WO2003039523A2 - Oligonucleotides modifies a l'aide de nouveaux analogues d'arn-l-alpha - Google Patents

Oligonucleotides modifies a l'aide de nouveaux analogues d'arn-l-alpha Download PDF

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WO2003039523A2
WO2003039523A2 PCT/IB2002/005080 IB0205080W WO03039523A2 WO 2003039523 A2 WO2003039523 A2 WO 2003039523A2 IB 0205080 W IB0205080 W IB 0205080W WO 03039523 A2 WO03039523 A2 WO 03039523A2
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oligonucleotide
group
rna
groups
optionally substituted
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Jesper Wengel
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Exiqon A/S
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Definitions

  • the present invention relates to the field of providing oligonucleotides modified with one or more ⁇ -L-configurated nucleoside analogues. Further, the invention relates to synthetic oligonucleotides capable of forming nucleobase-specific duplexes with complementary single stranded nucleic acids, and to the combination of ⁇ -L-RNA and LNA (Locked Nucleic Acid) nucleotides in the formation of said oligonucleotides. The invention also relates to the use of oligonucleotides comprising ⁇ -L-configurated nucleosides and nucleic acid derivatives comprising the same as therapeutic drags.
  • oligonucleotides are widely used compounds in disparate fields, such as molecular biology, DNA/RNA-based diagnostics, and therapeutics. To be useful for a range different applications, oligonucleotides have to satisfy a large number of different requirements. As therapeutics, for instance, a useful oligonucleotide must be able to penetrate the cell membrane, have good resistance to extra- and intracellular nucleases, and desirably have the ability to recruit endogenous enzymes like RNase H. In DNA-based diagnostics and molecular biology other properties are important.
  • oligonucleotides For instance, the ability of oligonucleotides to act as efficient substrates for a wide range of different enzymes evolved to act on natural nucleic acids, such as polymerases, kinases, ligases and phosphatases maybe desirable.
  • a fundamental property of oligonucleotides is their ability to recognize and hybridize sequence-specifically to complementary single stranded nucleic acids ("to pair"), employing either Watson-Crick hydrogen bonding (A-T and G-C) or other hydrogen bonding schemes, such as the Hoogsteen mode.
  • A-T and G-C Watson-Crick hydrogen bonding
  • affinity and specificity commonly used to characterize the hybridization properties of a given oligonucleotide.
  • Affinity is a measure of the binding strength of the oligonucleotide to its complementary target sequence (expressed as the thermostability (T m ) of the duplex). Each nucleobase that is paired adds to the thermostability of the duplex. Thus, affinity increases with increasing size (number of nucleobases) of the oligonucleotide. Specificity is a measure of the ability of the oligonucleotide to discriminate between a fully complementary and a mismatched target sequence. In other words, specificity is a measure of the loss of affinity associated with mismatched nucleobase pairs in the target. The term "specificity" may, in the present context, also be used to describe the ability of an oligonucleotide to discriminate between RNA and DNA based sequences.
  • oligonucleotides undergo a conformational transition in the course of hybridizing to a target sequence, from the relatively random coil structure of the single stranded state to the ordered structure of the duplex state.
  • conformational restriction has recently been applied to oligonucleotides in the search for analogues displaying improved hybridization properties compared to the unmodified (2'-deoxy) oligonucleotides.
  • LNAs Locked Nucleic Acids
  • WO 99/14226 and in various scientific publications, including Nielsen, R, Pfundheller, H. M., Olsen, C. E. and Wengel, J., J. Chem. Soc., Perkin Trans. 1, 1997, 3423; Nielsen, R, Pfundheller, H. M., Wengel, J., Chem. Commun., 1997, 9,825; Christensen, N. K., Petersen, M., Nielsen, P., Jacobsen, J. P. and Wengel, J., /. Am. Chem. Soc, 1998, 120, 5458; Koshkin, A.
  • ⁇ -L-LNA analogues are demonstrated to have an increased affinity towards a complementary strand when incorporated into an oligonucleotide.
  • Antisense oligonucleotides are useful as therapeutic agents. Briefly, an antisense drug operates by binding to the mRNA, thereby blocking or modulating its translation into protein. Thus, antisense drugs may be used to directly block the synthesis of disease causing proteins.
  • antisense drugs can also be used to activate genes rather than suppressing them by, for example, blocking the synthesis of a protein that otherwise suppresses RNA synthesis from the target gene.
  • the hybridizing oligonucleotide is thought to elicit its effect by either creating a physical block to the translation process or by recruiting a cellular enzyme (RNase H) that specifically degrades the mRNA part of the mRNA/antisense oligonucleotide duplex.
  • RNase H a cellular enzyme
  • nucleic acids that are antisense to the transcript of a specific gene such as a gene key to a pathogen or a deleterious human gene, such as those involved in certain cancers, could impair the expression of this gene, thereby disabling the particular disease state.
  • Antisense targets can be selected that are unique to the gene whose expression is to be controlled. Hence, only that gene's expression is inhibited. This is especially important for diseases like viral infections and cancers which employ normal cell functions as part of the disease process. It has been difficult to devise therapeutic strategies against these diseases without also disabling normal cell functioning. Antisense nucleic acids targeted toward a gene that is diverting these normal cell functions, however, can specifically impair the disease state without affecting cell function. An antisense approach, therefore, has the promise of fewer side effects and offers real therapeutic promise for certain cancers and viral diseases.
  • Another advantage to an antisense approach is that it inhibits the pathogenic process at the source of the disease. That is, it interferes with the formation of unwanted proteins, rather than stopping these proteins from functioning.
  • it is easier to identify nucleic acid targets than it is to identify protein targets.
  • oligonucleotides must satisfy a large number of different requirements to be useful as antisense drugs.
  • desirable properties of antisense oligonucleotide are binding with high affinity and specificity to its target mRNA, the ability to recruit RNase H, the ability to reach their site of action within the cell, resistant and to extra- and intracellular nucleases (both endo- and exo-nucleases), little or not toxicity at the relevant dose, and the ability to specifically hybridize to mRNA, leaving DNA unhybridized at physiological conditions.
  • oligonucleotides hybridize to RNA as well as DNA with comparative affinity, with an affinity slightly biased toward either the complementary RNA or the DNA.
  • an antisense oligonucleotide directed towards a target mRNA may unintentionally hybridize to genomic DNA, optionally via strand displacement, and cause toxic side effects.
  • antisense oligonucleotides that have greater affinity for RNA (e.g., mRNA) than DNA (e.g., genomic DNA).
  • An oligonucleotide selective towards RNA is also desirable for preparative purification of RNA from mixtures comprising DNA and RNA.
  • the object is to provide an oligonucleotide that selectively hybridizes to RNA.
  • An oligonucleotide showing this property may find various uses, including therapeutic and diagnostic utitlities.
  • a further object according to the present invention is to avoid or reduce toxic side effects of antisense or other gene-silencing oligonucleotides (e.g., double-stranded nucleic acids) by reducing or eliminating their affinity towards DNA.
  • a still further object of the present invention is to make available oligonucleotides which, in a single step, can selectively extract RNA from a sample containing a mixture of biological substances.
  • the invention provides an oligonucleotide (hereinafter termed " ⁇ -L-RNA modified oligonucleotide”) comprising at least one oligonucleotide analogue (hereinafter termed " ⁇ -L-RNA monomer”) of the general formula:
  • X is selected from -0-, -S-, -S(O)-, -S(0) 2 -, -N(R N* )-, -C(R 6 R 6* )-, -O- C(R 7 R 7* )-, -C(R 6 R 6* )-0-, -S-C(R 7 R 7* )-, -C(R 6 R 6* )-S-, -N(R N* )-C(R 7 R 7* )-, - C(R 6 R 6* )-N(R N* )-, and -C(R 6 R 6* )-C(R 7 R 7* )-;
  • B is selected from hydrogen, hydroxy, optionally substituted C -alkoxy, optionally substituted C ⁇ _ -alkyl, optionally substituted C ⁇ _ 4 -acyloxy, optionally protected nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups,
  • P* designates an internucleoside linkage to a preceding or successive monomer, or a 3'-terminal group.
  • R represents F, Cl, Br, I, SR", SeH, SeR", N(R N* ) 2 , OH, a protected hydroxy group, SH, a protected mercapto group, an optionally substituted linear or branched Ci- 1 - alkoxy, an optionally substituted linear or branched C 2 . 12 -alkenyloxy.
  • Each of the substituents R 1* , R 2* , R 3* , R 4 , R 5 , R 5* , R 6 , R 6* , R 7 , and R 7* is independently selected from hydrogen, optionally substituted linear or branched C ⁇ .
  • 6 -alkylaminocarbonyl mono- and di(C 1 . 6 -alkyl)amino-C ⁇ .
  • 6 - alkylaminocarbonyl Ci- ⁇ -alkylcarbonylamino, carbamido, C ⁇ _ 6 -alkanoyloxy, sulfono, C ⁇ _ 6 -alkylsulfonyloxy, nitro, azido, sulfanyl, C ⁇ .
  • R when present, represents a C ⁇ _ 6 -alkyl or phenyl group.
  • R N* when present, is selected from hydrogen and C ⁇ _ 4 -alkyl.
  • Oligonucleotides containing at least one monomer of the formula (I) may also comprise basic salts and acid addition salts thereof.
  • the nucleobase B may be selected from a variety of substituents. In one embodiment, B designates a naturally-occuring nucleobase selected from uracil- 1-yl, thymin-1-yl, adenin-9-yl, guanin-9-yl, cytosin-1-yl, and 5- methylcytosin-1-yl.
  • B is a non-naturally-occurring nucleobase.
  • the oligonucleotide according to the invention desirably contains at least one ⁇ -L-RNA monomer, wherein X is selected from the group consisting of -0-, -S-, -S(O)-, -S(0) 2 -, and -N(R N* )-. Desirably, X represents -0-.
  • the substituents R , R , R , R , R , and R may, in a desired embodiment independently represent hydrogen, C ⁇ _ -alkyl, or C ⁇ _ -alkoxy. In one embodiment, R represents hydrogen. In another embodiment, R represents a protected hydroxy group.
  • An exemplary hydroxy protecting group for R may be a linear or branched C ⁇ _ 6 -alkoxyl group or a silyloxy group substituted with one or more linear or branched C ⁇ _ 6 -alkyl groups.
  • the substituent R 2 is tert- butyldimethylsilyloxy.
  • the substituent P when representing a 5'-terminal group, suitably designates hydrogen, hydroxy, optionally substituted linear or branched C ⁇ _ 6 - alkyl, optionally substituted linear or branched C ⁇ . 6 -alkoxy, optionally substituted linear or branched C ⁇ .
  • P* when representing a 3'-terminal group, suitably is selected from hydrogen, hydroxy, optionally substituted linear or branched C ⁇ _ 6 -alkoxy, optionally substituted linear or branched C ⁇ _ 6 -alkylcarbonyloxy, optionally substituted aryloxy, and -W-A', wherein W is selected from -0-, -S-, and - N(R H ) ⁇ where R H is selected from hydrogen and C ⁇ _ 6 - alkyl, and where A' is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
  • P and P represent an internucleoside linkage between a successive and a preceding monomer
  • any internucleoside linkage independently is selected from -CH2-CO-NR H -, -CH2-NR H -0-, -S-CH2-O-, -
  • Most desirable as internucleoside linkage is -0-P(0)2-0-, which is the natural occuring linkage.
  • the present invention also provides an oligonucleotide comprising at least one ⁇ -L-LNA monomer and at least one ⁇ -L-RNA monomer.
  • oligonucleotides with this composition express an enhanced ability to hybridize selectively to RNA.
  • at least one ⁇ -L-LNA monomer and at least one ⁇ - L-RNA monomer are positioned relative to each other in any order.
  • at least one ⁇ -L-LNA monomer may be positioned in the oligonucleotide upstream or downstream relative to at least one ⁇ -L-RNA monomer.
  • it is desirable that at least one ⁇ -L-LNA monomer is placed adjacent to at least one ⁇ -L-RNA monomer in the oligonucleotide.
  • the oligonucleotide comprises a group or groups of at least one ⁇ -L-LNA monomer alternating with group(s) of at least one ⁇ -L-RNA, optionally with intervening natural occurring or synthetic nucleosides.
  • Each of the groups of ⁇ -L-LNA and ⁇ -L-RNA desirably comprise 1 to 10, more desirably, 2 to 8 nucleosides.
  • the alternating groups may be present in a block placed at the 3 ' or 5' end of the oligonucleotide, or the block may be situated in the central part of the oligonucleotide flanked by naturally occurring or synthetic nucleosides. More than one block may be present in the oligonucleotide.
  • alterations occur in the oligonucleotide typically 1 to 50 alternations, 1 to 25 alterations, 1 to 15 alterations, or 2 to 10 alterations are present.
  • the oligonucleotide may have any suitable length.
  • the oligonucleotide may have, e.g., 1-100 ⁇ -L-LNA monomers, 1-100 ⁇ -L-RNA monomers, and 0- 1000 nucleosides selected from naturally-occurring or synthetic nucleosides.
  • the total number of nucleotide monomer does not exceed 1000, and is not less than 3.
  • the number of nucleotide monomers is not above 100, and is not less than 8.
  • the total number of nucleotide monomers is at least 12. Most desirably, the total number of nucleotide monomers is 2 to 8.
  • the oligonucleotide is between 1000 to 2000, 2001 to 3000, 3001 to 5000, or 5001 to 10,000 nucelotides in length, inclusive.
  • the length of the nucleotide can be readily adjusted by one skilled in the art depending on the intended application of the oligonucleotide.
  • a suitable length of the nucleotides may be, e.g., 12 to 25 nucleoside monomers, while the length of the nucleotide in the study of expression of alternative splicing may be, e.g., between 50 and 100 nucleoside monomers.
  • the oligonucleotide comprises in a desired embodiment at least 3 ⁇ -L-
  • the LNA monomers and/or at least 3 ⁇ -L-RNA monomers may optionally be present as groups of two or more continuous ⁇ -L-LNA or ⁇ -L-RNA monomers.
  • the oligonucleotide comprises a sequence wherein ⁇ -L-LNA monomers alternate with ⁇ -L-RNA monomers.
  • the synthetic nucleotide monomer which optionally may be present in the oligonucleotide, may in one embodiment of the invention be ⁇ -D-LNA.
  • the synthetic nucleotide monomer is ⁇ -D-oxy-LNA.
  • a contiguous stretch of 2 to 30 ⁇ -oxy-LNA monomers in the oligonucleotide of the invention comprises at least one thymin-y-yl.
  • a contiguous stretch of 2 to 30 ⁇ -oxy-LNA monomers having at least one thymin-y-yl is exclusively of the ⁇ -L-RNA configuration.
  • an oligonucleotide of the invention may be advantageous to complex or to non- covalently or covalently bind an oligonucleotide of the invention to a compound selected from proteins, amplicons, enzymes, polysaccharides, antibodies, haptens, peptides, DNA, RNA, and peptide nucleic acids (PNAs).
  • a compound e.g., an intermediate compound useful in the synthesis of an oligonucleotide of the invention having the general formula II
  • X is selected from -0-, -S-, -S(O)-, -S(0) 2 - -N(R N* )-, -C(R 6 R 6* )-, -0- C(R 7 R 7* )-, -C(R 6 R 6* )-0-, -S-C(R 7 R 7* )-, -C(R 6 R 6* )-S-,-N(R N* )-C(R 7 R 7* )-, - C(R 6 R 6* )-N(R N* )-, and -C(R 6 R 6* )-C(R 7 R 7* )-.
  • R N* when present, is selected from hydrogen and Ci_ 4 -alkyl;
  • B is selected from hydrogen, hydroxy, optionally substituted C ⁇ - -alkoxy, optionally substituted C ⁇ - 4 -alkyl, optionally substituted C ⁇ _ 4 -acyloxy, optionally protected nucleobases;
  • L represents hydrogen or a hydroxy protection group; and M represents hydrogen or a hydroxy protection group.
  • A represents a phosphoramidite group of the general formula -0-P-(NR 8 R 8 )-R 9 , where R 8 and R independently are selected among linear or branched optional substituted C ⁇ _ 6 -alkyl and C ⁇ _ 6 -alkenyl, and R 9 is a phosphate protection group.
  • Each of the substituents R 1* , R 2* , R 3* ,R 4 , R 5 , R 5* , R 6 , R 6* , R 7 , and R 7 is independently selected from hydrogen, optionally substituted linear or branched C ⁇ _ ⁇ 2 -alkyl, optionally substituted linear or branched C 2 contend ⁇ 2 -alkenyl, optionally substituted linear or branched C - ⁇ 2 -alkynyl, hydroxy, C ⁇ - ⁇ 2 -alkoxy,
  • the compound of the formula II may also comprise basic salts and acid addition salts thereof.
  • the above intermediate may be referred to as " ⁇ -L-RNA amidite”.
  • the general formula II may comprise a 5- or 6-membered ring as a part of the nucleoside structure. Five membered rings are desirable in some applications because they are able to occupy essentially the same conformations as the native furanose ring of a naturally occurring nucleoside.
