washed using 3 x 200 ml sat. NaHCO3, 1 x 100 ml water. The combined aqueous phases were extracted with 2 x 100 ml 3 0 DCM . The organic phases were combined and dried over NaSO~, filtered and cnncPn~rated to a solid foam, obtaining 35 g of newly silylated product (slightly more than the theoretical yield).
The solid foam, 2 -O-Me, G(5'-ODMT, 3'-OtBDPS, N'IBU) was dissolved in 150 ml DCM and with magnetic stirring was treated all at once with 260 ml benzene sulfonic acid (0.1 Wo 95/13834 2 ~ ~ ~ 2 5 9 PCrrUS94/13387 M solution in 75~25 DCM/MeOH, 0 . 026 M, 0 . 67 eq. ) . Reac-tion proceeded for 10 minutes after which time a TLC in 5~
MeOH in DCM revealed that complete desilylation had occurred. The reaction was; ~ tPly quenched with 20 ml TEA at which time the solution changed f rom a deep clear amber color to a light clear yellow color. The solution was concentrated to a thick oil and then loaded onto 250 g silica gel equilibrated in 0 . 59~ MeOH in DCM.
The free trityl was removed with the same eluent and the product was then removed with 6~6 MeOX in DCM. The frac-tions crnt;3ln;n~ product were pooled and concentrated to obtain 21.8 g (98.5% pure by XPLC, 9196 yield overall) of the titled compound.
Exam~le 4 9 Pre~ara~ion of 2 -O-Me UC(5 -ODMT, N4IB~-3'CE Phos~hor-;:lm; dite Via UC, 3 ' -OH
980 mg 2'-O-Me 17C (5'DMT, 3'0X, N4IBU) MP(R~) dimer was rendered anhydrous with 3 x 10 ml ACN co-evaporations.
The resulting dry foam was then taken up in 10 ml anhy-drous ACN and to it was added 325 ~l TEA (2.32 mmol, 2.25 eq. ), followed by dropwise addition (via a 1 ml glass syr inge ) o f 4 6 0 ~ l 2 ' - cyanoethyl - N, N - di i s opropyl chloro -phosphoramidite (2 . 06 mmol, 2 . 0 eq. ) . The reaction was allowed to stir overnight, after which time a TLC and HPLC
showed the reaction to be complete. The reaction mix was concentrated and loaded onto a 1.5 x 20 cm column contain-ing 30 g of silica equilibrated in 3:1:1, EtOAc: DCM:ACN, with 1% TEA. The product was eluted in the same and the fractions with pure product were pooled and rrmr-pnt~ated to yield 600 mg of pure amidite.
Pharmaceutical compositions utilizing the compounds of the present invention, and methods of fo, ll~t;ng the same, are known in the art, and appropriate composition and formulation techniques are further described in U.S.

Wo 95113834 : PCTIUS94113387 2~ 7~2~9 Patent Application Serial Nos. 08/1~4, 013 and 08/154, 014.
Likewise, applicable methods of using the present com-pounds and compositionæ, for-example in l;;~n disease treatment, are disclosed in those applications, which are 5 incorporated herein by reference.
While the foregoing examples and description fiet forth the preferred embodiments and various ways of accomplishing the present invention, they are not ;n~n~
to be limiting as to the scope of the invention, which i5 10 as set forth in the following claims. Moreover, it will be recognized in view of the foregoing disclosure that the invention embraces alternative ~ 1; ts and structures that are the lawful equivalents of those described herein.