  • the embodiments, wherein X is selected from the group consisting of -0-, -S-, -S(O)-, -S(0) 2 -, and -N(R N* )- are desirable.
  • the aforementioned embodiment of the invention pertaining to use of -O- is interesting because the furanose ring is then identical to the one found in nature.
  • the intermediate wherein R , R , R , R , R , or R independently represent hydrogen, C ⁇ . 4 -alkyl or C ⁇ . 4 -alkoxy, notably hydrogen, is particularly desirable.
  • the hydroxy protection group M may be selected among any group having the ability to selectively protect a hydroxy group, e.g., a protecting group that ensures non-reactivity, in basic and neutral medium and which simultaneously has the ability to be removed (e.g., the ability to be removed in a suitable acidic medium) in preference to other protecting groups.
  • a protecting group that ensures non-reactivity, in basic and neutral medium and which simultaneously has the ability to be removed (e.g., the ability to be removed in a suitable acidic medium) in preference to other protecting groups.
  • the hydroxy protection group M is selected from trityl, 4,4'-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT); 9-(9-phenyl)xanthenyl (pixyl); ethoxycarbonyloxy, phenylazo-phenyloxycarbonyloxy, tetrahydropyranyl (THP), 9-fluorenyl-methoxycarbonyl (Fmoc), methoxytetrahydropyranyl (MTHP); trimethylsilyl (TMS), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triethylsilyl, phenyldimethylsilyl; benzyloxycarbonyl; 2- bromobenzyloxycarbonyl; tert-butyl ethers; methyl ether; acetals; acetyl; or halogen substituted
  • the protection group L may be selected among groups having the ability to selectively protect, e.g., ensure non-reactivity of, a hydroxy protecting group in certain acid or neutral media, while allowing for removal in an appropriate medium, desirably in preference to other protecting groups.
  • Desirable substituents L include 2'-0-triisopropylsilyloxymethyl (TOM) and 2'-0-t- butyldimethylsilyl (TBDMS).
  • TOM 2'-0-triisopropylsilyloxymethyl
  • TDMMS 2'-0-t- butyldimethylsilyl
  • TBDMS and TOM may be removed with tetrabutylammoniumfluoride (TBAF) or another suitable deprotecting reagent.
  • TBAF tetrabutylammoniumfluoride
  • the A and the OM group have changed places, i.e., the A substituent is arranged at the 5' position of the formula of the general formula II, and the OM substituent is at the 3' position.
  • A may be hydroxy or protected hydroxy
  • M may be -P-(NR 8 R 8* )-R 9 .
  • the substituent B comprises a variety of possible moieties, which may contain chemical groups reactive under the conditions prevailing in chemical oligonucleotide synthesis. Such reaction sensitive groups are desirably protected to keep side reactions to a mininum.
  • the nucleobase is suitably selected among cytosin-1-yl having the amino group at the 4 position protected, adenin-9-yl having the amino group at the 6 position protected, and gaunin-9-yl having the amino group at the 2 position.
  • the amino protecting group may be independently selected among acetyl (Ac), phenoxyacetyl (Pac), isopropylphenoxyacetyl (iPr-Pac), benzoyl (Bz), and dimethylformamidine (Dmf).
  • the optionally protected nucleobase is selected from the group consisting of 4-N-acetylcytosin-l-yl, 6-N-phenoxyacetyladenin-9-yl, 6-N-benzoyladenin-9-yl, 2-N-isopropylphenoxyacetylgaunin-9-yl, 2-N- dimethylform-amidinegaunin-9-yl, thymin- 1 -yl, 5-methyl-5-N-benzoyl- cytosine, 5-N-benzoylcytosine, and uracil- 1-yl.
  • the phosphate protection group R 9 is selected among groups having the ability to remain in position under acid conditions, while being removed under basic conditions. Desirable phosphate protection groups are selected from 2- cyanoethyloxy, 2,2,2-trichloro- 1 , 1 -dimethyl ethyloxy, j ⁇ -nitrophenylethyloxy, methoxy, methylthio, and allyloxy.
  • the invention provides a method for the synthesis of a population of nucleic acids (e.g., a population of nucleic acids of the invention) on a solid support.
  • This method involves the reaction of a plurality of nucleoside phosphoramidites with an activated solid support (e.g., a solid support with an activated linker) and the subsequent reaction of a plurality of nucleoside phosphoramidites with activated nucleotides or nucleic acids bound to the solid support.
  • an activated solid support e.g., a solid support with an activated linker
  • the solid support or the growing nucleic acid bound to the solid support is activated by illumination, a photogenerated acid, or electric current.
  • one or more spots or regions e.g., a region with an area of less than 1 cm 2 , 0.1 cm 2 ,
  • 0.01 cm , 1 mm , or 0.1 mm that desirably contains one particular nucleic acid monomer or oligomer) on the solid support are irradiated to produce a photogenerated acid that removes the 5'-OH protecting group of one or more nucleic acid monomers or oligomers to which a nucleotide is subsequently added.
  • an electric current is applied to one or more
  • 9 9 spots or regions e.g., a region with an area of less than 1 cm , 0.1 cm , 0.01
  • one or more spots or regions e.g., a region with an area of less than 1 cm 2 , 0.1 cm 2 ,
  • 0.01 cm , 1 mm , or 0.1 mm that desirably contains one particular nucleic acid monomer or oligomer) on the solid support are irradiated to remove a photosensitive protecting group of one or more nucleic acid monomers or oligomers to which a nucleotide is subsequently added.
  • the solid support e.g., chip, coverslip, microscope glass slide, quartz, or silicon
  • the process includes the steps of (a) providing a nucleoside unit attached to a solid support through a base-labile bond, the nucleoside being protected on the 5'-position with an acid-labile group; (b) treating the solid support, attached to a nucleoside according to step (a) or (g), with an acid to remove the 5'-protection group; (c) adding a proton donating activator and a successive nucleoside monomer comprising at the 3 '-position a phosphoramidite group and at the 5'- position an acid-labile protection group; (d) reacting the nucleoside attached to the solid support with the successive nucleoside phosphorami
  • one, more, or all hydroxy and exocyclic amino group(s) of the first nucleoside attached to the support, and any subsequently added nucleoside monomer may be protected. Desirably, all the groups prone to reaction under the conditions prevailing during the synthesis of the oligo-nucleotide are protected.
  • an acid is added to remove the 5' protection group, the nucleoside directly attached to the solid support or the nucleotide indirectly attached to the solid support through one or more nucleotide monomer(s), an acid is added. Desirably, the acid is 2,2-dichloroacetic acid or 2,2,2-trichloroacetic acid.
  • the reaction medium of step (b) is, in general, a non-aquaeous media, such as dichloromethane, trichloromethane, or toluene.
  • the proton donating activator of step (c) is desirably lH-tetrazole, 4,5-dicyano imidazole or a pyridinium salt.
  • any free hydroxy group is optionally capped.
  • the capping of 5'-hydroxy groups in step (e) is accomplished by reacting the 5'-hydroxy groups with acetic anhydride for the formation of acetyl groups.
  • N- methylimidazole is also present during the reaction.
  • the sequence of a cycle is usually terminated with an oxidation step to oxidize the phosphite group to the corresponding phosphate group.
  • a suitable oxidizing agent is iodine.
  • the invention features a pharmaceutical composition that includes one or more of the nucleic acids and/or alpha-L-RNA monomers of the invention (e.g., an ⁇ -L-RNA nucleoside or nucleotide) and a pharmaceutically acceptable carrier, such as one of the carriers described herein.
  • a pharmaceutically acceptable carrier such as one of the carriers described herein.
  • the invention features a population of two or more nucleic acids of the invention.
  • the populations of nucleic acids of the invention may contain any number of unique molecules.
  • the population may contain as few as 10, 10 2 , 10 4 , or 10 5 unique molecules or as many as 10 , 10 , 10 or more unique molecules.
  • at least 1, 5, 10, 50, 100 or more of the polynucleotide sequences are a non- naturally-occurring sequence.
  • at least 20, 40, or 60% of the unique polynucleotide sequences are non-naturally-occurring sequences.
  • the nucleic acids are all the same length; however, some of the molecules may differ in length. Desirable Nucleic Acids for Any of the Above Aspects
  • the length of one or more nucleic acids is between 15 and 150 nucleotides, 5 and 100 nucleotides, 20 and 80 nucleotides, or 30 and 60 nucleotides in length, inclusive.
  • the nucleic acid is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 nucleotides or at least 60, 70, 80, 90, 100, 120, or 130 nucleotides in length.
  • the oligonucleotide has one or more nucleotides with universal bases.
  • nucleotides with universal bases can be used to increase the thermal stability of the oligonucleotides.
  • the oligonucleotide has a universal base located at the 5' or 3' terminus of the nucleic acid.
  • one or more (e.g., 2, 3, 4, 5, 6, or more) universal bases are located at the 5' and 3' termini of the oligonucleotide. Desirably, all of the oligonucleotides in the population have the same number of universal bases.
  • Desirable universal bases include inosine, 3- nitropyrrole, 5-nitroindole, pyrene and pyridyloxazole derivatives, pyrenyl, pyrenylmethylglycerol moieties, pyrrole, diazole or triazole moieties, all of which may be optionally substituted, and other groups e.g.
  • Other desirable universal bases include, pyrrole, diazole or triazole moieties, all of which may be optionally substituted.
  • the oligonucleotide has one or more nucleotides with universal bases.
  • nucleotides with universal bases can be used to increase the thermal stability of the oligonucleotides.
  • the oligonucleotide has a universal base located at the 5' or 3' terminus of the nucleic acid.
  • one or more (e.g., 2, 3, 4, 5, 6, or more) universal bases are located at the 5' and 3' termini of the oligonucleotide. Desirably, all of the oligonucleotides in the population have the same number of universal bases.
  • Desirable universal bases include inosine, 3-nitropyrrole, 5-nitroindole, pyrene and pyridyloxazole derivatives, pyrenyl, pyrenylmethylglycerol moieties, pyrrole, diazole or triazole moieties, all of which may be optionally substituted, and other groups e.g.
  • Other desirable universal bases include, pyrrole, diazole or triazole moieties, all of which may be optionally substituted.
  • the nucleic acids are covalently bonded to a solid support.
  • the nucleic acids are in a predefined arrangement.
  • the first population has at least 10; 100; 1,000; 5,000; 10,000; 100,000; or 1,000,000 different nucleic acids.
  • Desirable LNA units have a carbon or hetero alicyclic ring with four to six ring members, e.g. a furanose ring, or other alicyclic ring structures such as a cyclopentyl, cycloheptyl, tetrahydropyranyl, oxepanyl, tetrahydrothiophenyl, pyrrolidinyl, thianyl, thiepanyl, piperidinyl, and the like.
  • at least one ring atom of the carbon or hetero alicyclic group is taken to form a further cyclic linkage to thereby provide a multi-cyclic group.
  • the cyclic linkage may include one or more, typically two atoms, of the carbon or hetero alicyclic group.
  • the cyclic linkage also may include one or more atoms that are substituents, but not ring members, of the carbon or hetero alicyclic group.
  • Other desirable LNA units are compounds having a substituent on the 2'- position of the central sugar moiety (e.g., ribose or xylose), or derivatives thereof, which favors the C3'-endo conformation, commonly referred to as the North (or simply N for short) conformation.
  • These LNA units include ENA (2'-0,4'-C-ethylene-bridged nucleic acids such as those disclosed in WO 00/47599) units as well as non-bridged riboses such as 2'-F or 2'-0-methyl.
  • the invention features a method for detecting the presence of one or more target nucleic acids in a sample.
  • This method involves incubating a nucleic acid sample with one or more nucleic acids of the invention under conditions that allow at least one target nucleic acid to hybridize to at least one of the nucleic acids of the invention. Desirably, hybridization is detected for at least 2, 3, 4, 5, 6, 8, 10, or 12 target nucleic acids.
  • the method further includes contacting the target nucleic acid with a second nucleic acid or a population of second nucleic acids that binds to a different region of the target molecule than the first nucleic acid.
  • the method further involves identifying one or more hybridized target nucleic acids and/or determining the amount of one or more hybridized target nucleic acids.
  • the method further includes determining the presence or absence of nucleic acid of interest (e.g., an mRNA) in the sample and/or determining the presence or absence of a mutation, deletion, and/or duplication in the nucleic acid of interest.
  • the mutation, deletion, and/or duplication is indicative of a disease, disorder, or condition, such as cancer.
  • the invention features a method of detecting a nucleic acid of a pathogen (e.g., a nucleic acid in a sample such as a blood or urine sample from a mammal).
  • a nucleic acid probe of the invention e.g., a probe specific for an mRNA from a particular pathogen or family of pathogens
  • the probe is desirably at least 60, 70, 80, 90, 95, or 100% complementary to a nucleic acid of a pathogen (e.g., a bacteria, virus, or yeast such as any of the pathogens described herein).
  • Hybridization between the probe and a nucleic acid in the sample is detected, indicating that the sample contains the corresponding nucleic acid from a pathogen.
  • the method is used to determine what strain of a pathogen has infected a mammal (e.g., a human) by determining whether a particular nucleic acid is present in the sample.
  • the probe has a universal base in a position corresponding to a nucleotide that varies among different strains of a pathogen, and thus the probe detects the presence of a nucleic acid from any of a multiple of pathogenic strains.
  • the method also includes identifying the hybridized target nucleic acid and/or determining the amount of hybridized target nucleic acid.
  • the target nucleic acids are labeled with a fluorescent group.
  • the first nucleic acid standard is labeled with a different fluorescent group. The fluorescence of the target nucleic acids and the first nucleic acid standard can be detected simultaneously or sequentially.
  • the invention features a method for amplifying a target nucleic acid molecule.
  • the method involves (a) incubating a first nucleic acid of the invention with a target nucleic acid under conditions that allow the first nucleic acid to bind the target nucleic acid; and (b) extending the first nucleic acid with the target nucleic acid as a template.
  • the method further involves contacting the target nucleic acid with a second nucleic acid (e.g., a second nucleic acid of the invention) that binds to a different region of the target nucleic acid than the first nucleic acid.
  • the sequence of the target nucleic acid is known or unknown.
  • oligonucleotides which are complementary to a specific target nucleic acid (e.g., an mRNA).
  • a specific target nucleic acid e.g., an mRNA
  • oligonucleotides with a modified backbone such as LNA or phosphorothioate
  • the invention provides a method for inhibiting the expression of a target nucleic acid in a cell.
  • the method involves introducing into the cell a nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid.
  • the introduced nucleic acid has a nucleotide sequence that is essentially complementary to a region of desirably at least 20 nucleotides of the target nucleic acid.
  • the cell is in a human.
  • the invention provides a method for preventing, stabilizing, or treating a disease, disorder, or condition associated with a target nucleic acid in a mammal (e.g., a human patient).
  • This method involves introducing into the mammal (e.g., a human patient) a nucleic acid of the invention in an amount sufficient to specifically attenuate expression of the target nucleic acid, wherein the introduced nucleic acid has a nucleotide sequence that is essentially complementary to a region of desirably at least 20 nucleotides of the target nucleic acid.
  • the invention provides a method for preventing, stabilizing, or treating a pathogenic infection in a human patient by introducing into said patient a nucleic acid of the invention in an amount sufficient to specifically attenuate expression of a target nucleic acid of a pathogen.
  • the introduced nucleic acid has a nucleotide sequence that is essentially complementary to a region of desirably at least 20 nucleotides of the target nucleic acid.
  • the oligonucleotide according to the present invention may be used for a variety of applications.
  • the ability of the nucleotide to discriminate between DNA and RNA enhances its use for antisense or other gene silencing technology.
  • the present invention therefore includes the use of an oligonucleotide according to the invention for the manufacture of a pharmaceutical composition for the treatment, stabilization, or prevention of a disease, disorder, or infection.
  • the invention includes a method of treating a subject having, or suspected of having, a disease, disorder, or infection comprising administering to the subject an amount of an oligonucleotide of the invention that which is effective to treat, stabilize, or prevent the disease, disorder, or infection.
  • oligonucleotide of the present invention Most desirable is the treatment of human disease by the oligonucleotide of the present invention.
  • exemplary conditions that can be treated or prevented in humans include acute hepatic failure, autoimmune disorders, blood disorders, bone disorders, cancer, including bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, cancer of the head and neck, lung cancer, metastatic cancer, liver cancer, leukemia, ovarian cancer, prostate cancer, renal cell carcinoma, sarcoma, and skin cancer, cardiovascular disease, gastrointestinal tract disorders, infectious disease, including HIN inherited autosomal disease, mesothelioma, myopathies, neurological disorders, and neuropathy.
  • the introduced nucleic acid is single stranded or double stranded.
  • nucleic acids may be administered in a single dose or multiple doses.
  • the doses may be separated from one another by, for example, one week, one month, one year, or ten years. It is to be understood that, for any particular subject, specific dosage regimes should be adjusted over time according to the individual need and the 0 professional judgment of the person administering or supervising the administration of the compositions.
  • Optimum dosages for gene silencing applications may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC 50 values found to be effective in in vitro and in vivo 5 animal models. In general, dosage is from 0.001 ug to 100 g per kg of body weight (e.g., 0.001 ug/kg to 1 g/kg), and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years (U.S.P.N. 6,440,739). Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the o drug in bodily fluids or tissues.
  • oligonucleotide is administered in maintenance doses, ranging from 0.001 ug to 100 g per kg of body weight (e.g., 0.001 ug/kg to 1 g/kg), once or more daily, to once every 20 years.
  • 5 conventional treatments may be used in combination with the nucleic acids of the present invention.
  • Suitable carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the composition can be adapted for the mode of administration and can be in the form of, for example, a pill, tablet, capsule, spray, powder, or liquid.
  • the pharmaceutical composition contains one or more pharmaceutically acceptable additives suitable for the selected route and mode of administration.
  • compositions may be administered by, without limitation, any parenteral route including intravenous, intra-arterial, intramuscular, subcutaneous, intradermal, intraperitoneal, intrathecal, as well as topically, orally, and by mucosal routes of delivery such as intranasal, inhalation, rectal, vaginal, buccal, and sublingual.
  • the pharmaceutical compositions of the invention are prepared for administration to vertebrate (e.g., mammalian) subjects in the form of liquids, including sterile, non- pyrogenic liquids for injection, emulsions, powders, aerosols, tablets, capsules, enteric coated tablets, or suppositories.
  • the invention features a method of modulating the ability of a target oligonucleotide to act as a substrate for one or more nucleic acid enzymes.
  • This method involves hybridizing an oligonucleotide of invention to a target oligonucleotide.
  • one or more ⁇ -L- RNA monomers with the oligonucleotide modulate the ability of the oligonucleotide to act as a substrate for nucleic acid active enzymes.
  • the oligonucleotide has a region with substantial complementarity to a target oligonucleotide and also has a region of nucleotides that recruits a nucleic acid active enzyme, such as RNaseRNase H.
  • a nucleic acid active enzyme such as RNaseRNase H.
  • the oligonucleotide of the invention consists of DNA monomers and at least one ⁇ - L-RNA monomer and the target oligonucleotide is a single-stranded or double- stranded RNA sequence, the hybridization of which by the oligonucleotide of the invention results in a sequence-specific strand displacement.
  • the oligonucleotide of the invention is 5 -d(GTC TCT A( ⁇ L U)G GAC CT), 5 -d(GTC ( ⁇ L U)CT ATG GAC CT), or 5 -d(G( ⁇ L U)C TCT ATG GAC CT).
  • the combination of the selective hybridization of the oligonucleotide of the invention to a target gene to be silenced and the decomposition of the complementary strand by RNase H can be a powerful tool for antisense applications.
  • RNA e.g., mRNA
  • an oligonucleotide that has a region with substantial complementarity to a corresponding region in an RNA of interest is contacted with the RNA of interest under conditions that allow hybridization between the oligonucleotide and the RNA of interest.
  • the hybridized RNA is isolated.
  • the isolation step includes a method selected from the group consisting of filtration, affinity chromatography, ion exchange chromatography, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, chromatofocusing, centrifugation, high pressure liquid chromatography, and dialysis.
  • the oligonucleotide of the invention is bound to a solid support.
  • a one-step recovery of the mRNA from a sample e.g., as disclosed in US patent No. 6,303,315 (One step sample preparation and detection of nucleic acids in complex biological samples) which is incorporated herein by reference.
  • the invention features a method for binding an oligonucleotide to a target squence in a dsRNA molecule.
  • An oligonucleotide of the invention is contacted with a dsRNA molecule under conditions that allow the oligonucleotide to hybridize or bind to the dsRNA molecule by way of strand displacement or triple helix formation.
  • the resistance of an analyzing or purification system to degradation by one or more nucleic acid-active enzymes is increased by administering an oligonucleotide of the invention to the analyzing or purification system.
  • the selective hybridization of the oligonucleotides of the invention to RNA can also be used to detect one or more mismatches in a target RNA sequence when the oligonucleotide comprises a region with substantial complementarity to the corresponding region in the target RNA and, when hybridized to the target RNA oligonucleotide having one or more mismatches, exhibits a reduction in T m compared to the T m obtained for the hybridization of the oligonucleotide of the invention to an RNA oligonucleotide that has 100% complementarity.
  • Oligonucleotides have been described not only as catalysts in self- modififying biological reactions, but also in such diverse reactions a Diels- Alder reactions, glycosidic bond formations, alkylations, acylations, amide bond formations, or in the hydrolysis of phosphodiester linkages or bonds.
  • the oligonucleotides of the invention can be used as a catalyst in a biological or chemical process (e.g., a catalyst for the hydrolysis of a phosphodiester linkage or bond).
  • the invention provides a method for catalyzing a biological or chemical reaction. This method involves administering an oligonucleotide of the invention to a reaction mixture in an amount sufficient to increase the rate of the reaction.
  • a nucleic acid probe or primer specifically hybridizes to a target nucleic acid but does not substantially hybridize to non-target molecules, which include other nucleic acids in a cell or biological sample having a sequence that is less than 99, 95, 90, 80, or 70% identical or complementary to that of the target nucleic acid.
  • the amount of the these non-target molecules hybridized to, or associated with, the nucleic acid probe or primer, as measured using standard assays is 2-fold, desirably 5-fold, more desirably 10-fold, and most desirably 50-fold lower than the amount of the target nucleic acid hybridized to, or associated with, the nucleic acid probe or primer.
  • the amount of a target nucleic acid hybridized to, or associated with, the nucleic acid probe or primer, as measured using standard assays is 2-fold, desirably 5- fold, more desirably 10-fold, and most desirably 50-fold greater than the amount of a control nucleic acid hybridized to, or associated with, the nucleic acid probe or primer.
  • the nucleic acid probe or primer RNA is substantially complementary (e.g., at least 80, 90, 95, 98, or 100% complementary) to a target nucleic acid or a group of target nucleic acids from a cell.
  • the probe or primer is homologous to multiple RNA or DNA molecules, such as RNA or DNA molecules from the same gene family. In other embodiments, the probe or primer is homologous to a large number of RNA or DNA molecules. In desirable embodiments, the probe or primer binds to nucleic acids which have polynucleotide sequences that differ in sequence at a position that corresponds to the position of a universal base in the probe or primer. Examples of control nucleic acids include nucleic acids with a random sequence or nucleic acids known to have little, if any, affinity for the nucleic acid probe or primer. In some embodiments, the target nucleic acid is an RNA, DNA, or cDNA molecule.
  • LNA Locked Nucleoside Analogues
  • nucleoside analogues e.g., bicyclic nucleoside analogues, e.g., as disclosed in WO 9914226
  • LNA nucleoside and LNA nucleotide e.g., LNA nucleoside and LNA nucleotide.
  • monomeric LNA may, e.g., refer to the monomers LNA A, LNA T, LNA C, or any other LNA monomers.
  • LNA unit is meant an individual LNA monomer (e.g., an LNA nucleoside or LNA nucleotide) or an oligomer (e.g., an oligonucleotide or nucleic acid) that includes at least one LNA monomer.
  • LNA units as disclosed in WO 99/14226,WO 0056746, WO 0056748, and WO 0066604 are in general particularly desirable modified nucleic acids for incorporation into an oligonucleotide of the invention and includes ENA (2'0,4'C-ethylene-bridged nucleic acids). Additionally, the nucleic acids may be modified at either the 3 ' and/or 5' end by any type of modification known in the art.
  • either or both ends may be capped with a protecting group, attached to a flexible linking group, attached to a reactive group to aid in attachment to the substrate surface, etc.
  • Desirable LNA units and their method of synthesis also are disclosed in WO 0056746, WO 0056748, WO 0066604, Morita et al, Bioorg. Med. Chem. Lett. 12(l):73-76, 2002; Hakansson et al, Bioorg. Med. Chem. Lett. ll(7):935-938, 2001; Koshkin et al, J. Org. Chem. 66(25):8504- 8512, 2001; Kvaerno et al, J. Org. Chem.
  • Exemplary 5', 3', and/or 2' terminal groups include -H, -OH, halo (e.g., chloro, fluoro, iodo, or bromo), optionally substituted aryl, (e.g., phenyl or benzyl), alkyl (e.g, methyl or ethyl), alkoxy (e.g., methoxy), acyl (e.g.
  • acetyl or benzoyl aroyl, aralkyl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, nitro, cyano, carboxy, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acylamino, aroylamine, alkylsulfonyl, arylsulfonyl, heteroarylsulfonyl, alkylsulfinyl, arylsulfinyl, heteroarylsulfinyl, alkylthio, arylthio, heteroarylthio, aralkylthio, heteroaralkylthio,amidino, amino, carbamoyl, sulfamoyl, alkene, alkyne, protecting groups (e.g., silyl, 4,4'-dimethoxytrityl, monomethoxytrityl, or trity
  • a “modified base” or other similar term refers to a composition (e.g., a non-naturally occuring nucleobase or nucleosidic base) which can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine) and/or can pair with a non-narurally occurring nucleobase or nucleosidic base.
  • the modified base provides a T m differential of 15, 12, 10, 8, 6, 4, or 2°C or less as described herein.
  • Exemplary modified bases are described in EP 1 072 679 and WO 97/12896.
  • nucleobase is meant the naturally occurring nucleobases adenine
  • nucleobases such as xanthine, diaminopurine, 8-oxo-N - methyladenine, 7-deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 - ethano-2,6-diaminopurine, 5-methylcytosine (mC), 5-(C -C )-alkynyl-cytosine, 5-fluorouracil, 5-bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4- triazolopyridin, isocytosine, isoguanine, inosine and the "non-narurally occurring" nucleobases described in Benner et al., U.S.
  • nucleobase thus includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof. Further naturally and non-naturally occurring nucleobases include those disclosed in U.S. Pat. No. 3,687,808 (Merigan, et al.), in Chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T. Crooke and B. Lebleu, CRC Press, 1993, in Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613-722 (see especially pages 622 and 623, and in the
  • nucleosidic base or “base unit” is further intended to include compounds such as heterocyclic compounds that can serve like nucleobases including certain "universal bases” that are not nucleosidic b.ases in the most classical sense but serve as nucleosidic bases.
  • universal bases are 3-nitropyrrole, optionally substituted indoles (e.g., 5-nitroindole), and optionally substituted hypoxanthine.
  • Other desirable universal bases include, pyrrole, diazole or triazole derivatives, including those universal bases known in the art.
  • a "substituted" group such as a nucleobase or nucleosidic base and the like may be substituted by other than hydrogen at one or more available positions, typically 1 to 3 or 4 positions, by one or more suitable groups such as those disclosed herein.
  • suitable groups that may be present on a "substituted” group include e.g.
  • alkanoyl such as a C ⁇ _ 6 alkanoyl group such as acyl and the like; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5, or 6 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to 12 carbon, or 2, 3, 4, 5 or 6 carbon atoms; alkoxy groups including those having one or more oxygen linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms, or 1, 2, 3, 4, 5 or 6 carbon atoms; alkylsulfinyl groups including those moieties having one or more s
  • universal base is meant a naturally-occurring or desirably a non- naturally occurring compound or moiety that can pair with a natural base (e.g., adenine, guanine, cytosine, uracil, and/or thymine), and that has a T m differential of 15, 12, 10, 8, 6, 4, or 2°C or less as described herein.
  • a natural base e.g., adenine, guanine, cytosine, uracil, and/or thymine
  • T m is used in reference to the "melting temperature.”
  • the melting temperature is the temperature at which 50% of a population of double- stranded nucleic acid molecules becomes dissociated into single strands.
  • the equation for calculating the T m of nucleic acids is well-known in the art.
  • a nucleic acid compound that has a T m differential of a specified amount means the nucleic acid exhibits that specified T m differential when incorporated into a specified 9-mer oligonucleotide with respect to the four complementary variants, as defined immediately below.
  • a T m differential provided by a particular modified base is calculated by the following protocol (steps a) through d)): a) incorporating the modified base of interest into the following oligonucleotide 5'-d(GTGAMATGC), wherein M is the modified base; b) mixing 1.5 x 10 "6 M of the oligonucleotide having incorporated therein the modified base with each of 1.5x10 "6 M of the four oligonucleotides having the sequence 3'-d(CACTYTACG), wherein Y is A, C, G, T, respectively, in a buffer of lOmM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0; c) allowing the oligonucleotides to hybridize; and d) detecting the T m for each of the four hybridized nucleotides by heating the hybridized nucleotides and observing the temperature at which the maximum of the first derivative of the melting
  • a T m differential for a particular modified base is determined by subtracting the highest T m value determined in steps a) through d) immediately above from the lowest T m value determined by steps a) through d) immediately above.
  • Monomers are referred to as being "complementary” if they contain nucleobases that can form hydrogen bonds according to Watson-Crick base- pairing rales (e.g., G with C, A with T, or A with U) or other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, and pseudoisocytosine with G.
  • Watson-Crick base- pairing rales e.g., G with C, A with T, or A with U
  • other hydrogen bonding motifs such as for example diaminopurine with T, inosine with C, and pseudoisocytosine with G.
  • substantially complementarity is meant having a sequence that is at least 60, 70, 80, 90, 95, or 100% complementary to that of another sequence. Sequence complementarity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.
  • the term “homology” refers to a degree of complementarity. There can be partial homology or complete homology (i.e., identity). A partially complementary sequence that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid is referred to using the functional term "substantially homologous.”
  • substantially homologous refers to a probe that can hybridize to a strand of the double-stranded nucleic acid sequence under conditions of low stringency, e.g. using a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.
  • SSC sodium citrate
  • substantially homologous refers to a probe that can hybridize to (i.e., is the complement of) the single-stranded nucleic acid template sequence under conditions of low stringency, e.g. using a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.
  • a hybridization buffer comprising 20% formamide in 0.8M saline/0.08M sodium citrate (SSC) buffer at a temperature of 37°C and remaining bound when subject to washing once with that SSC buffer at 37°C.
  • corresponding unmodified reference nucleobase is meant a nucleobase that is not part of an LNA unit and is in the same orientation as the nucleobase in an LNA unit.
  • mutation is meant an alteration in a naturally-occurring or reference nucleic acid sequence, such as an insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or missense mutation.
  • the amino acid sequence encoded by the nucleic acid sequence has at least one amino acid alteration from a naturally-occurring sequence.
  • selecting substantially partitioning a molecule from other molecules in a population.
  • the partitioning provides at least a 2-fold, desirably, a 30-fold, more desirably, a 100-fold, and most desirably, a 1, 000- fold enrichment of a desired molecule relative to undesired molecules in a population following the selection step.
  • the selection step may be repeated a number of times, and different types of selection steps may be combined in a given approach.
  • the population desirably contains at least 10 9 molecules, more desirably at least 10 11 , 10 13 , or 10 14 molecules and, most desirably, at least 10 15 molecules.
  • a “population” is meant more than one nucleic acid.
  • a “population” according to the invention desirably means more than 10 1 , 10 2 , 10 3 , or 10 4 different molecules .
  • target nucleic acid or “nucleic acid target” is meant a particular nucleic acid sequence of interest.
  • the “target” can exist in the presence of other nucleic acid molecules or within a larger nucleic acid molecule.
  • solid support any rigid or semi-rigid material to which a nucleic acid binds or is directly or indirectly attached.
  • the support can be any porous or non-porous water insoluble material, including without limitation, membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, strips, plates, rods, polymers, particles, microparticles, capillaries, and the like.
  • the support can have a variety of surface forms, such as wells, trenches, pins, channels and pores.
  • an “array” is meant a fixed pattern of at least two different immobilized nucleic acids on a solid support. Desirably, the array includes at least 10 2 , more desirably, at least 10 3 , and, most desirably, at least 10 4 different nucleic acids.
  • antisense nucleic acid is meant a nucleic acid, regardless of length, that is complementary to a coding strand or mRNA of interest. In some embodiments, the antisense molecule inhibits the expression of only one nucleic acid, and in other embodiments, the antisense molecule inhibits the expression of more than one nucleic acid.
  • the antisense nucleic acid decreases the expression or biological activity of a nucleic and or encoded protein by at least 20, 40, 50, 60, 70, 80, 90, 95, or 100%.
  • An antisense molecule can be introduced, e.g., to an individual cell or to whole animals, for example, it may be introduced systemically via the bloodstream.
  • a region of the antisense nucleic acid or the entire antisense nucleic acid is at least 70, 80, 90, 95, 98, or 100% complimentary to a coding sequence, regulatory region (5' or 3' untranslated region), or an mRNA of interest.
  • the region of complementarity includes at least 5, 10, 20, 30, 50, 75,100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in the antisense nucleic acid.