WO 9~113834 ~ 1 7 6 2 ~ 9 PCTIUS94/13387 ~ , . . .
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A MEDIUM TYPE: Floppy disk B COMPllTER: IBM PC, t i hl rl C OPER~TING SYST13M: PC-DOS/MS-DOS
D~ SOFTW'ARE: PatemtIn Release #l o, Version #1 2s (vi ) CURRE~IT APPLICATION DATA
(A) APPLICATION NUMBER: US 08/239,177 (B) FILING DATE: 04-MAY-1994 (C) CLASSIFICATION: 03B1/0712 (viii) ATTORN~EY/AGENT lN~U.. _.IlUN:
(A) NAME: Meier, P~ul EI
(B) REGISTRATION NUMSER: 32,274 (C) ~sr:;~L~u~;/DOCRET NUMBER. Z07/174 (ix) TEL ù IN I r .~7~TO~ lNrU.~ lUN:
(A) TELEPhONE 213/489-1600 (B) TELEFAX: il3/955-0440 (C) TELEX: 67-3510 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQ-~ENCE ~r7~v~ T~ lr~
A LENGTE~: 15 base pairs B TYPE: ~ucleic acid C ~ q: single Dl TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii~ nY~ L:
(iv) ANTI-SENSE: yes ( ix ) FEATURE:
(A) NAME/};EY: CT oligomers 2286-1, 2288-1, 2287-1, 2781-1, 2782-1, 3253-1, 2768-1, 2793-1 2760-1 2784-1, 2795-1, 2765-1, 2792-1 (C) l~llrl~llUN MÉTI~OD: synthesig P~Pr~; C
(D) OTE~ER lNr~ :1, lUN: ~ 1 l y to synthetic RNA
target WO 95/13834 = ` ,~ i PCI/IJS94113387 ~ 7~2~
-~Xi) SEQUENCE DESC~IPTION: SEQ ID NO:1:
~1.1~l~1 CTCTA 1 (3) INFORMATION FOR SEQ ID NO:2:
(i) SEQ~ENCE ~TDDr~
~ A. LENGTH: 15 ba8e Pair8 ~ B TYPE: nUC1eiC aCid I C STRr~T~RI~ ~C: 8ing1e ~'D, TOPOLOGY: 1inear (ii) MOLECV~E TYPE: Other nUC1eiC aCid (iii) ~YJ'U~r~11W~L: nO
(iV) ANTI-SENSE: YeS
( iX ) FEATVRE:
(~) NAME/KEY: CU O1igOmer (C) 1~ 11n1~ TION METHOD: SYntheSiS Prr r;
(Xi) SEQUENCE ~ llUN: SEQ ID NO:2:
- ll CUCUA . 15 (4) INFORMATION FOR SEQ ID NO:3:
(i) SEQVENCE ~7r~r-~'T~I~TCTICS:
IA) LENGTH: 19 baSe Pair8 IB) r'PE: nUC1eiC aCid C ) S~ r A- ing1 e D) TOPOLOGY: 1inear (ii) MOLEC~LE TYPE: Other nUC1eiC aCid (iii) ~Y~U,~1~L: nO
(iV) ANTI-SENSE: YeS
( iX) FEATURE:
(A) NAME/KEY: O1igOmerS 1634-1, 2570-1 (C) 1L~ L1~1~ Jn METHOD: SYntheSiS PYrPr; a (D) OTHER INFORMATION: ~ _ 1 Pn1Pn~:~rY tO 5YnthetiC RNA
3 0 target (Xi) SEQVENCE DESCRIPTION: SEQ ID NO:3:

(5) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENOE ~r~ 11U~;
IA,~ LENGTH: 19 baSe Pair8 B I TYPE: nUC1eiC aCid I C I CTAr : Sing1e D TOPOLOGY: 1inear (ii) MOLECIILE TYPE: Other nUO1eiC aCid (iii) ~Y~u~ll~L: nO
(iV) ANTI-SENSE: Ye8 WO 95/13834 2 1 ~ ~ 2 S 9 PCT/US94/133~7 ( ix ) PEATURE:
~A) NAME/KEY: oligomers 2624-1, Z571-l (C) LL)~ TION M~THOD: synthesis experiments (D) OTEIER lNl~ ~J~N:, ,1. Ary to 3ynthetic RNA
target (xi) SEOUENCE l~L:b~ llUl~: SEQ ID NO:4:
~1.ll.~1~ CATGTTGTC 19 (6) lNr~ T FOR SEQ ID NO:5:
( i ) SE QUENCE ~T~ T cTIcs:
A) LENGI~I: 17 base pairs i3) TYPE: nucleic acid C) slrR~NnFn~cc Gingle D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) liY~Ul~llC:AL: no (iv) ANTI-SENSE: ye6 ) FEATURE:
lX (A) NAME/REY: GAG oligomer (C) lL~wllr'L~TION MET~OD: 3ynthesiG oYr ~;
(xi) SEQUENCE L)~;~GKI~lluN: SEQ ID NO:5:
r~ AGGAAGG 17 (7) 1N~I _ilUN FOR SEQ ID NO:6:
( i ) SE QtlENCE ~T~ T~ G ~ . l L ~
Al LENGTE~: 20 base pairs ~B TYPE: nucleic acid 'C STRG~T~Rn~ - ..c: single ID TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) hY~ul~lL~L: no (iv) ANTI-SENSE: yes ( ix ) FEATURE:
(A) NAME/REY: oligomers 3130-~, 2566-1, 2567-1, 2687-3169-1, 3214-1, 3257-1, 3256-1, 2681-1, 2498-1, 3130-3 (C) LlJ~NlL~LW~TION M~TEIOD: syntheGi5 oYr~or; ' -(D) OTHER LN~ T~l~: cleave target mRNA and inhibit mRNA T ~An c l A ~-; nn (xi) SEQUENOE L)~3CKL~lLUN: SEQ ID NO:6:
GTCTTCCATG CAi~ lCC 20 4 0 ( 8 ) INFORMATION FOR SEQ ID NO: 7:

(i) SEQ~ENCE ~r~T~ T~ilLu~:
A LENGTE~: 24 base pairG
B TYPE: nucleic acid ~ C. sTl~D~Tr~T:nNFcc: single 1:) TOPOLOGY: linear -WO 95/13834 ~ PCT/US94/13387 ~7~2~
-(ii~ MOLECULE TYPE: other nucleic acid ~iii) ~Y~U~ UAL: no (iv) ANTI-SENSE: yeq ( ix ) FEATURE:
(A) NAME/KEY: oligomers 32~8-1, 3260-1, XV-l (C) lL~ TION MET~OD: aynthesis experiments (D) OT~ER INFORMATION: inhibit target mRNA trAnqlAt;nn (xi) SEQUENCE L~ :l~Kl~llUN: 8EQ ID NO:7:
CATGGTAGCT TCCTT~GCTC CTGC 24 ( 9 ) INFORMATION FOR SEQ ID NO: 8:
(i) SEQ;IENOE r~T7~D~rT~DTCTICS:
A I LENGTII: 24 base pairs B ~ TYPE: nucleic acid C ST~ ~: single D TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) ~Y~Ul~;ll~L: no (iv) A`wTI-SENSE: yes ( ix ) FEATURE:
(A) NAME/KEY: oligomers 3261-1, 3262-1, XV-2 (C) lY~c.wll~ TION METE~OD: aynthesis experiments (D) OTE}ER INFORMATION: ~nhibit target mRNA trAnqlAt~nn (xi) SEQUENCE U~ Kl~llUI~: SEQ ID NO:8:
Wl~l~iU~ GTGATCTTCT TCTC ==~= 24 (10) lN~ --Tr-~r POR SEQ ID NO:9:
(i) SEQ'~ENCE rTT~D~rT~DTqTT~.C
A LENGT~I: 24 base pairs B TYPE: nucleic acid c sTDr~n~cc: single 3 0 D TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) IIY~ul~ll AL: no (iv) A~TI-SENSE: yes (ix) FEATURE:
(A) NAME/KEY: oligomers 3269-1, 3270-1, XV-6 (C) lU ~ ATION METEIOD: synthe8i8 f.rr~r~ ~ q (D) OTXER INFORMATION: inhibit target mD~NA trAnq1At;nn (xi) SEQUENCE l~Kll:'llUN: SEQ ID NO:9:

(11) lNr~ ~Tt-l~ FOR SEQ ID NO:10:
(i) SEQ~ENCE r~D~ -llu~
(A) LENGTE~ base pairs (B) TYPE: nucleic acid WO 95tl3834 2 1 7 6 2 S 9 PCTIUS94/13387 (c~ sT~ n~qc: single (D) TOPOLOGY linear (ii) MOLEC~LE TYPE other nucleic acid (iii) ~Y~o~ L: no 5 (iv) ANTI-SENSE ye6 ( iX ) FEATURE
(A) NAME/KEY 01igOmerq 2323-1, 22~3-1, 2252-1 (C) 11~ 1~IION MET~OD synthesis experiment_ (D) OTEIE}~ mT~)N: . ,1- ~ y to synthetic RNA
target (Xi) SEQIJENCE l,/~ ~KJ ~llOh SE~ ID NO 10 P~ AGAGT 15 (12) INFORMATION FOR SEQ ID NO:11:
( i ) SEQUENCE r~
,A. LENGTE~ 17 base pairs B ~ TYPE nucleic acid C sT~ nNRcc: single ID TOPOLOGY: linear (ii) MOLECULE TYPE other llucleic acid (iii) ~Y~ l~L: no (iV) ANTI-SENSE: ye~
(iX) FEATURE
(A) NAME/~EY GT oligomers 2517-1, 2516-1 (C) IDENTIFICATION METHOD ~ynthesis '-~T''r;
(D) OTEER l~r~ 1~: 1~ Ary to synthetic RNA
target (Xi) SEQUENCE L)~ ~Kll'llO~J SEQ ID NO 11 (13) INFORMATION FOR SEQ ID NO:12:
3 0 ( i ) SEQ-JENCE ~
A LENGT~ 19 ba3e pairs 'B TYPE nucleic acid C, ST~ : single D. TOPOLOGY linear (ii) MOLECULE TYPE other nucleic acid ( i i i ) ~ y ~ L: no - (iV) ANTI-SENSE: yes ( iX) FEATURE
(A) NAME/}~EY oligomers 2688-1, 2662-2 4 0 (C) 1~ ION METIIOD 3ynthegis nT~--1 q (D) OT~ER lNI''~ mTfl~ ,1 ` y to synthetic RNA
target (Xi~ SEQUENCE Jl;~Kl~lloDl SEQ ID NO 12 ; TTTGAGGTT 19 (14) INFORMATION FOR SEQ ID NO:13:
~i) SEQT~ENCE f~~ rTT;~l~TcTIcs:
A LENGT~I: l9 base pairs B TYPE: nucleic acid C l sT~ nN~cc: single ID: TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) ~YPOTEIETIC~L: no (iv) AaTI-SENSE yes (ix) FEATT~RE:
(A) NAME/REY: oligomers 2625-1 2574-1 (C) lJ~rl~:ATION METlIOD: synthesis ~r~n~n~
(D) OTE~ER INFORMATION: complementary to synthetic RNA
target (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GCTTCCATCT TCCTCGTCC . - 19 ( 15 ) INFORMATION FOR SEQ ID NO :14:
(i) SE:QJENCE CT~D~ I 'iLl~'i A~ LENGTEI: 39 base pa~rs B TYPE: nucleic acid C ~ . c: single D I TOPOLOGY: linear (ii) MOLECULE TYPE: mR~A
(iii) ~YPOTE~ETICAL: no (iv) A~TI-SENSE: no ( ix ) FEATURE:
(A) NAME/XEY: wild-type CAT gene portion (as mRNA) (D) OTHER INFORMATION: pG1036 insert (as TnKNA) (xi) SEQllENCE 1~ Kl~llUN: SEQ ID NO:14:
3 0 GCCUAUUITCC ~ 7` r GIlrlTT~ I~UGAGAAUA 3 9 ( 16 ) INFORMATION FOR SEQ ID NO :15:
(i) SEQUENCE ~T~
A~ LENGTE~: 120 base pairs B TYPE: nucleic acid C sT~ n--~C: single D TOPOLOGY: linear (ii) MOLECT~LE TYPE: ToKNA
(iii) hY~ ~L: no (iv) A~TI-SENSE: no (ix) FEATT~RE:
(A) NAME/XEY: CAT gene portion with intron (as mKNA) (D) OT~;ER INFORMATION: pG1035 insert (as mRNA) (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:15-WO 95/13834 ~! ~ 7 6 2 ~ 9 : PCTIUS94113387 TT~TTrTUrCrTTD U;.~UCCWADA r.~;rTr.DrlTr.Dr TT7~~TT~ rrJc r~rP~~~ TT7~TrDrTUr.~ 60 UTT~'`~Tr~U'`" rD~ rrr~ TT~TTrTTTTrr~r~ ~WWCAGGG riTjTT~TTUr~ 70 (17) lN~( - rrM FOR SEQ ID NO:16:
(i) SEQ',JENCE r~TrnD~
1 A LENGT~: 54 base pairs B I TYPE: nucleic acid C~ STD~'-)T~nMrqq: 3inyle I D'~ TOPOLOGY: linear (ii) MOLEWLE TYPE: TnD~NA
10 (iii) liY~ol~ll~L: no (iv) AMTI-SENSE: no (ix) FEATURE:
(A) NAME/KEY: ~qild-type CAT gene portion ~as T~D~NA) (xi) SEQUENOE IJ~O~ll:'~lUN: SEQ ID NO:16:
T~UrD~~-`-- rTTT D~~'`7`~~ UA~A ArLTG GAG A~A A~A AUC AW GGA UAU ACC 51 Met Glu LYB LyG Ile Ser Gly Tyr Thr Thr ao 1O
(18) INFORMATION FOD~ SEQ ID NO:17:
(i) S~QJENCE rr~nDDr~T.DTqTICS
A LENGTE~: 54 base pairs B: TYPE : nucleic acid C sTDDMnr~nMr.~.qq siLyle D I TOPOLOGY: linear (ii) MOLEWLE TYPE: T~D.NA
(iii) ~Y~ols~ll~ ~L: no (iv) AMTI-SEN8E: no (ix) FEATURE:
(A) NAME/~tEY: pG1040 insert (as ToD~NA) (Xi) SEQrlJENcE J~ N: SEQ ID NO:17:
rGrr~ rTTDD~~7~rr~ UACC ArTG GAG AAG AAG AlTC ACU GGA UAU ACC 51 Met Glu Lys Lys Ile Ser Gly Tyr Thr Thr (19) lNr~l --TOM FOR SEQ ID NO:18:
(i) SEQ~7CE rS~DD~ . ~ TI~
(A) LENGTE}: 24 base pairs (B) TYPE: nucleic acid (C) STD~ : single WO 95/13834 ~ 2 5 9 PCT/US94/13387 (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: other nucleic acid (iii) nY~uln~ AL: no (iv) ANTI-SENSE: yes ( ix) FEATURE:
(A) NAME/KEY: oligomers 3264-1, XV-5 (C) l~ lUATION METXOD: synthetic oTn.~rlm~ntc (D) OThER INFORMATION: inhibit target mRNA trAnqlAt;rn (xi) SEQUENOE DESCRIPTION: SEQ ID NO:18:
10 CACTCACCTT T~r~rr7~ T~ GGCC 24 (20) INFOKMATION FOR SEQ ID NO:1g:
(i) SEQ-~ENCE rT7DrJ~rTr.'r~TqTICS:
A LENGTX: 24 ~ase pairG
B I TYPE: nucleic acid IC sTr~r~nNr~lcc: single IDI TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (iii) XYPOTHETICAL: no (iv) ANTI-SENSE: yes 20 (ix) FE~TURE:
(A) NAME/KEY: oligomers 3265-1, 3266-1, J~V-7 (C) llr~ ATION METXOD: synthetic experiments (D) OTHER lNr~ mTr,N: inhibit target mRNA trAnqlAt;rn (xi) SEQUENOE Ll~sb~KI~IluN: SEQ ID NO:19:
25 CCCTGAGAGA r7 r~ `r~rr, TTCG 24 (21) lN~'Unl_.llUN FOR SEQ ID NO:20:
(i) SEQ-~ENCE r~rT~r~rTr~T~TcTIcs:
A LENGTX: 54 base pairs B TYPE: nucleic acid ~C ~,~ r~ N~:.`. 5: single ~D TOPOLOGY: linear (ii) MOLECULE TYPE: m.RNA
(iii) nYl~Ul~~ AL: no (iv) ANTI-SENSE: no 35 (ix) FEATUKE:
(A) NAME/KEY: pG1042 mismatch insert (as m.~NA) (D) OTHER lNlC -lUN: controlled mismatch oligomer screening (xi) SEQUENCE YJK~UKll~llUN: SEQ ID NO:2~:

~rrJr~r7~r~ UrJl3r-cr~7lr-r UACC ~7G GAC AGG A~G A~U ACG GGA UAU ACC 51 Met Asp Arg ~yc Ile Thr Gly Tyr Thr WO gS/13834 ~ PCTIUS9~/1338 1~1 ACC
Thr 54 (22) INFO~MATION FOR SEQ ID NO:21:
(i) SEQ~ENCE rT-Tr~n;~rTT.~TiT~TIcs~
A LENGT~I: 54 base pair3 f' B TYPE: nucleic acid C sTT~r~TnT~nNlpcc: single ID TOPOLOGY: linear (ii) MOLECULE TYPE: TCRNA
( iii) t- yJ~ ~L: yes (iv) ANTI-SENSE: no (ix) FEATURE:
(A) NAME/}~EY: mismatch insert (as m~NA) (D) OT}~ER INFORMATION: controlled mismatch oligomer screening (xi) SEQT~ENCE Lm;~s~l~ll~N: SEQ ID NO:21:
p~-T-TfTrDr-r~D~- C'TTD anrT T ' '` urrrDTTG~ '' P D~ TTrD rTT~-rT ~TT T~r CACC 54 (23) INFORMPTION FOR SEQ ID NO:22:
(i) SEQTJENCE r~TDT rrTT~T~TC TICS:
A LENGTEI: 54 base pair~
B: TYPE: nucleic acid C sTT~r : single D TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA
(iii) ~iY~Jl~lL~AL: yes (iv) ANTI-SENSE: no (ix) FEATURE:
(A) NAT~E/KEY: mismatch insert (as mRNA) 3 0 (D) OT}~ER lNr'~ _.I1UN: controlled mismatch oligomer screenLng ~xi) SEQUENCE DESC~IPTION: SEQ ID NO:22:
DrTJ13rD~::Dn ~TTT.7~"--T7~r ur~rrDTT~rpr D7~"T7~"~TTrD rTT~ TT~TTDr CACC 54 ~24) lNr~ ~TrN FOR SEQ ID NO:23:
3 5 ~ i ) SEQ~ENCE riTT~ n D ' . . .~ I '. 11~:
I A LENGT~: 54 base pair6 B TYPE: nucleic acid ' STT~r ~: single D TOPOLOGY: linear ~ii) MOLECULE TYPE: mRNA
Y~Clrlr;l l~L: yes ( iv) ANTI - SENSE: no ( ix ) FEATURE:
(A) NAMi~/XEY: mismatch insert (as mRNA) WO 95/13834 2 ~ ~ 2 ~ 9 PCTIUS94/13387 (D) OTdER INFORMDTION: controlled mismatch oligomer screening (xi) SEQUENCE Lll:;~Kl~llUN: SEQ ID NO:23:
rrrTqrDr~r. rTTDD--rTDDrr urrrT~TTrr~-- Pr--~r`--7'TTrD rTTr~ATT7~T~r CACC 54 ~25) INFORMATION~ FOR SEQ ID NO:24:
(i) SEQ-~ENCE r~TD~Dl . r.~ I .'ill~:
A LENGTd: 54 base pairs B TYPE: nucleic acid ' C b ~ C: single ~D TOPOLOGY: linear (ii) MOLECULE TYPE: mRNA
(iii) nY~ULd~~ L: yes ( iv) ANTI - SENSE: no ( ix) FEATURE:
(A) NAME/EEY: mismatch insert (as mRNA) (D) OTdER INFORMATION: controlled mismatch oligomer screening (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Dr.lTr,rDr-7~- rTT~ Trrrr urrrDTT--~- r7~ rTrD rTT--"TT~rT~- CACC 54 (26) INFORMATION FOR SEQ ID NO:25:
(i) SEQ'~ENCE rlT~D- ~ .Ll~
A LENGTd: 54 baoe pairs B I TYPE: nucleic acid lc sT~r c: single 2!~ D: TOPOI,OGY: linear (ii) MOLECULE TYPE: mRNA
(iii) dYfUld~ L: yes (iv~ ANTI-SENSE: no ( ix) FEATURE:
(A) NAME/KEY: mismatch insert (as mRNA) (D) OT}~ER INFORMATION: controlled mismatch oligomer screening (xi) SEgTJENCE J~ lrllUN: SEQ ID NO:25:
Dr~UGrr~ -- rTT--~--TT~ r TTrrrDTTrr.Dr. D~ rr~rTrD rTT----~rT~rT~-- CACC 54 (27) IN-FOR~TION FOR SEQ ID NO:26:
(i) SEQUBNCE r~TDT~DrTFTlTcTIcs (A) LENGTd: 21 base pairs (B) TYPE: nucleic acid (C) ::, : single (D) TOPOLOGY: linear (ii) MOLEC~lLE TYPE: other nucleic acid ( iii ) d~ ~u ~ d~ l~ AL: yes -~iv~ ANTI-SENSE: yes (ix) FEATUE:
(A) NAME/KEY: mismatch oligomer ~D) OTE~ER INFORMATION: mismatch oligomer to target m.~NA SEQ ID NOS: 21-25 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