  • the antisense molecule is less than 200, 150, 100, 75, 50, or 25 nucleotides in length. In other embodiments, the antisense molecule is less than 50,000; 10,000; 5,000; or 2,000 nucleotides in length. In certain embodiments, the antisense molecule is at least 200, 300, 500, 1000, or 5000 nucleotides in length.
  • the number of nucleotides in the antisense molecule is contained in one of the following ranges: 5-15 nucleotides, 16-20 nucleotides, 21-25 nucleotides, 26-35 nucleotides, 36-45 nucleotides, 46-60 nucleotides, 61-80 nucleotides, 81-100 nucleotides, 101-150 nucleotides, or 151-200 nucleotides, inclusive.
  • the antisense molecule may contain a sequence that is less than a full-length sequence or may contain a full-length sequence.
  • double stranded nucleic acid is meant a nucleic acid containing a region of two or more nucleotides that are in a double stranded conformation.
  • the double stranded nucleic acids consists entirely of LNA units or a mixture of LNA units, ribonucleotides, and/or deoxynucleotides.
  • the double stranded nucleic acid may be a single molecule with a region of self-complimentarity such that nucleotides in one segment of the molecule base-pair with nucleotides in another segment of the molecule.
  • the double stranded nucleic acid may include two different strands that have a region of complimentarity to each other.
  • the regions of complimentarity are at least 70, 80, 90, 95, 98, or 100% complimentary.
  • the region of the double stranded nucleic acid that is present in a double stranded conformation includes at least 5, 10, 20, 30, 50, 75,100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in the double stranded nucleic acid.
  • Desirable double stranded nucleic acid molecules have a strand or region that is at least 70, 80, 90, 95, 98, or 100% identical to a coding region or a regulatory sequence (e.g., a transcription factor binding site, a promoter, or a 5' or 3' untranslated region) of a nucleic acid of interest.
  • the double stranded nucleic acid is less than 200, 150, 100, 75, 50, or 25 nucleotides in length. In other embodiments, the double stranded nucleic acid is less than 50,000; 10,000; 5,000; or 2,000 nucleotides in length.
  • the double stranded nucleic acid is at least 200, 300, 500, 1000, or 5000 nucleotides in length.
  • the number of nucleotides in the double stranded nucleic acid is contained in one of the following ranges: 5-15 nucleotides, 16-20 nucleotides, 21-25 nucleotides, 26-35 nucleotides, 36-45 nucleotides, 46-60 nucleotides, 61-80 nucleotides, 81-100 nucleotides, 101-150 nucleotides, or 151-200 nucleotides, inclusive.
  • the double stranded nucleic acid may contain a sequence that is less than a full-length sequence or may contain a full-length sequence.
  • the double stranded nucleic acid inhibits the expression of only one nucleic acid, and in other embodiments, the double stranded nucleic acid molecule inhibits the expression of more than one nucleic acid.
  • the nucleic acid decreases the expression or biological activity of a nucleic acid of interest or a protein encoded by a nucleic acid of interest by at least 20, 40, 50, 60, 70, 80, 90, 95, or 100%.
  • a double stranded nucleic acid can be introduced, e.g., to an individual cell or to whole animals, for example, it may be introduced systemically via the bloodstream.
  • the double stranded nucleic acid or antisense molecule includes one or more LNA nucleotides, one or more universal bases, and/or one or more modified nucleotides in which the 2' position in the sugar (e.g., riobse or xylose) contains a halogen (such as flourine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the double stranded nucleic acid or antisense molecule in vitro or in vivo compared to the corresponding double stranded nucleic acid or antisense molecule in which the corresponding 2' position contains a hydrogen or an hydroxyl group.
  • a halogen such as flourine group
  • alkoxy group such as a methoxy group
  • the double stranded nucleic acid or antisense molecule includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage.
  • linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages.
  • the double strandwd or antisense molecule is purified.
  • a factor is substantially pure when it is at least 50%, by weight, free from proteins, antibodies, and naturally-occurring organic molecules with which it is naturally associated. Desirably, the factor is at least 75%, more desirably, at least 90%, and most desirably, at least 99%, by weight, pure.
  • a substantially pure factor may be obtained by chemical synthesis, separation of the factor from natural sources, or production of the factor in a recombinant host cell that does not naturally produce the factor.
  • Nucleic acids and proteins may be purified by one skilled in the art using standard techniques such as those described by Ausubel et al (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 2000).
  • the factor is desirably at least 2, 5, or 10 times as pure as the starting material, as measured using polyacrylamide gel electrophoresis, column chromatography, optical density, HPLC analysis, or western analysis (Ausubel et al, supra).
  • Desirable methods of purification include immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immunoaffinity purification, and panning with a plate-bound antibody.
  • treating, stabilizing, or preventing a disease, disorder, or condition is meant preventing or delaying an initial or subsequent occurrence of a disease, disorder, or condition; increasing the disease-free survival time between the disappearance of a condition and its reoccurrence; stabilizing or reducing an adverse symptom associated with a condition; or inhibiting or stabilizing the progression of a condition.
  • at least 20, 40, 60, 80, 90, or 95% of the treated subjects have a complete remission in which all evidence of the disease disappears.
  • the length of time a patient survives after being diagnosed with a condition and treated with a nucleic acid of the invention is at least 20, 40, 60, 80, 100, 200, or even 500% greater than (i) the average amount of time an untreated patient survives or (ii) the average amount of time a patient treated with another therapy survives.
  • treating, stabilizing, or preventing cancer is meant causing a reduction in the size of a tumor, slowing or preventing an increase in the size of a tumor, increasing the disease-free survival time between the disappearance of a tumor and its reappearance, preventing an initial or subsequent occurrence of a tumor, or reducing an adverse symptom associated with a tumor.
  • the number of cancerous cells surviving the treatment is at least 20, 40, 60, 80, or 100% lower than the initial number of cancerous cells, as measured using any standard assay.
  • the decrease in the number of cancerous cells induced by administration of a nucleic acid of the invention is at least 2, 5, 10, 20, or 50- fold greater than the decrease in the number of non-cancerous cells.
  • the number of cancerous cells present after administration of a nucleic acid of the invention is at least 2, 5, 10, 20, or 50- fold lower than the number of cancerous cells present prior to the administration of the compound or after administration of a buffer control.
  • the methods of the present invention result in a decrease of 20, 40, 60, 80, or 100% in the size of a tumor as determined using standard methods.
  • At least 20, 40, 60, 80, 90, or 95% of the treated subjects have a complete remission in which all evidence of the cancer disappears.
  • the cancer does not reappear or reappears after at least 5, 10, 15, or 20 years.
  • Exemplary cancers that can be treated, stabilized, or prevented using the above methods include prostate cancers, breast cancers, ovarian cancers, pancreatic cancers, gastric cancers, bladder cancers, salivary gland carcinomas, gastrointestinal cancers, lung cancers, colon cancers, melanomas, brain tumors, leukemias, lymphomas, and carcinomas. Benign tumors may also be treated or prevented using the methods and nucleic acids of the present invention.
  • infection is meant the invasion by a pathogen (e.g., a bacteria, yeast, or virus).
  • a pathogen e.g., a bacteria, yeast, or virus.
  • At bacterial infection may be due to gram positive and/or gram negative bacteria.
  • the bacterial infection is due to one or more of the following bacteria: Chlamydophila pneumoniae, C. psittaci, C. abortus, Chlamydia trachomatis, Simkania negevensis, Parachlamydia acanthamoebae, Pseudomonas aeruginosa, P. alcaligenes, P. chlororaphis, P. fluorescens, P. luteola, P. mendocina, P.
  • a pathogen e.g., a bacteria, yeast, or virus.
  • ducreyi Pasteurella multocida, P. haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, C.jejuni, C. coli, Borrelia burgdorferi, V. cholerae, V. parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhea, N. meningitidis, Kingella dentrificans, K. kingae, K. oralis, Moraxella catarrhalis, M. atlantae, M. lacunata, M. nonliquefaciens, M. osloensis, M.
  • a nucleic acid is administered in an amount sufficient to prevent, stabilize, or inhibit the growth of a pathogenic bacteria or to kill the bacteria.
  • the viral infection relevant to the methods of the invention is an infection by one or more of the following viruses: West Nile virus (e.g., Samuel, “Host genetic variability and West Nile virus susceptibility,” Proc. Natl. Acad. Sci.
  • West Nile virus e.g., Samuel, "Host genetic variability and West Nile virus susceptibility,” Proc. Natl. Acad. Sci.
  • Figure 1 is a gel electrophoresis that represents the ability of RNase H (from E. coli) to cleave the RNA strand of a duplex between oligomers containing a single ⁇ -L-RNA monomer and complementary RNA strand.
  • the hybrid containing compound 34 [5'-d(GTC TCT A( ⁇ L U)G GAC CT)] can be found in lanes marked "1" on the gel
  • the hybrid containing compound 35 [5'- d(GTC ( ⁇ L U)CT ATG GAC CT)] can be found in lanes marked "2" on the gel
  • the hybrid containing compound 36 [5'-d(G( ⁇ L U)C TCT ATG GAC CT)] can be found in lanes marked "3" on the gel.
  • the lanes marked with an asterisk were control experiments in which no RNase was used.
  • the figure shows that the modified oligonucleotides 34-36 support RNase H cleavage when hybridized to the 32 P-labelled complementary RNA sequence, with oligomer 34 degraded less efficiently than oligomers 35 and 36.
  • the RNA sequence is 5'-r(AGG UCC AUA GAG AC).
  • the DNA reference sequence is
  • FIG. 1 shows the reaction scheme for the preparation of compounds 21, 22
  • Figure 3 shows the reaction scheme for the preparation of compounds 24, 25, 26, 28, 30 and 32.
  • Figure 4 shows the reaction scheme for the preparation of compounds 38, 39, 40, 41, 42, 43, and 44.
  • Figure 5 shows the structure of an exemplary compound of the invention.
  • Figure 6 shows the structures of selected nucleotide monomers: DNA (T), LNA (T L ), pyrene DNA (Py), 2'-OMe-RNA [2'-OMe(T)] 3 abasic LNA, phenyl LNA, and pyrenyl LNA.
  • Other exemplary universal bases and methods for synthesizing them are disclosed in U.S.S.N. 10/235,683 and IB02/03911, which are hereby incorporated by reference.
  • the nucleotide monomers forming part of the oligonucleotide of the present invention comprises a moiety B.
  • the substituent B may designate a group which, when the oligomer is complexing with DNA or RNA, is able to interact (e.g., by hydrogen bonding or covalent bonding or electronic interaction) with DNA or RNA, especially nucleobases of DNA or RNA.
  • the substituent B may designate a group which acts as a label or a reporter, or the substituent B may designate a group (e.g., hydrogen) which is expected to have little or no interactions with DNA or RNA.
  • the substituent B is desirably selected from hydrogen, hydroxy, optionally substituted C ⁇ _ 4 -alkoxy, optionally substituted C ⁇ profession -alkyl, optionally substituted C ⁇ _ -acyloxy, nucleobases, DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands.
  • nucleobase covers naturally occurring nucleobases as well as non-naturally occurring nucleobases.
  • nucleobase includes not only the known purine and pyrimidine heterocycles, but also heterocyclic analogues and tautomers thereof.
  • nucleobases are adenine, guanine, thymine, cytosine, uracil, purine, xanthine, diaminopurine, 8-oxo-N 6 -methyladenine, 7- deazaxanthine, 7-deazaguanine, N 4 ,N 4 -ethanocytosin, N 6 ,N 6 -ethano-2,6- diaminopurine, 5-methylcytosine, 5-(C -C )-alkynylcytosine, 5-fiuorouracil, 5- bromouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine and the "non-naturally occurring" nucleobases described in Benner et al., U.S.
  • nucleobase is intended to cover every and all of these examples as well as analogues and tautomers thereof.
  • Especially interesting nucleobases are adenine, guanine, thymine, cytosine, 5-methylcytosine, and uracil, which are considered as the naturally occurring nucleobases in relation to therapeutic and diagnostic application in humans.
  • DNA intercalator means a group which can intercalate into a DNA or RNA helix, duplex or triplex.
  • functional parts of DNA intercalators are acri dines, anthracene, quinones, such as anthraquinone, indole, quinoline, isoquinoline, dihydroquinones, anthracyclines, tetracyclines, methylene blue, anthracyclinone, psoralens, coumarins, ethidium-halides, dynemicin, pyrene, metal complexes such as 1 , 10-phenanthroline-copper, tris(4,7-diphenyl- 1 , 10-phenanthroline)rathenium- cobalt, enediynes, such as calcheamicin, porphyrins, distamycin, netropcin, viologen, or daunomycin.
  • acridines quinones
  • quinones such as anthra
  • DNA intercalators comprise as the functional part in the helix, duplex or triplex formation, a moiety of 2 to 6 fused aromatic rings.
  • fused aromatic rings are naphthyl, anthracenyl, phen-anthrenyl, pyrenyl, chrysenyl, benzanthracenyl, dibenzanthracenyl, benzopyrenyl, pyrenyl.
  • the DNA intercalators of the invention promise to be a so-called universal nucleobase, that is a nucleobase which pairs with any of the natural occurring nucleobases with a similar binding energy.
  • photochemically active groups covers compounds which are able to undergo chemical reactions upon irradiation with light.
  • functional groups hereof are quinones, especially 6-methyl-l,4-naphtoquinone, anthraquinone, naphtoquinone, and 1,4-dimethyl- anthraquinone, diazirines, aromatic azides, benzophenones, psoralens, diazo compounds, and diazirino compounds.
  • thermochemically reactive group is defined as a functional group which is able to undergo thermochemically-induced covalent bond formation with other groups.
  • functional parts thermochemically reactive groups are carboxylic acids, carboxylic acid esters such as activated esters, carboxylic acid halides such as acid fluorides, acid chlorides, acid bromides, and acid iodides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulfides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, and boronic acid derivatives.
  • chelating group means a molecule that contains more than one binding site and frequently binds to another molecule, atom or ion through more than one binding site at the same time.
  • functional parts of chelating groups are iminodiacetic acid, nitrilotriacetic acid, ethylenediamine tetraacetic acid (EDTA), and aminophosphonic acid.
  • reporter group means a group which is detectable either by itself or as a part of an detection series. Examples of functional parts of reporter groups are biotin, digoxigenin, fluorescent groups (groups which are able to absorb electromagnetic radiation, e.g.
  • dansyl (5- dimethylamino)-l-naphthalenesulfonyI
  • DOXYL Noxyl-4,4- dimethyloxazolidine
  • PROXYL N-oxyl-2,2,5,5-tetramethyl-pyrrolidine
  • TEMPO N-oxyl-2,2,6,6-tetramethylpiperidine
  • dinitrophenyl acridines, coumarins, Cy3 and Cy5 (trade-marks for Biological Detection Systems, Inc.), erytrosine, coumaric acid, umbelliferone, texas red, rhodamine, tetra-methyl rhodamine, Rox, 7-nitrobenzo-2-oxa-l -diazole (NBD), pyrene, fluorescein, Europium, Ruth
  • ligand means something which binds.
  • Ligands can comprise functional groups such as: aromatic groups (such as benzene, pyridine, naphtalene, anthracene, and phenanthrene), heteroaromatic groups (such as thiophene, furan, tetrahydrofuran, pyridine, dioxane, and pyrimidine), carboxylic acids, carboxylic acid esters, carboxylic acid halides, carboxylic acid azides, carboxylic acid hydrazides, sulfonic acids, sulfonic acid esters, sulfonic acid halides, semicarbazides, thiosemicarbazides, aldehydes, ketones, primary alcohols, secondary alcohols, tertiary alcohols, phenols, alkyl halides, thiols, disulfides, primary amines, secondary amines, tertiary amines, hydrazines, epoxides, maleimides, C ⁇ -C 2 o
  • DNA intercalators photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands correspond to the "active/functional" part of the groups in question.
  • DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands are typically represented in the form M-K- where M is the "active/functional" part of the group in question and where K is a spacer through which the "active/functional" part is attached to the 5- or 6- membered ring.
  • the group B in the case where B is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands, has the form M-K-, where M is the "active/functional" part of the DNA intercalator, photochemically active group, thermochemically active group, chelating group, reporter group, and ligand, respectively, and where K is an optional spacer comprising 1-50 atoms, desirably 1-30 atoms, and in particular 1-15 atoms, between the 5- or 6-membered ring and the "active/functional" part.
  • spacer means a thermo-chemically and photochemically non-active distance-making group and is used to join two or more different moieties of the types defined above. Spacers are selected on the basis of a variety of characteristics including their hydrophobicity , hydrophilicity, molecular flexibility and length (e.g. see Hermanson et. al., "Immobilized Affinity Ligand Techniques", Academic Press, San Diego, California (1992), p. 137). Generally, the length of the spacers are less than or about 400, in some applications desirably less than 100 A.
  • the spacer thus, comprises a chain of carbon atoms optionally interrupted or terminated with one or more heteroatoms, such as oxygen atoms, nitrogen atoms, and/or sulfur atoms.