(28) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE ~Tr~rTT~.T~TICS:
A) LENGTE~: 20 base pairs B) TYPE: nucleic acid C) ~ .c: single D) TOPOLOGY: liLear (ii) MO~ECULE rYPE: other nucleic acid l~i (iii) XYPOrHETICAL: no (iv) ANTI-SENSE: no (ix) F13ATU.~E:
(A) NAME/~EY: PNA target oligomer (C) ll~ ~TION METHOD: synthetic experiment (D) OTHER INFORMATION: target ~or oligomers 26B1-1 (xi) SEQ~ENCE J~ UN: SEQ ID NO:27:
r~ .TT~ ~TT7~r~T~r~ ~ 20

Claims (39)

What is claimed is:

1. An oligonucleoside compound for effecting RNaseH-mediated cleavage of a target ribonucleic acid sequence, comprising an RNaseH-activating region and a non-RNaseH-activating region, wherein the RNaseH-activating region comprises a segment of at least three consecutive 2'-unsubstituted nucleosides linked by charged internucleoside linkage structures, the non-RNaseH-activating region comprises a segment of at least two linked nucleosides, at least one of the linkages in said non-RNaseH-activating region being chirally-selected, and wherein the base sequence of the oligo-nucleoside compound is complementary to a target region of the target ribonucleic acid sequence.