  • the spacer K may comprise one or more amide, ester, amino, ether, and or thioether functionalities, and optionally substituted aromatic or mono/polyunsaturated hydrocarbons, polyoxyethylene such as polyethylene glycol, oligo/polyamides such as, polyalanine, polyglycine, polylysine, and , ⁇ peptides in general, oligosaccharides, oligo/polyphosphates.
  • the spacer may consist of combined units thereof.
  • the length of the spacer may vary, taking into consideration the desired or necessary positioning and spatial orientation of the "active/functional" part of the group in question in relation to the 5- or 6-membered ring.
  • the spacer includes a chemically cleavable group. Examples of such chemically cleavable groups include disulfide groups cleavable under reductive conditions and peptide fragments cleavable by peptidases.
  • K designates a single bond so that the "active/functional" part of the group in question is attached directly to the 5- or 6-membered ring.
  • the substituent B in the general formula (I) is selected from the naturally-occurring nucleobases adenine (A), guanine (G), cytosine (C), thymine (T), 5-methylcytosine, and uracil (U).
  • the designation "5'- terminal” refers to the position corresponding to the 5' carbon atom of a ribose moiety in a nucleoside, with “3 '-terminal” referring to the position corresponding to the 3' carbon atom of a ribose moiety in a nucleoside.
  • P designates the radical position for an internucleoside linkage to another monomer, or a 5'- terminal group.
  • internucleoside linkage or 5'- terminal group may include the substituent R 5 (or equally applicable, the substituent R ) thereby forming a double bond to the group P.
  • R 5 or equally applicable, the substituent R
  • the term "monomer” may relate to naturally occurring nucleosides, non-naturally occurring nucleosides, LNAs, PNAs, as well as ⁇ -L-RNA.
  • the term “successive monomer” relates to the neighboring monomer in the 5 '-terminal direction and the term “preceding monomer” relates to the neighboring monomer in the 3 '-terminal direction.
  • Such successive and preceding monomers seen from the position of an ⁇ -L- RNA monomer, may be naturally-occurring or non-naturally-occurring nucleosides, or even further ⁇ -L-RNA monomers.
  • oligomer means an oligonucleotide modified by the incorporation of one or more ⁇ -L-RNA monomers.
  • oligonucleotide which is the same as “oligomer” which is the same as “oligo” means a successive chain of nucleoside monomers connected via internucleoside linkages.
  • R" is selected from C ⁇ _ 6 -alkyl and phenyl.
  • An especially desirable linkage includes the natural phosphodiester (-0-P(0) 2 -0-) linkage. Further illustrative examples are given in Mesmaeker et. al., Current Opinion in Structural Biology 1995, 5, 343-355 and Susan M. Freier and Karl- Heinz Altmann, Nucleic Acids Research, 1997, vol 25, pp 4429-4443. The left-hand side of the intemuceoside linkage is bound to the 5-membered ring as substituent P* at the 3 '-position, whereas the right-hand side is bound to the 5'- position of a preceeding monomer.
  • the group P may also designate a 5'- terminal group in the case where the LNA in question is the 5 '-terminal monomer.
  • Examples of such 5 '-terminal groups are hydrogen, hydroxy, optionally substituted C ⁇ _ 6 -alkyl, optionally substituted C ⁇ .
  • the terms "monophosphate”, “diphosphate”, and “triphosphate” mean groups of the formula: -0-P(0) 2 -0-, - 0-P(0) 2 -0-P(0) 2 -0-, and 0-P(0) 2 -0-P(0) 2 -0-P(0) 2 -0, respectively.
  • the group P designates a 5 '-terminal group selected from monophosphate, diphosphate and triphosphate.
  • the triphosphate variant is especially interesting as a enyme substrate.
  • the group P* may designate a 3'-terminal group in the case where the ⁇ -L-RNA in question is the 3 '-terminal monomer.
  • 3'-terminal groups are hydrogen, hydroxy, optionally substituted C ⁇ . 6 -alkoxy, optionally substituted C ⁇ _ 6 -alkylcarbonyloxy, optionally substituted aryloxy, and -W-A', wherein W is selected from -0-, -S-, and -N(R H )- where R H is selected from hydrogen and C ⁇ . 6 -alkyl, and where A' is selected from DNA intercalators, photochemically active groups, thermochemically active groups, chelating groups, reporter groups, and ligands (where the latter groups may include a spacer as defined for the substituent B).
  • alkyl alkyl
  • alkenyl alkynyl
  • optionally substituted means that the group in question may be substituted one or several times, desirably 1-3 times, with group(s) selected from hydroxy (which when bound to an unsaturated carbon atom may be present in the tautomeric keto form), C ⁇ . 6 -alkoxy (i.e., Q. 6 -alkyl-oxy), C 2 - 6 -alkenyloxy, carboxy, oxo (forming a keto or aldehyde functionality), C ⁇ _ 6 -alkoxycarbonyl, C ⁇ .
  • hydroxy which when bound to an unsaturated carbon atom may be present in the tautomeric keto form
  • C ⁇ . 6 -alkoxy i.e., Q. 6 -alkyl-oxy
  • C 2 - 6 -alkenyloxy carboxy
  • oxo forming a keto or aldehyde functionality
  • the substituents are selected from hydroxy, C ⁇ _ 6 -alkoxy, carboxy, C ⁇ _ 6 -alkoxycarbonyl, C 1 . 6 -alkylcarbonyl, formyl, aryl, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- and di pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, piperidinyl, coumaryl, furyl, quinolyl, benzothiazolyl, benzotriazolyl, benzodiazolyl, benzo-oxozolyl, phthalazinyl, phthalanyl, triazolyl, tetrazolyl, isoquinolyl, acridinyl, carbazolyl, dibenzazepinyl, indolyl, benzopyrazolyl, phenoxazonyl.
  • aryl and "heteroaryl
  • “optionally substituted” means that the group in question may be substituted one or several times, desirably 1-5 times, in particular 1-3 times) with group(s) selected from hydroxy (which when present in an enol system may be represented in the tau-tomeric keto form), C ⁇ _ 6 -alkyl, C ⁇ _ 6 -alkoxy, oxo (which may be represented in the tautomeric enol form), carboxy, Ci_ 6 -alkoxycarbonyl, C ⁇ _ 6 -alkylcarbonyl, formyl, aryl, aryloxy, aryloxycarbonyl, arylcarbonyl, heteroaryl, amino, mono- and di(C ⁇ _ 6 - alkyl)amino; carbamoyl, mono- and di(C ⁇ _ 6 -alkyl)-aminocarbonyl, amino-C ⁇ _ 6 -alkyl-aminocarbonyl, mono- and di(Ci_ 6 -alkyl)amin
  • aryl and heteroaryl representing substituents may be substituted 1-3 times with C ⁇ _ 4 - alkyl, C ⁇ _ -alkoxy, nitro, cyano, amino or halogen.
  • Desirable examples are hydroxy, C ⁇ . 6 -alkyl, C ⁇ _ 6 -alkoxy, carboxy, Ci_ 6 -alkoxycarbonyl, C ⁇ _ 6 - alkylcarbonyl, aryl, amino, mono- and di(C ⁇ _ 6 -alkyl)amino, and halogen, wherein aryl may be substituted 1-3 times with C ⁇ _ 4 -alkyl, C ⁇ . 4 -alkoxy, nitro, cyano, amino or halogen.
  • Halogen includes fluoro, chloro, bromo, and iodo.
  • salts include acid addition salts and basic salts.
  • acid addition salts are hydrochloride salts, sodium salts, calcium salts, potassium salts, etc.
  • basic salts are salts where the (remaining) counter ion is selected from alkali metals, such as sodium and potassium, alkaline earth metals, such as calcium, and ammonium ions ( + N(R g ) 3 R h , where each of R s and R h independently designates optionally substituted C ⁇ . 6 -alkyl, optionally substituted C 2 .
  • the oligonucleotide of the present invention may optionally include at least one LNA monomer in addition to the at least one ⁇ -L-RNA monomer.
  • the LNA monomer is generally disclosed in WO 99/14226 (the entire content of which is hereby incorporated by reference).
  • the LNA monomer appears in several configurational stractures. Particularly, the LNA monomer appears in a ⁇ -L-LNA or a ⁇ -D-LNA configuration. While any of the ⁇ -D-LNA analogeous disclosed in WO 99/14226 are incorporated herein and may be a part of the the oligo-nucleotide of the invention, desirable ⁇ -D-LNA monomers include those of the following formula III
  • X represent oxygen, sulfur, amino, carbon or substituted carbon, and desirably is oxygen;
  • B is as disclosed for formula I above;
  • R 1 , R 2 , R 3 , R 5 and R 5* are hydrogen;
  • P designates the radical position for an inter-nucleoside linkage to another monomer (e.g., a successive monomer) or a 5'-terminal group,
  • R 3 is an internucleoside linkage to a another monomer (e.g., a preceeding monomer) or a 3 '-terminal group;
  • R 2 and R 4 together designate -0-CH 2 -, -S-CH 2 -, or
  • ⁇ -D-LNA monomers of formula III where R 2* and R 4* together designates a linkage -0-CH 2 - are sometimes referred to as " ⁇ -D-oxy-LNA” or, for short, "oxy-LNA”; units of formula III where R 2* and R 4* contain sulfur are sometimes referred to as "thio-LNA”; and units of formula III where R 2 and R 4* contain nitrogen are sometimes referred to as "amino-LNA".
  • oxy-LNA units are desired modified nucleic acid residues of oligonucleotides of the invention.
  • ⁇ -L-LNA monomer which in a desirable embodiment of the invention is present in the oligonucleotide, is disclosed in the international patent application, publication No. WO 00/66604, the entire content of which is incorporated herein by reference.
  • Desirable ⁇ -L-LNA monomers include such having the general formula IV:
  • X represents oxygen, sulfur, amino, carbon or substituted carbon, and desirably is oxygen
  • B is as disclosed for formula I above
  • R , R , R , R and R 5 are hydrogen
  • P designates the radical position for an internucleoside linkage to another monomer (e.g., successive monomer), or a 5'-terminal group
  • P is an internucleoside linkage to another monomer (e.g., precceeding monomer), or a 3 '-terminal group
  • R 2* and R 4* together designate -0-CH 2 -, -S-CH 2 -, -NH-CH 2 -, where the hetero atom is attached in the 2'- position, or a linkage of -(CH 2 ) n -, where n is 2, 3 or 4, desirably 2.
  • Desirable modified bases are covalently linked to the 1 '-position of a furanosyl ring, particularly to the 1 '-position of a 2',4'-linked furanosyl ring, especially to the 1 '-position of a 2'-O,4'-C-methylene-beta-D-ribofuranosyl ring.
  • modified bases contain one or more carbon alicyclic or carbocyclic aryl units, i.e. non-aromatic or aromatic cyclic units that contain only carbon atoms as ring members.
  • Modified bases that contain carbocyclic aryl groups are generally desirable, particularly a moiety that contains multiple linked aromatic groups, particularly groups that contain fused rings.
  • optionally substituted polynuclear aromatic groups are especially desirable such as optionally substituted naphthyl, optionally substituted anthracenyl, optionally substituted phenanthrenyl, optionally substituted pyrenyl, optionally substituted chrysenyl, optionally substituted benzanthracenyl, optionally substituted dibenzanthracenyl, optionally substituted benzopyrenyl, with substituted or unsubstituted pyrenyl being particularly desirable.
  • Such carbon alicyclic and/or carbocyclic aryl modified bases can increase hydrophobic interaction with neighboring bases of an oligonucleotide. Those interactions can enhance the stability of a hybridized oligo pair, without necessity of interactions between bases of the distinct oligos of the hybridized pair.
  • Such hydrophobic interactions can be particularly favored by platelike stacking of neighboring bases, i.e. intercalation.
  • intercalation will be promoted if the base comprises a moiety with a relatively planar extended structure, such as provided by an aromatic group, particularly a carbocyclic aryl group having multiple fused rings. This is indicated by the increases in T m values exhibited by oligos having LNA units with pyrenyl nucleobases relative to comparable oligos having LNA units with naphthyl nucleobases.
  • Modified bases that contain one or more heteroalicyclic or heteroaromatic groups also are suitable for use in LNA units, particularly such non-aromatic and aromatic groups that contains one or more N, O or S atoms as ring members, particularly at least one sulfur atom, and from 5 to about 8 ring members. Also desirable is a nucleo base that contains two or more fused rings, where at least one of the rings is a heteroalicyclic or heteroaromatic group containing 1, 2, or 3 N, O, or S atoms as ring members.
  • modified bases that contain 2, 3, 4, 5, 6, 7 or 8 fused rings, which may be carbon alicyclic, heteroalicyclic, carbocyclic aryl and or heteroaromatic; more desirably modified bases that contain 3, 4, 5, or 6 fused rings, which may be carbon alicyclic, heteroalicyclic, carbocyclic aryl and/or heteroaromatic, and desirably the fused rings are each aromatic, particularly carbocyclic aryl.
  • the base is not an optionally substituted oxazole, optionally substituted imidazole, or optionally substituted isoxazole modified base.
  • suitable modified bases for use in LNA units in accordance with the invention include optionally substituted pyridyloxazole, optionally substituted pyrenylmethylglycerol, optionally substituted pyrrole, optionally substituted diazole and optionally substituted triazole groups.
  • Desirable modified bases of the present invention when incorporated into an oligonucleotide containing all LNA units or a mixture of LNA and DNA or RNA units will exhibit substantially constant T m values upon hybridization with a complementary oligonucleotide, irrespective of the bases present on the complementary oligonucleotide.
  • one or more of the common RNA or commonly used derivatives thereof such as 2'-0-methyl, 2'-fluoro, 2'-allyl, and 2'-0- methoxyethoxy derivatives are combined with at least one nucleotide with a universal base to generate an oligonucleotide having between five to 100 nucleotides.
  • Modified nucleic acid compounds may comprise a variety of nucleic acid units e.g. nucleoside and/or nucleotide units.
  • an LNA nucleic acid unit has a carbon or hetero alicyclic ring with four to six ring members, e.g., a furanose ring, or other alicyclic ring structures such as a cyclopentyl, cycloheptyl, tetrahydropyranyl, oxepanyl, tetrahydrothiophenyl, pyrrolidinyl, thianyl, thiepanyl, piperidinyl, and the like.
  • At least one ring atom of the carbon or hetero alicyclic group is taken to form a further cyclic linkage to thereby provide a multi-cyclic group.
  • the cyclic linkage may include one or more, typically two atoms, of the carbon or hetero alicyclic group.
  • the cyclic linkage also may include one or more atoms that are substituents, but not ring members, of the carbon or hetero alicyclic group.
  • an alicyclic group as referred to herein is inclusive of group having all carbon ring members as well as groups having one or more hetero atom (e.g. N, O, S or Se) ring members.
  • the disclosure of the group as a "carbon or hetero alicyclic group” further indicates that the alicyclic group may contain all carbon ring members (i.e. a carbon alicyclic) or may contain one or more hetero atom ring members (i.e. a hetero alicyclic). Alicyclic groups are understood not to be aromatic, and typically are fully saturated within the ring (i.e. no endocyclic multiple bonds).
  • the alicyclic ring is a hetero alicyclic, i.e., the alicyclic group has one or more hetero atoms ring members, typically one or two hetero atom ring members such as O, N, S or Se, with oxygen being often desirable.
  • the one or more cyclic linkages of an alicyclic group may be comprised completely of carbon atoms, or generally more desirable, one or more hetero atoms such as O, S, N or Se, desirably oxygen for at least some embodiments.
  • the cyclic linkage will typically contain one or two or three hetero atoms, more typically one or two hetero atoms in a single cyclic linkage.
  • the one or more cyclic linkages of a nucleic acid compound of the invention can have a number of alternative configurations and/or configurations.
  • cyclic linkages of nucleic acid compounds of the invention will include at least one alicyclic ring atom.
  • the cyclic linkage may be disubstituted to a single alicyclic atom, or two adjacent or non-adjacent alicyclic ring atoms may be included in a cyclic linkage.
  • a cyclic linkage may include a single alicyclic ring atom, and a further atom that is a substituent but not a ring member of the alicyclic group.
  • desirable cyclic linkages include the following: C-l', C-2'; C-2', C-3'; C-2', C-4'; or a C-2*, C-5' linkage.
  • a cyclic linkage will typically comprise, in addition to the one or more alicyclic group ring atoms, 2 to 6 atoms in addition to the alicyclic ring members, more typically 3 or 4 atoms in addition to the alicyclic ring member(s).