2. The oligonucleoside compound of claim 1 wherein said RHaseH-activating region comprises between five and about nine consecutive linked nucleosides.

3. The oligonucleoside compound of claim 2 wherein the charged linkage structures in said RNaseH-activating region are selected from the group consisting of phospho-diester linkages, phosphorodithioate linkages and phos-phorothioate linkages.

4. The oligonucleoside compound of claim 2 wherein the segment of charged linkage structures in said RNaseH-activating region comprises a mixed charged linkage sequence including at least two different charged linkage structures.

5. The oligonucleoside compound of claim 4 wherein said mixed charged linkage sequence is repeated at least twice in the RNaseH-activating region.

6. The oligonucleoside compound of claim 3 wherein said RNaseH-activating region comprises a plurality of phosphorothioate linkages.

7. The oligonucleoside compound of claim 2 wherein said segment of chirally-selected nucleosides in the non-RNase-activating region comprises at least four linked nucleosides, and further comprises a plurality of Rp-selected linkage structures.

8. The oligonucleoside compound of claim 7 wherein at least about 40% of the total number of linkage struc-tures in said chirally-selected nucleoside segment are Rp linkage structures.

9. The oligonucleoside compound of claim 7 wherein at least about 75% of the asymmetric linkage structures in said chirally-selected nucleoside segment are Rp linkage structures.

10. The oligonucleoside compound of claim 7 wherein substantially all of the asymmetric linkage structures in said chirally-selected nucleoside segment are Rp linkage structures.

11. The oligonucleoside compound of claim 7 wherein the segment of chirally-selected linkage structures in said non-RNaseH-activating region comprises a mixed chiral linkage sequence including at least two different linkage structures, at least one of which is asymmetric.

12. The oligonucleoside compound of claim 11 wherein said mixed chiral linkage sequence is repeated at least twice in the non-RNaseH-activating region.

13. The oligonucleoside compound of claim 11 wherein said different linkage structures in the mixed chiral linkage sequence are selected from the group consisting of:
Rp-methylphosphonate and phosphodiester linkage;
Rp-methylphosphonate and racemic methylphos-phonate linkages;
Rp-methylphosphonate and phosphorothioate linkages;
Rp-methylphosphonate and phosphorodithioate linkages; and Rp-methylphosphonate and alkylphosphonothioate linkages.

14. The oligonucleoside compound of claim 11 wherein said different linkage structures in the mixed chiral linkage sequence are selected from the group consisting of MP(R)/DE
2'OMeMP(R)/2'OMeDE
MP (R)/2'OMeNP
MP(R) enriched 2'OMeMP (R) enriched MP(R)/PS
2'OMeMP (R)/2'OMePS
MP(R)/PS2 2'OMeMP(R)/2'OMePS2 2'OMeMP/2'OMeDE
MP/2'OMeDE
MP(R)/PAm 2'OMeMP(R)/2'OMePam 2'OMeMP/2'OMePAm MP/2'OMePAm MP(R)/TE
2'OMeMP (R)/2'OMeTE
2'OMeMP/2'OMeTE
MP/2'OMeTE
MP(R)/MPS
2'OMeMP(R)/2'OMeMPS
2'OMeMP/2'OMeMPS
MP/2'OMeMPS
MP(R)/PF
2'OMeMP (R)/2'OMePF
2'OMeMP/2'OMePF
MP/2'OMePF
MP(R)/PBH3 2'OMeMP(R)/2'OMePBH3 2'OMeMP/2'OMePBH3 MP/2'OMePBH3 MP(R)/RSi 2'OMeMP (R)2'OMeRSi 2'OMeMP/2'OMeRSi MP/2'OMeRSi MP(R)/CH2 2'OMeNP(R)/2'OMeCH2 2'OMeNP/2'OMeCH2 and MP/2'OMeCH2, or from the foregoing mixed linkage structure combinations wherein at least one MP or MP(R) linkage structure therein is replaced, respectively, with an MPS or MPS (R) linkage structure, an AAP or AAP(R) linkage structure, or an AAPS
or AAPS (R) linkage structure.

15. The oligonucleoside compound of claims 11, 13 or 14 wherein one or both of the nucleosides linked by said different linkage structures in the mixed chiral linkage sequence are 2'-substituted nucleosides.