  • modified nucleic acids for use oligonucleotides of the invention include locked nucleic acids as disclosed in W099/ 14226 (which include bicyclic and tricyclic DNA or RNA having a 2'-4' or 2'-3' sugar linkages); 2'-deoxy-2'-fluoro ribonucleotides; 2'-0-methyl ribonucleotides; 2'- O-methoxyethyl ribonucleotides; peptide nucleic acids; 5-propynyl pyrimidine ribonucleotides; 7-deazapurine ribonucleotides; 2,6-diaminopurine ribonucleotides; and 2-thio-pyrimidine ribonucleot
  • LNA units as disclosed in WO 99/14226 are in general particularly desirable modified nucleic acids for incorporation into an oligonucleotide of the invention. Additionally, the nucleic acids may be modified at either the 3 ' and/or 5' end by any type of modification known in the art. For example, either or both ends may be capped with a protecting group, attached to a flexible linking group, attached to a reactive group to aid in attachment to the substrate surface, etc. Desirable LNA units also are disclosed in WO 0056746, WO 0056748, and WO 0066604. Desirable syntheses of pyrene-LNA monomers is shown in the following Schemes 1 and 2. In the below Schemes 1 and 2, the compound reference numerals are also referred to in the examples below.
  • modified nucleic acids may be employed, including those that have 2'-modification of hydroxyl, 2 '-O-methyl, 2'-fluoro, 2'- trifluoromethyl, 2'-0-(2-methoxyethyl), 2'-0-aminopropyl, 2'-0-dimethyl- amino-oxyethyl, 2'-0-fluoroethyl or 2'-0-propenyl.
  • the nucleic acid may further include a 3' modification, desirably where the 2'- and 3 '-position of the ribose group is linked.
  • the nucleic acid also may contain a modification at the 4'-position, desirably where the 2'- and 4'-positions of the ribose group are linked such as by a 2'-4* link of -CH 2 -S-, -CH 2 -NH-, or -CH 2 -NMe- bridge.
  • the nucleotide also may have a variety of configurations such as ⁇ -D- ribo, ⁇ -D-xylo, or ⁇ -L-xylo configuration.
  • the internucleoside linkages of the units of oligos of the invention may be natural phosphorodiester linkages, or other linkages such as -0-P(0) 2 -0-, -0-P(0,S)-0-, -0-P(S) 2 -0-, -NR H -P(0) 2 -0-, -0-P(0,NR H )-0-, -O-P0(R")-O-, -0-PO(CH 3 )-0-, and -0-PO(NHR N )-0-, where R H is selected from hydrogen and C ⁇ - 4 -alkyl, and R" is selected from C ⁇ _ 6 -alkyl and phenyl.
  • a further desirable group of modified nucleic acids for incorporation into oligomers of the invention include those of the following formula:
  • B is a modified base as discussed above e.g. an optionally substituted carbocyclic aryl such as optionally substituted pyrene or optionally substituted pyrenylmethylglycerol, or an optionally substituted heteroalicylic or optionally substituted heteroaromatic such as optionally substituted pyridyloxazole.
  • Other desirable universal bases include, pyrrole, diazole or triazole moieties, all of which may be optionally substituted.
  • R is hydrogen.
  • R designates the radical position for an internucleoside linkage to a successive monomer, or a 5'-terminal group, such internucleoside linkage or 5'- terminal group optionally including the substituent R 5 , R 5 being hydrogen or included in an internucleoside linkage.
  • R is a group P* which designates an internucleoside linkage to a preceding monomer, or a 3'-terminal group.
  • Z is selected from -0-, -S-, and -N(R a )-
  • R a and R b each is independently selected from hydrogen, optionally substituted C ⁇ _ 6 -alkyl, optionally substituted C 2 _ 6 -alkenyl, hydroxy, C ⁇ .
  • Modified nucleobases and nucleosidic bases may comprise a cyclic unit (e.g. a carbocyclic unit such as pyrenyl) that is joined to a nucleic unit, such as a l'-position of furasonyl ring through a linker, such as a straight of branched chain alkylene or alkenylene group.
  • Alkylene groups suitably having from 1 (i.e. -CH 2 -) to about 12 carbon atoms, more typically 1 to about 8 carbon atoms, still more typically 1 to about 6 carbon atoms.
  • Alkenylene groups suitably have one, two or three carbon-carbon double bounds and from 2 to 12 carbon atoms, more typically 2 to 8 carbon atoms, still more typically 2 to 6 carbon atoms.
  • Desirable LNA units include those that contain a furanosyl-type ring and one or more of the following linkages: C-l', C-2'; C-2', C-3'; C-2', C-4'; or a C- 2', C-5' linkage.
  • a C-2', C-4' is particularly desirable.
  • desirable LNA units are compounds having a substituent on the 2'- position of the central sugar moiety (e.g., ribose or xylose), or derivatives thereof, which favors the C3'-endo conformation, commonly referred to as the North (or simply N for short) conformation.
  • Exemplary LNA units include ENA (2'-0,4'-C-ethylene-bridged nucleic acids such as those disclosed in WO 00/47599 or those illustrated below) units as well as non-bridged riboses such as 2'-F or 2'-0-methyl.
  • the oligonucleotide has at least one LNA unit with a modified base as disclosed herein.
  • Suitable oligonucleotides also may contain natural DNA or RNA units (e.g., nucleotides) with natural bases, as well as LNA units that contain natural bases.
  • the oligonucleotides of the invention also may contain modified DNA or RNA, such as 2'-0-methyl RNA, with natural or modified nucleobases (e.g., pyrene).
  • Desirable oligonucleotides contain at least one of and desirably both of 1) one or more DNA or RNA units (e.g., nucleotides) with natural bases, and 2) one or more LNA units with natural bases, in addition to LNA units with a modified base.
  • the nucleic acid does not contain a modified base.
  • Oligonucleotides of the invention desirably contain at least 50 percent or more, more desirably 55, 60, 65, or 70 percent or more of non-modified or natural DNA or RNA units (e.g., nucleotides) or units other than LNA units based on the total number of units or residues of the oligo.
  • a non-modified nucleic acid as referred to herein means that the nucleic acid upon incorporation into a 10-mer oligomer will not increase the T m of the oligomer in excess of 1°C or 2°C. More desirably, the non-modified nucleic acid unit (e.g., nucleotide) is a substantially or completely "natural" nucleic acid, i.e.
  • RNA ⁇ -D-ribose
  • DNA ⁇ -D-2-deoxyribose
  • Oligonucleotides of the invention suitably may contain only a single modified (i.e. LNA) nucleic acid unit, but desirably an oligonucleotide will contain 2, 3, 4 or 5 or more modified nucleic acid units.
  • an oligonucleotide contains from about 5 to about 40 or 45 percent modified (LNA) nucleic acid units, based on total units of the oligo, more desirably where the oligonucleotide contains from about 5 or 10 percent to about 20, 25, 30 or 35 percent modified nucleic acid units, based on total units of the oligo.
  • Typical oligonucleotides that contain one or more LNA units with a modified base as disclosed herein suitably contain from 3 or 4 to about 200 nucleic acid repeat units, with at least one unit being an LNA unit with a modified base, more typically from about 3 or 4 to about 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 or 150 nucleic acid units, with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 LNA units with a modified base being present.
  • particularly desirable oligonucleotides contain a non- modified DNA or RNA unit at the 3' terminus and a modified DNA or
  • the modified base is at the 3' terminal position of a nucleic acid primer, such as a primer for the detection of a single nucleotide polymorphism.
  • a nucleic acid primer such as a primer for the detection of a single nucleotide polymorphism.
  • Other particularly desirable nucleic acids have an LNA unit with or without a modified base in the 5' and/or 3 ' terminal position.
  • oligonucleotides that do not have an extended stretches of modified DNA or RNA units, e.g. greater than about 4, 5 or 6 consecutive modified DNA or RNA units. That is, desirably one or more non- modified DNA or RNA will be present after a consecutive stretch of about 3, 4 or 5 modified nucleic acids.
  • oligonucleotides that contain a mixture of LNA units that have non-modified or natural nucleobases (i.e., adenine, guanine, cytosine, 5-methyl-cytosine, uracil, or thymine) and LNA units that have modified bases as disclosed herein.
  • Particularly desirable oligonucleotides of the invention include those where an LNA unit with a modified base is interposed between two LNA units each having non-modified or natural bases (adenine, guanine, cytosine, 5- methyl-cytosine, uracil, or thymine.
  • the LNA "flanking" units with natural base moieties may be directly adjacent to the LNA with modified base moiety, or desirably is within 2, 3, 4 or 5 nucleic acid units of the LNA unit with modified base.
  • Nucleic acid units that may be spaced between an LNA unit with a modified base and an LNA unit with natural nucleobasis suitably are DNA and or RNA and or alkyl-modif ⁇ ed RNA/DNA units, typically with natural base moieties, although the DNA and or RNA units also may contain modified base moieties .
  • oligonucleotides of the present invention are comprised of at least about one universal base. Oligonucleotides of the present can also be comprised, for exmple, of between about one to six 2'-Ome-RNA unit, at least about two LNA units and at least about one LNA pyrene unit. Exemplary Target Nucleic Acids
  • target nuclei acids may be suitably single-stranded or double-stranded RNA; however, single-stranded RNA targets, suchs as mRNAs are desirable.
  • single-stranded RNA targets suchs as mRNAs are desirable.
  • sequence of the target polynucleotide e.g., Peyman and Uhnann, Chemical Reviews, 90:543-584, 1990; Crooke, Ann. Rev. Pharmacol Toxicol, 32:329-376 (1992); and Zamecnik and Stephenson, Proc. Nail. Acad. Sci, 75:280-284 (1974).
  • Desirable mRNA targets include the 5' cap site, tRNA primer binding site, the initiation codon site, the mRNA donor splice site, and the mRNA acceptor splice site, e.g., Goodchild et al., U.S. Patent 4,806,463.
  • DNA synthesizer using the phosphoramidite approach involves the use of nuclosides derivatized on the 3' position of the furanose ring with a phosphoramidite group and on the 5' position of the furanose ring with an acid-labile hydroxy protection group.
  • the phosphoramidite group has the general formula: -0-P-(NR 8 R 8* )-R 9 .
  • R 8 and R 8* may be same or different.
  • R 8 and R 8 are selected from linear and branched optional substituted C ⁇ , 6 -alkyl and C ⁇ . 6 -alkenyl.
  • R 8 and R 8* also can form alone or together a morpholino group (- N(CH 2 CH 2 ) 2 0).
  • R 8 and R 8* represent ethyl, or isopropyl. Allyl is also a possibility.
  • R 9 is a phosphate protection group.
  • R 9 is methoxy. This method has been improved by K ⁇ ster (US 4,725,677), where a protecting group that can be liberated by ⁇ -elimination is suggested. It has also been suggested to use ⁇ -elimination for protecting group removal.
  • the phospho protection groups can be removed with suitable means following the synthesis of the oligonucleotide.
  • the specific steps for removal of the protection groups depends largely on the nature of the protection group. Most of the above groups may be removed under appropriate basic conditions. However, the allyloxy groups may require treatment with paladium.
  • the acid-labile hydroxy protection group of the 5' position of the furanose ring can be selected among a large group of compounds.
  • Illustrative examples of hydroxy protection groups are optionally substituted trityl, such as 4,4'-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), or trityl, optionally substituted 9-(9-phenyl)xanthenyl (pixyl), optionally substituted ethoxycarbonyloxy, phenylazophenyloxycarbonyloxy, tetrahydropyranyl (THP), 9-fluorenyl-methoxycarbonyl (Fmoc), methoxytetrahydropyranyl
  • MTHP silyloxy, such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), tert- butyldimethylsilyl (TBDMS), triethylsilyl, or phenyldimethylsilyl, benzyloxycarbonyl or substituted benzyloxy-carbonyl ethers, such as 2-bromo benzyloxycarbonyl, tert-butylethers, alkyl ethers, such as methyl ether, acetals (including two hydroxy groups), acyloxy such as acetyl, halogen substituted acetyls, such as chloroacetyl or fluoroacetyl, isobutyryl, pivaloyl, benzoyl or substituted benzoyls, methoxymethyl (MOM), and benzyl ethers or substituted benzyl ethers, such as 2,6-dichlorobenzyl (2,6-
  • any chemical group (including any nucleobase), which is reactive under the conditions prevailing in chemical oligonucleotide synthesis, is optionally functional-group-protected as known in the art.
  • hydroxy protection groups are optionally substituted trityl, such as 4,4'-dimethoxytrityl (DMT), 4-monomethoxytrityl (MMT), or trityl, optionally substituted 9-(9-phenyl)xanthenyl (pixyl), optionally substituted ethoxycarbonyloxy, phenylazophenyloxycarbonyloxy, tetrahydropyranyl (THP), 9-fluorenylmethoxycarbonyl (Fmoc), methoxytetrahydropyranyl (MTHP), silyloxy, such as trimethylsilyl (TMS), triisopropylsilyl (TIPS), tert-butyldimethylsilyl (TBDMS), triethylsilyl, or phenyldimethylsilyl, benzyloxycarbonyl or substituted benzyloxycarbonyl ethers, such as 2-bromobenz
  • the hydroxy group may be protected by attachment to a solid support optionally through a linker.
  • amino protection groups are Fmoc (fluorenylmethoxycarbonyl), BOC (tert-butyloxycarbonyl), trifluoroacetyl, allyloxycarbonyl (alloc or AOC), benzyloxycarbonyl (Z or Cbz), substituted benzyloxycarbonyls, such as 2-chlorobenzyloxycarbonyl (2-C1Z), monomethoxytrityl (MMT), dimethoxytrityl (DMT), phthaloyl, and 9-(9- phenyl)-xanthenyl (pixyl).
  • carboxy protection groups are allyl esters, methyl esters, ethyl esters, 2-cyanoethyl esters, trimethylsilylethyl esters, benzyl esters (OBn), 2-adamantyl esters (O-2-Ada), cyclohexyl esters (OcHex), 1,3-oxazolines, oxazoler, 1,3-oxazolidines, amides, and hydrazides.
  • allyl esters methyl esters, ethyl esters, 2-cyanoethyl esters, trimethylsilylethyl esters, benzyl esters (OBn), 2-adamantyl esters (O-2-Ada), cyclohexyl esters (OcHex), 1,3-oxazolines, oxazoler, 1,3-oxazolidines, amides, and hydrazides.
  • mercapto protecting groups are trityl (Trt), acetamidomethyl (Acm), trimethylacetamidomethyl (Tacm), 2,4,6- trimethoxybenzyl (Tmob), tert-butylsulfenyl (StBu), 9-fluorenylmethyl (Fm), 3-nitro-2-pyridinesulfenyl (Npys), and 4-methylbenzyl (Meb).
  • Trt trityl
  • Acm acetamidomethyl
  • Tacm trimethylacetamidomethyl
  • Tmob 2,4,6- trimethoxybenzyl
  • StBu tert-butylsulfenyl
  • Fm 9-fluorenylmethyl
  • Npys 3-nitro-2-pyridinesulfenyl
  • Meb 4-methylbenzyl
  • Illustrative examples are benzoyl, isobutyryl, tert-butyl, tert-butyloxycarbonyl, 4-chloro-benzyloxycarbonyl, 9- fluorenylmethyl, 9-fluorenylmethyloxycarbonyl, 4-methoxybenzoyl, 4- methoxytriphenylmethyl, optionally substituted triazolo, p-toluenesulfonyl, optionally substituted sulfonyl, isopropyl, optionally substituted amidines, optionally substituted trityl, phenoxyacetyl, optionally substituted acyl, pixyl, tetrahydropyranyl, optionally substituted silyl ethers, and 4- methoxybenzyloxycarbonyl.
  • the group B in a monomeric LNA is selected from nucleobases and protected nucleobases.
  • compound 2 was treated with aqueous sodium hydroxide, affecting stereochemical inversion of the hydroxy substituent at C-2, presumably through an anhydro nucleoside intermediate, to yield compound 3.
  • aqueous sodium hydroxide is added and the temperature is raised to remove the protection group 1,1,3,3-tetraisopropyldisiloxane of the 3'- and 5'-position.
  • compound 4 is obtained and isolated.
  • Compound 4 is subsequently treated with 4,4'-dimethoxytrityl chloride (DMTC1) in anhydrous pyridine to obtain compound 5, which has the 5'-hydroxy group protected with DMT.
  • DMTC1 4,4'-dimethoxytrityl chloride
  • Compound 5 was treated with a combination of imidazole and tert- butyldimethylsilyl chloride (TBDMSC1) in anhydrous pyridine to produce compound 6, which has the 2'-hydroxy group protected with TBDMS and the 3'-hydroxy group unprotected.
  • phosphoramidite (compound 7) was made by treating compound 6 with NC(CH 2 ) 2 ⁇ P(Cl)N(z-Pr) 2 in a mixture of anhydrous dichloromethane and NN-diisopropylethylamine.
  • phosphoramidite derivatives like 7 of other (properly protected) nucleobases e.g., uracil- 1-yl, cytosin-1-yl. 5-methyl- cytosin-1-yl, guanin-9-yl and adenin-9-yl.
  • ⁇ -L-ribofuranosyl pyrimidine nucleosides such as l-( ⁇ -L-ribofuranosyl)uracil 23 and l-( ⁇ -L-ribofuranosyl)cytosine 25
  • l-( ⁇ -L-ribofuranosyl)uracil 23 and l-( ⁇ -L-ribofuranosyl)cytosine 25 A. L. Weis, C. T. Goodhue, K. Shanmuganathan, WO 96/13512.
  • oligonucleotides and polynucleotides of the invention may be produced using the polymerisation techniques of nucleic acid chemistry well known to a person of ordinary skill in the art of organic chemistry.
  • Phosphoramidite chemistry S. L. Beaucage and R. P. Iyer, Tetrahedron, 1993, 49, 6123; S. L. Beaucage and R. P. Iyer, Tetrahedron, 1992, 48, 2223
  • other standard chemistry e.g., H-phosphonate chemistry (F. Seela, K. W ⁇ mer and H. Rosemeyer, Helv. Chim.
  • phosphotriester chemistry or enzymatic synthesis may be used.
  • standard coupling conditions and the phosphoramidite approach were used, but for some monomers of the invention, longer coupling time, and/or repeated couplings with fresh reagents, and/or use of more concentrated coupling reagents were used.
  • activators more active than 1 H-tetrazole could also be used to increase the rate of the coupling reaction.
  • An example of such activator is pyridine hydrochloride (V. K. Rajwanshi, A.E. Hakansson, B. M. Dahl, and J. Wengel, Chem. Commun. 1999, 1395-1396).
  • the first step for synthesis of the oligonucleotides of the invention according to the phosphoramidite approach is initiated by attaching the first nucleoside monomer to a solid support at the 3 '-position of the sugar moeity.
  • the linkage between the 3 '-hydroxy group of the nucleoside and the support is desirably base-labile to make it possible to detach the oligonucleotide under mild alkaline conditions, such as 50:50 aqueous methyl: ammonium hydroxide mixture (AMA), after the formation of the oligonucleotide.
  • AMA aqueous methyl: ammonium hydroxide mixture
  • the first nucleotide is generally protected on all hydroxy groups, e.g., on the 2'-position (in case of RNA), and 5'-position.
  • the protection of the 5'-position is performed with a acid labile group, such as 4,4'- dimethoxytrityl (DMT).
  • DMT 4,4'- dimethoxytrityl
  • any exocyclic amino groups on the nucleobase are also protected, desirably with base-labile acyl groups.
  • nucleosides Commercially, several solid supports containing 5'-0-DMT- and base protected nucleosides are available.
  • the starting nucleoside is dT (deoxyribose thymin-1-yl), which does not need protection of the nucleobase (no exocyclic amino groups present) and does not contain a 2'-hydroxy group to be protected.
  • the dT group is attached to the support through a base-labile ester linkage.
  • the second step involves the removal of the acid-labile DMT group from the 5 '-position of the first nucleoside to form the hydroxy group available for reaction.
  • the most commonly used reagent for this detritylation is a diluted solution of dechloroacetic acid in dichloromethane. However, it is also possible to replace dichloromethane with toluene.
  • the third step includes the addition of a phosphoramidite derivative and an activator.
  • the activator may be tetrazole, a pyridinium salt, or a similar proton-donating compound.
  • the phosphoramidite derivative comprises the subsequent monomer protected at the 5'-position with DMT or similar acid labile protection group.
  • the 3'-position of the ribose moeity comprises the phosphoramidite group, which is composed of a phosphine group bound to the 3'-carbon of the ribose moiety through a oxygen, where the phosphine group is further connected to an amino group and a phosphate protecting group.
  • the amino group is mono- or disubstituted with a linear or branched alkyl or alkenyl having 1 to 6 carbon atoms.
  • a desirable amino group is N,N- diisopropylamine.
  • the phosphate-protecting group may be selected in accordance with the specific process. Currently, it is desirable to use ⁇ - cyanoethyloxy, ⁇ -cyano-2-butenyloxy, methoxy, or allyloxy. In a desirable embodiment used in the experiments reported herein, the phosphoramidite group is N,N-diisopropylamino-2-cyano-ethyloxyphosphino.
  • the second and the third step may be combined. That is, the addition of the acid, such as trichloroacetic acid, may occur simultaneous with the addition of the phosphoramidite derivative and the activator.
  • the acid such as trichloroacetic acid
  • the third step results in a condensation of the phosphoramadite derivative with the first nucleoside attached to the support.
  • the generally accepted mechanism for the condensation involves catalysis (e.g., nucleophilic catalysis) by tetrazole or similar activator for the phosphoramidite coupling reaction. It is believed that a proton is donated by the activator to the phosphorus atom to produce a phosphinium ion.
  • the protonation reaction is followed by nucleophilic displacement of the amino group with tetrazolide or similar activator. Reaction with the 5'-hydroxy group of the support bound first nucleoside generates the dinucleoside phosphite.
  • any unreacted hydroxy groups may exist.
  • any unreacted hydroxy groups are capped, e.g., with acetyl groups.
  • An acetylation of the 5'-hydroxy groups may be performed by adding acetic anhydride and N- methylimidazole.
  • the fifth step of the process involves oxidation of the phosphite triester resulting from step three to the corresponding phosphodiester. This oxidation can be executed by addition of aqueous iodine or a similar oxidation agent. If further monomers are to be added to the dinucleotide, steps two through five are repeated.
  • the oligonucleotide When the oligonucleotide has reached the desired length and order of sequence, the oligonucleotide is released from the support and the phosphorus and nucleobase protecting groups are removed by addition of a mild base, such as aqueous ammonia.
  • a mild base such as aqueous ammonia.
  • the DMT protection group of the 5'-hydroxy group of the last added nucleotide can be removed prior or subsequent to purification using an appropriate acid.
  • the purification of the oligonucleotide is suitably performed on a laboratory scale by reversed-phase HPLC or anion exchange chromatography.
  • An additional aspect of the present invention is to furnish procedures for oligonucleotide analogues containing ⁇ -L-RNA linked by non-natural internucleoside linkages.
  • synthesis of the corresponding phosphorothioate or phosphoramidate analogues is possible using strategies well-established in the field of oligonucleotide chemistry (Protocols for Oligonucleotides and Analogs, vol 20, (Sudhir Agrawal, ed.), Humana Press, 1993, Totowa, NJ; S. L. Beaucage and R. P.
  • an ⁇ -L-RNA modified oligonucleotide may comprise normal nucleosides (i.e.
  • incorporation of ⁇ -L-RNA modulates the ability of the oligonucleotide to act as a substrate for nucleic acid active enzymes.
  • solid-support materials having immobilized thereto an optionally nucleobase-protected and optionally 5'-hydroxy protected ⁇ -L-RNA are especially interesting as material for the synthesis of ⁇ -L-RNA modified oligo-nucleotides where an LNA monomer is included at the 3' end.
  • the solid support material is CPG or a polymeric material, e.g., polystyrene.
  • the CPG is a readily (commercially) available CPG material onto which a 3'-functionalized, optionally nucleobase-protected and optionally 5'-hydroxy protected ⁇ -L-RNA is linked using the conditions stated by the supplier for that parti-cular material.
  • BioGenex Universial CPG Support BioGenex, U.S.A.
  • the 5'-hydroxy protecting group may be a DMT group.
  • the 3'-functional group can be selected by one skilled in the art with due regard to the conditions applicable for the CPG material in question.
  • composition of each of the monomers, the order of such monomers, and the total number of monomers of the oligonucleotide of the invention constitute the design of a nucleotide.
  • the composition of most-desired monomers, and notably the ⁇ -L-RNA monomer, is described above.
  • the number, order, and presence of further nucleosides may be of importance in the design of an oligonucleotide intended to hybridize to a specific target. It should be noticed that a successful design of an oligonucleotide of the invention toward a specific target oligonucleotide does not necessarily mean that a like design would be successful toward a different complementary target nucleoside.
  • the at least one ⁇ -L-RNA monomer may, in principle, be placed anywhere in the oligonucleotide, e.g., in the central part, close to or at the 3' end, or close to or at the 5' end.
  • ⁇ -L-RNA monomers may appear as single ⁇ -L-RNA monomers dispersed throughout the oligonucleotide, or the ⁇ - L-RNA monomers can be grouped in blocks, or can appear as mixtures of single and grouped ⁇ -L-RNA monomers.
  • T m melting temperature
  • oligonucleotides comprising ⁇ -L- RNA monomers sometimes showed a lower hybridization tendency compared to the corresponding DNA reference. It may be possible to increase the hybridization power by incorporating LNA monomers into the oligonucleotide.
  • the LNA monomer may have the "normal" ⁇ -D configuration or the ⁇ -L- configuration.
  • the LNA monomer may occur in the nucleotide, in addition to the ⁇ -L-RNA monomer(s), in any appropriate number.
  • the LNA monomer may appear in the oligonucleotide as single entities spread over the entire oligonucleotide or may be present in groups of 2 or more LNA monomers.
  • the one or more LNA adjacent to the one or more ⁇ -L-RNA monomer in the oligonucleotide. It is believed that the adverse effect on the affinity shown by the ⁇ -L-RNA monomer in some oligomers herein is counteracted by the close presence of the LNA.
  • ⁇ -L-RNA monomers and the LNA monomers are present in groups, it is desirable to find not just an increase in the affinity, but also an increase in preferred hybridization to the RNA complement. Most desirable is when no hybridization with the DNA complement is detected. Moreover, it is desirable that the above effect for oligonucleotides containing blocks of ⁇ -L- RNA monomers and LNA monomers are apparent for blocks of ⁇ -L-LNA as well as for ⁇ -D-LNA. A similar effect, that yields a high discriminating power and an acceptable affinity, is obtained when single ⁇ -L-RNA monomers alternate with single ⁇ -L-LNA monomers.
  • the present invention discloses the surprising fact that novel ⁇ -L-RNA monomers, when incorporated into oligomers, dramatically increase the discrimination power toward hybridization to a complementary single stranded RNA oligonucleotide. It is possible to design an ⁇ -L-RNA modified oligonucleotide which displays an ability to hybridize to the corresponding RNA complement only, while it is not possible to measure any hybridization under the prevailing conditions with the corresponding DNA complement. Furthermore, the oligonucleotides of the invention seem to be unable to be digested by phosphodiesterases. The above properties of the oligonucleotide of the invention, suggest a variety of applications within the diagnostic and therapeutic field.
  • the application of an antisense or RNAi; approach is very promising.
  • the antisense approach pertains to the inhibition or destruction of mRNA before it is translated into a peptide or protein.
  • One of the complications with previous oligonucleotides have been that they, besides binding to the mRNA, also bind to DNA in the cell.
  • the binding to genomic DNA may de detrimental for the treated organism because it may alter the expression of the genes at or in the vicinity of the part of the genomic DNA to which the oligonucleotide has hybridized. This unintended hybridization to genomic DNA may be fully or partly avoided by the oligonucleotides of the present invention due to the preferred hybridization to RNA.
  • a therapeutic agent comprising this oligonucleotide may be present in a biological environment with little or no degradation. This implies that the therapeutic agent, once administered to the organism, may have a long active period in which it can exert the therapeutic affect. It has been demonstrated that oligomers containing one ⁇ -L-RNA modified nucleotide have the ability to recruit RNase H. Thus, when the mRNA is captured in the biological environment by hybridization to the ⁇ -L-RNA modified nucleotide of the invention, digestion of the captured mRNA is conducted by RNase H. In other words, the oligonucleotide of the invention holds the target mRNA in a vice-like grip, allowing the RNase H to degrade the target mRNA.
  • RNAzymes function as specific RNA endonucleases by binding to predetermined sequences in an RNA and cleaving the phosphodiester backbone (R. R. Breaker, G. F. Joyce, Chem. Biol 199 , 1, 223). Highly efficient, sequence-specific cleavage of RNA is a prerequisite for the use of DNAzymes both as therapeutic antisense oligonucleotides and as general tools for manipulation of RNA.
  • DNAzymes are derivatives of the 31 -nucleotide " 10-23 " oligomer, which was originally isolated by in vitro selection (S. W. Santoro, G. F. Joyce, Proc. Natl Acad. Sci. USA 1997, 94, 4262). This DNAzyme attains its high specificity through hybridization of its two binding arms to complementary sequences immediately adjacent to the point of cleavage in the RNA substrate. It has recently been shown that incorporation of ⁇ -L-LNA nucleotide monomers into the binding arms of a DNAzyme markedly increase the efficiency of RNA cleavage (B. Vester, L. B. Lundberg, M. D. S ⁇ rensen, B. R. Babu, S.
  • DNAzymes containing ⁇ -L-RNA modified nucleotide(s) should therefore find use because of the RNA selectivety of oligomers containing ⁇ -L-RNA modified nucleotide(s) as described above for more traditional antisense oligomers.
  • the ⁇ -L-RNA modified nucleotide may also find application in oligomers designed to strand-invade dsRNA targets.
  • the RNA selective hybridization should make the targeting of both RNA strands possible by applying not only the oligomer containing ⁇ -L-RNA modified nucleotide(s) but also an oligomer, e.g. a DNA oligomer, not able to hybridize to the oligomer containing ⁇ -L-RNA modified nucleotide(s).
  • the ⁇ -L-RNA modified nucleotide may also find application in the combat of RNA-based vimses. It is believed that the oligonucleotide of the invention can selectively bind to viral RNA at various stages of the viral life cycle and therefore hamper the development of the disease caused by the virus. Other classes of cellular RNAs, e.g., tRNA, rRNA, snRNA, scRNA, which have not previously attracted much attention as targets for the use of antisense or RNAi technology may potentially be regulated by the ⁇ -L-RNA modified oligonucleotides of the invention. Furthermore, double stranded RNAs are known to inhibit the growth of several types of cancers and virases. The ⁇ -L-RNA modified oligonucleotides of the invention may have potential applications within these therapeutic fields.
  • oligonucleotide arrays are immobilized in a predetermined pattern on a solid support such that the presence of a particular mutation in the target nucleic acid can be revealed by the position on the solid support where it hybridizes.
  • One important prerequisite for the successful use of arrays of different oligonucleotides in the analysis of nucleic acids is that they are all specific for their particular target sequence under the single applied hybridization condition.
  • the array of oligonucleotides hybridize to DNA as well as RNA target nucleic acid in a biological sample because the traditional oligonucleotide probes composed of DNA and/or RNA monomers do not substantially discriminate between RNA and DNA.
  • the consequence is that the signal to noise ratio decreases, especially for crude biological samples, which may neccesiate a purification step prior to the sample analysis.
  • the present invention alleviates the problem associated with the higher noise using conventional oligonucleotide probes and provides an oligonucleotide which may be designed with high RNA/DNA discriminating power.
  • oligonucleotides of the invention may decrease the need for a purification of the biological sample comprising the target nucleic acid prior to sample testing.
  • the acceptable affinity and high specificity toward RNA is exploited in the sequence specific capture and purification of natural or synthetic nucleic acids.
  • the natural RNA or the synthetic RNA analogous are contacted with the ⁇ -L-RNA modified oligonucleotide immobilized on a solid surface. In this case hybridization and capture occurs simultaneously.
  • the captured RNA may be, for instance, detected, characterised, quantified, or amplified directly on the surface by a variety of methods well known in the art, or it may be released from the surface before such characterisation or amplification occurs by subjecting the immobilized ⁇ -L-RNA modified oligonucleotide and captured nucleic acid to dehybridizing conditions, such as heat or low ionic strength buffers.
  • the solid support may be chosen from a wide range of polymer materials such as, for example, CPG (controlled pore glass), polypropylene, polystyrene, polycarbonate, or polyethylene and it may take a variety of forms such as, for example a tube, a microtiter plate, a stick, a bead, or a filter.
  • CPG controlled pore glass
  • polypropylene polypropylene
  • polystyrene polystyrene
  • polycarbonate polyethylene
  • polyethylene polyethylene
  • it may take a variety of forms such as, for example a tube, a microtiter plate, a stick, a bead, or a filter.
  • the ⁇ -L-RNA modified oligonucleotide may be immobilized to the solid support via its 5' or 3' end (or via the terminus of linkers attached to the 5 ' or 3' end) by a variety of chemical or photochemical methods usually employed in the immobilization of oligonucleotides or by non-covalent coupling such as for instance via binding of a biotinylated ⁇ -L-RNA modified oligonucleotide to immobilized streptavidin.
  • One desirable method for immobilizing ⁇ -L-RNA modified oligonucleotides on different solid supports is by photochemical linkage using a photochemically active anthraquinone covalently attached to the 5' or 3' end of the modified oligonucleotide (optionally via linkers) as described in (WO 96/31557).
  • the present invention also provide a surface carrying an ⁇ -L-RNA modified oligonucleotide.
  • the conventional method for recovery of RNA from a biological sample involves the treatment of the sample with a phenolic solvent to precipitate the RNA, subsequent centrifugation, and then dissolution of the RNA pellet. This method is time consuming and laborious.
  • the present invention provides the possibility of immobilizing the ⁇ -L-RNA modified oligonucleotide on a support in a suitable vessel, like a column, charging the vessel with a biological sample to hybridize the sample RNA to the probes, and subsequently eluting the sample RNA with a suitable buffer.
  • the method may be designed to capture a specific known RNA or a pool of mRNA.
  • the ⁇ -L-RNA modified oligonucleotide carries a ligand covalently attached to either the 5' or 3' end.