16. The oligonucleoside compound of claim 15 wherein both of the nucleosides linked by said different linkage structures in the mixed chiral linkage sequence are 2'-substituted nucleosides.

17. The oligonucleoside compound of claim 15 wherein said 2'-substituents are selected from the group consist-ing of alkoxy, allyloxy and halo substituents.

18. The oligonucleoside compound of claim 17 wherein said 2'-substituents are methoxy substituents.

19. The oligonucleoside compound of claims 1, 7, 11, 13 or 14 wherein said RNaseH-activating region is at one terminal portion of the compound and said non-RNaseH-activating region is at the other terminal portion of the compound.

20. The oligonucleoside compound of claims 1, 7, 11, 13 or 14 comprising a second non-RNaseH-activating region, and wherein said RNaseH-activating region is flanked in the compound by the first and second non-RNaseH-activating regions.

21. The oligonucleoside compound of claim 20 wherein said second non-RNaseH-activating region comprises at least four linked nucleosides, and further comprises a plurality of Rp-selected linkage structures.

22. The oligonucleoside compound of claim 21 wherein the internucleoside linkage structures and optional 2' -substituents in said second non-RNaseH-activating region are selected from among those defined for said first non-RNaseH-activating region.

23. An oligonucleoside compound for effecting RNaseH-mediated cleavage of a target ribonucleotide acid sequence, comprising an RNaseH-activating region and a non-RNaseH-activating region, wherein the RNaseH-activating region comprises a segment of at least three consecutive 2'-unsubstituted nucleosides linked by charged internucleoside linkage structures, the non-RNaseH-activating region comprises a segment including an alternating sequence of racemic internucleoside linkages, said sequence comprising (a) a racemic lower alkylphosphonate, lower alkyl-phosphonothioate or amino- (lower alkylene)-phospho-nate linkage structure alternating with (b) a nega-tively-charged phosphate ester, phosphorothioate or phosphorodithioate linkage structure, and wherein the base sequence of the oligonu-cleoside compound is complementary to a target region of the target ribonucleic acid sequence.

24. The oligonucleoside compound of claim 23 wherein said RHaseH-activating region comprises between five and about nine consecutive linked nucleosides.

25. The oligonucleoside compound of claim 24 wherein the charged linkage structures in said RNaseH-activating region are selected from the group consisting of phospho-diester linkages, phosphorodithioate linkages and phos-phorothioate linkages.

26. The oligonucleoside compound of claim 25 wherein said RNaseH-activating region comprises a plurality of phosphorothioate linkages.

27. The oligonucleoside compound of claim 24 wherein said lower alkyl or alkylene portion is selected from methyl and methylene.

28. The oligonucleoside compound of claims 24 or 27 wherein said negatively-charged linkage structure is a phosphodiester linkage structure.

29. The oligonucleoside compound of claims 24 or 27 wherein one or more of the nucleosides linked in said alternating linkage structure are 2'-substituted nucleo-side residues.

30. The oligonucleoside compound of claim 29 wherein said alternating linkage sequence comprises a 2'-substi-tuted phosphodiester-linked nucleoside residue.

31. The oligonucleoside compound of claim 29 wherein said 2'-substituents are selected from the group consist-ing of alkoxy, allyloxy and halo substituents.

32. The oligonucleoside compound of claim 31 wherein said 2'-substituents are methoxy substituents .

33. The oligonucleoside compound of claim 23 wherein said RNaseH-activating region is at one terminal portion of the compound and said non-RNaseH-activating region is at the other terminal portion of the compound.

34. The oligonucleoside compound of claim 23 com-prising a second non-RNaseH-activating region, and wherein said RNaseH-activating region is flanked in the compound by the first and second non-RNaseH-activating regions.

35. The oligonucleoside compound of claim 29 wherein the internucleoside linkage structures and optional 2'-substituents in said second non-RNaseH-activating region are selected from among those defined for said first non-RNaseH-activating region.

36. The oligonucleoside compound of claim 34 wherein the internucleoside linkage structures in said second non-RNaseH-activating region are selected from among those defined for said first non-RNaseH-activating region.

37. The oligonucleoside compound of claim 36 wherein one or more of the nucleosides linked in one or more of said alternating linkage structures are 2'-substituted nucleoside residues.

38. A pharmaceutical composition comprising an effective amount of an oligonucleoside compound of claims 1 or 23 and a pharmaceutically acceptable carrier.

39. A method of inhibiting translation of a target ribonucleic acid sequence in a cell or a multicellular organism comprising administering to said cell or organism an oligonucleoside compound of claims 1 or 23.