  • the ⁇ -L-RNA modified oligonucleotide is contacted with the natural or synthetic nucleic acids in solution after which the hybrids formed are captured onto a solid support carrying molecules that can specifically bind the ligand.
  • ⁇ -L-RNA modified oligonucleotides designed with the purpose of high specificity are used as primers in the sequencing of nucleic acids or as primers in any of the several well known amplification reactions, such as the PCR reaction.
  • the products of the amplification reaction can be analysed by a variety of methods applicable to the analysis of amplification products generated with normal DNA/RNA primers.
  • the ⁇ -L-RNA modified oligonucleotide primers are designed to sustain a linear amplification, the resulting amplicons will carry single stranded ends that can be targeted by complementary probes without denaturation. Such ends could, for example, be used to capture amplicons by other complementary ⁇ -L-RNA modified oligonucleotides attached to a solid surface.
  • probes have been developed for real- time detection of amplicons generated by target amplification reactions.
  • One such class of probes have been termed "Molecular Beacons”. These probes are synthesised as partly self-complementary oligonucleotides containing a fluorophore at one end and a quencher molecule at the other end. When free in solution, the probe folds up into a hairpin structure (guided by the self- complimentary regions) which positions the quencher in sufficient closeness to the fluorophore to quench its fluorescent signal. Upon hybridization to its target nucleic acid, the hairpin opens, thereby separating the fluorophore and quencher and giving off a fluorescent signal.
  • Taqman probes Another class of probes have been termed "Taqman probes". These probes also contain a fluorophore and a quencher molecule. Contrary to the Molecular Beacons, however, the quencher's ability to quench the fluorescent signal from the fluorophore is maintained after hybridization of the probe to its target sequence. Instead, the fluorescent signal is generated after hybridization by physical detachment of either the quencher or fluorophore from the probe by the action of the 5' exonuclease activity of a polymerase which has initiated synthesis from a primer located 5' to the binding site of the Taqman probe.
  • ⁇ - L-RNA monomers in combination with LNA monomers are used to improve production and subsequent performance of Taqman probes and Molecular Beacons by reducing their size whilst retaining the required affinity.
  • the pharmaceutical preparations of the present invention comprise novel oligonucleotides and/or their physiologically tolerated salts in addition to pharmaceutically unobjectionable excipients and/or auxiliary substances.
  • the pharmaceutical preparations of the present invention also include novel nucleoside analogues of the formula II and pharmaceutically acceptable derivatives thereof including physiologically tolerated salts and esters in addition to pharmaceutically unobjectionable excipients and/or auxiliary substances. These analogues may be especially valuable as viral replication inhibitors.
  • the oligonucleotides and/or their physiologically tolerated salts can be administered to animals, desirably mammals, and in particular humans, as pharmaceuticals on their own, in mixtures with each other, or in the form of pharmaceutical preparations which permit topical, percutaneous, parenteral, or enteral use and which comprise, as the active constituent, an effective dose of at least one oligonucleotide in addition to customary pharmaceutically unobjectionable excipients and auxiliary substances.
  • the preparations normally comprise from about 0.1 to 90% by weight of the therapeutically active compound.
  • a topical use is desirable.
  • infusions and oral administration are desirable.
  • oral administration is desirable.
  • the pharmaceutical products are prepared in a manner known per se (e.g., Remingtons Pharmaceutical Sciences, Mack Publ. Co., Easton, PA), with pharmaceutically inert inorganic and/or organic excipients being used.
  • Lactose, corn starch and or derivatives thereof, talc, stearic acid and/or its salts, etc. can, for example, be used for preparing pills, tablets, coated tablets and hard gelatin capsules.
  • excipients for soft gelatin capsules and/or suppositories are fats, waxes, semisolid and liquid polyols, natural and/or hardened oils, etc.
  • suitable excipients for preparing solutions and or syrups are water, sucrose, invert sugar, glucose, polyols, etc.
  • Suitable excipients for preparing injection solutions are water, alcohols, glycerol, polyols, vegetable oils, etc.
  • Suitable excipients for microcapsules, implants and/or rods are mixed polymers of glycolic acid and lactic acid.
  • liposome formulations which are known to those skilled person (N. Weiner, Drug Develop Ind Pharm 15 (1989) 1523; “Liposome Dermatics, Springer Verlag 1992), for example, HVJ Liposomes (Hayashi, Gene Therapy 3 (1996) 878), are suitable.
  • Dermal administration can also be effected, for example, using ionophoretic methods and/or by means of electroporation.
  • use can be made of lipofectins and other carrier systems, for example, those which are used in gene therapy.
  • a pharmaceutical preparation can also comprise additives, such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizing agents, emulsifiers, preservatives, sweeteners, dyes, flavorings or aromatizing agents, thickeners, diluents, buffering substances, solvents and/or solubilizing agents, agents for achieving a slow release effect, salts for altering the osmotic pressure, coating agents, or antioxidants.
  • additives such as fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilizing agents, emulsifiers, preservatives, sweeteners, dyes, flavorings or aromatizing agents, thickeners, diluents, buffering substances, solvents and/or solubilizing agents, agents for achieving a slow release effect, salts for altering the osmotic pressure, coating agents, or antioxidants.
  • oligonucleotides may also comprise two or more different oligonucleotides and/or their physiologically tolerated salts and, furthermore, in addition to at least one oligonucleotide, one or more different therapeutically active ingredients.
  • the dose can vary within wide limits and is to be adjusted to the individual circumstances in each individual case.
  • R 1 OH
  • R 2 I f 6
  • R 1 DMT
  • R 2 H
  • R 3 TBDMS vi C
  • R 1 - DMT
  • ⁇ R2 ⁇ P(N(/-Pr) 2 )0(CH 2 ) 2 CN
  • J TBDMS resorption, phagocytosis, immune response, signal transduction, and the metastasis of neoplastic cells.
  • Such a pharmaceutical composition can be used for the treatment and prevention of cancer and metastasis of cancer, the treatment and prevention of osteoporosis, the treatment of ocular diseases, chronic inflammation, psorasis, restenosis, and in support of wound healing.
  • the reaction mixture was cooled in an ice bath and the reaction quenched by addition of water (10 ml).
  • the solvent was removed under reduced pressure and the residue was dissolved in water (150 ml) which was extracted with dichloromethane (3 x 70 ml).
  • the combined organic phase was washed successively with saturated aqueous sodium hydrogen carbonate (2 x 100 ml) and brine (2 x 100 ml), and dried (MgS0 ).
  • the solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography (0-2% CH 3 OH in CH 2 C1 2 ) to give nucleoside 2 as a white solid material (6.59 g; 84%).
  • nucleoside 2 To at stirred solution of nucleoside 2 (297 mg; 0.51 mmol) in ethanol (6 ml) was added water (1.5 ml) and 2M aqueous sodium hydroxide (1.03 ml). The solution was stirred for 24 hours at room temperature to afford a ribo- configurated nucleoside 3 as intermediate.
  • the intermediate 3 was not isolated but treated with further 2 M aqueous sodium hydroxide (2.06 ml).
  • the reaction mixture was stirred at 65 °C overnight.
  • Example 3 l-[5-0-(4,4 '-Dimethoxytrityl)- ⁇ -L-ribofuranosyl]thymine (5).
  • nucleoside 4 413 mg; 1.60 mmol
  • anhydrous pyridine 15 ml
  • 4,4 '-dimethoxytrityl chloride 650 mg; 1.92 mmol
  • the solution was stirred for 24 hours, additional 4,4 '-dimethoxytrityl chloride (271 mg; 0.80 mmol) was added, and after 2 hours, the reaction was quenched with water (40 ml).
  • the mixture was extracted with dichloromethane (3 x 40 ml) and the combined organic phase was dried (MgS0 4 ).
  • nucleoside 5 was obtained by silica gel column chromatography (3-6% CH 3 OH in CH 2 C1 2 with 0.5%> pyridine) to give nucleoside 5 as a white solid material (834 mg; 93%).
  • nucleoside 5 To a stirred solution of nucleoside 5 (559 mg; 0.997 mmol) in anhydrous pyridine (20 ml) was added imidazole (407 mg; 5.98 mmol) and tert- butyldimethylsilyl chloride (450 mg; 2.99 mmol). The reaction mixture was stirred for 14 hours whereupon saturated aqueous sodium hydrogen carbonate (60 ml) was added.
  • Example 5B 2-0,2'-0-Anhydro-l-( ⁇ -L-ribofuranosyl)uracil (22).
  • a mixture oxazoline 21 (1.00 g, 5.75 mmol) in 96% aqueous EtOH (10 mL) and methyl propiolate (1.69 g, 20.11 mmol) was heated under reflux for 2 h. After cooling to room temperature, the mixture was evaporated to dryness under reduced pressure and then co-evaporated (under reduced pressure) several times with 96% aqueous EtOH to give 1.05 g of nucleoside 22 (81%) after recrystallization from 96% aqueous EtOH.
  • nucleoside 22 (2.17 g, 9.60 mmol) in 0.2 N aqueous hydrochloric acid (10 mL) was refluxed for 1 h. After cooling to room 5 temperature, the solution was neutralized using Amberlyst IRA 410 [OH " ]. The resin was filtered off and washed with approximately 40 °C warm H 2 0. The combined filtrates were evaporated to dryness under reduced pressure. The residue was chromatographed on a silica gel column using AcOEt/MeOH (85:15, v/v) to afford 1.80 g (77%) of nucoeoside 23.
  • Acetic anhydride (2.32 mL, 24.5 mmol) was added to a solution of nucleoside 23 (1.71 g, 7.00 mmol) in anhydrous pyridine (10 mL). The reaction mixture was stirred at room temperature for 12 h. After addition of o methanol (5 mL) and stirring for additional 10 min, the mixture was evaporated under reduced pressure to near dryness. The residue was disolved in ethyl acetate (20 mL) and washing was performed using first a saturated aqueous solution of sodium hydrogencarbonate (15 mL) and then brine (15 mL). The separated organic phase was dried over anhydrous sodium sulfate, filtered and 5 evaporated to dryness under reduced pressure.
  • the Lawesson reagent (1.80 g, 4.45 mmol) was added to a stirred solution of compound 24 (2.06 g, 5.57 mmol) in anhydrous 1,2-dichloroethane (50 mL). The reaction mixture was heated under reflux for 4 h and then cooled to room temperature. Methanol (20 mL) was added and the mixture was evaporated to dryness under reduced presuure to give a residue (the crude thiouracile derivative). This residue was immediately disolved in a saturated solution of ammonia in methanol (100 mL) and the resulting mixture was heated at 100 °C for 3 h in an autoclave. After cooling to room temperature, the mixture was evaporated to dryness under reduced pressure.
  • nucleoside 25 (1.00 g, 4.11 mmol) in anhydrous pyridine (20 mL) at 0 °C was added trimethylchlorosilane (3.13 mL, 24.67 mmol).
  • benzoyl chloride (2.38 mL, 20.56 mmol) was added.
  • H 2 0 10 mL was added and stirring was continued for 5 min.
  • Aqueous ammonia (10 mL, 29%, w/w) was added and the mixture was stirred at room temperature for 15 min.
  • 4,4 '-Dimethoxytrityl chloride (0.43 g, 1.3 mmol) was added to a solution of compound 23 (0.26 g, 1.07 mmol) in anhydrous pyridine (5 mL). The reaction mixture was stirred at room temperature for 12 h whereupon methanol was added (2 mL). After stirring for additional 10 min, the mixture was poured into a saturated aqueous solution of sodium hydrogencarbonate (25 mL). Extraction was performed using chloroform (3 x 20 mL), and the combined organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure.
  • 4,4 '-Dimethoxytrityl chloride (0.33 g, 1.0 mmol) was added to a solution of compound 26 (0.12 g, 0.49 mmol) in anhydrous pyridine (5 mL). The reaction mixture was stirred at room temperature for 12 h whereupon methanol was added (2 mL). After stirring for additional 10 min, the mixture was poured into a saturated aqueous solution of sodium hydrogencarbonate (25 mL). Extraction was performed using chloroform (3 x 20 mL), and the combined organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure.
  • the 5'-0-(4,4'-dimethoxytrityl)- ⁇ -L-ribonucleoside 27 (3.20 g, 5.90 mmol) and imidazole (1.04 g, 15.2 mmol) were disolved in anhydrous pyridine (60 mL).
  • tert-Butyldimethylsilyl chloride (1.15 g, 7.6 mmol) was added and the solution was stirred at room temperature for 24 h.
  • the reaction mixture was then poured into a saturated aqueous solution of sodium hydrogencarbonate (120 mL) and extraction was performed using chloroform (3 x 80 mL).
  • Example 5J 4-7V Benzoyl-l-(2-0-te ⁇ -butyldimethylsilyl-5-6>-(4,4'-dimethoxytrityl)- ⁇ -L- ribofuranosyl)cytosine (30).
  • the 5'-0-(4,4'-dimethoxytrityl)- ⁇ -L-ribonucleoside 28 (0.44 g, 0.81 mmol) and imidazole (0.14 g, 2.10 mmol) were disolved in anhydrous pyridine (10 mL).
  • tert-Butyldimethylsilyl chloride (0.19 g, 1.05 mmol) was added and the resulting mixture was stirred at room temperature for 24 h.
  • the reaction mixture was then poured into a saturated aqueous solution of sodium hydrogencarbonate (25 mL) and extraction was performed using chloroform (3 x 20 mL).
  • nucleoside 29 (0.26 g, 0.40 mmol) in dichloromethane (10 mL) at room temperature was added NJY- diisopropylethylamine (0.69 mL, 3.95 mmol). After dropwise addition of 2- cyanoethyl N,N'-diisopropylphosphoramidochloridite (0.38 mL, 1.98 mmol), the mixture was stirred at room temperature for 15 h. Dichloromethane (20 mL) and triethylamine (5 mL) were added and the mixture was washed with saturated aqueous sodium hydrogencarbonate (25 mL).
  • furanose 38 (9.84 g, 19.00 mmol) in THF (150 mL) at room temperature was added a solution of 1 M TBAF (38.0 mL, 38.0 mmol). The mixture was stirred at room temperature for 12 h and was then evaporated to dryness under reduced pressure. The residue was dissolved in ethyl acetate (200 mL) and washing was performed using brine (2 x 100 mL). The separated organic phase was dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure.
  • the layer of 0 Celite 545 was washed with chloroform (2 x 50 mL) and the combined filtrate was washed successively with a saturated aqueous solution of sodium hydrogencarbonate (3 x 100 mL) and brine (2 x 100 mL), dried over anhydrous sodium sulfate, filtered and evaporated to dryness under reduced pressure.
  • the residue was purified by column chromatography using a stepwise gradient of 5 methanol (0-5%», v/v) in chloroform as eluent to afford 0.87 g of nucleoside 42 (88%) as a white solid material.
  • nucleoside 42 (0.64 g, 1.06 mmol) was dissolved in a mixture of MeOH (16 mL) and saturated methanolic ammnonia (16 mL). The mixture was stirred 5 0 °C for 1.5 h and then evaporated to dryness under reduced pressure. The residue was coevaporated with 96% EtOH (50 mL) and purified by column chromatography using a stepwise gradient of methanol (0-5%, v/v) in chloroform as eluent to afford 0.55 g of nucleoside 43 (92%) as a white solid material. 13 C NMR (CDC1 3 ) ⁇ 63.9, 72.7, 80.6, 82.3, 91.4, 123.2, 128.0, 128.1,
  • Nucleoside 43 (0.45 g, 0.80 mmol) was dissolved in a mixture of anhydrous dichloromethane (20 mL) and anhydrous pyridine (4 mL). The stirred solution was cooled to -30 °C and trifluoromethanesulfonic anhydride (0.35 mL, 2.15 mmol) was added. After 1.5 h, the reaction mixture was allowed to warm to 0 °C and a saturated aqueous solution of sodium hydrogencarbonate (10 mL) was added followed by addition of dichloromethane (60 mL).
  • the oligomers were synthesized on an automated DNA synthesizer using the phosphoramidite approach (Camthers, M. H. Ace Chem. Res. 1991, 24, 278). Building blocks 7, 31 and 32 were used for the synthesis of the ⁇ -L- RNA oligomers, e.g., 11, 12 and 16-20 and 33-36.
  • the stepwise coupling yield for amidite 7 was approximately 98%, for amidite 31 approximately 94%, and for amidite 32 approximately 91% (20 min coupling time; lH-tetrazole as activator) using procedures described in Rajwanshi, V. K.; Hakansson, A. E.; Dahl, B. M.; Wengel, J. Chem. Commun. 1999, 1395.
  • the residue was desilylated for 20 h at 55 °C or 20 h at RT using a method described by Wincott, E; DiRenzo, A.; Schaffer, C; Grimm, S.; Tracz, D.; Workman, C; Sweedler, D.; Gonzalez, C; Scaringe, S.; Usman, N. Nucleic Acids Res. 1995, 23, 2677. Desilylation of the oligomers was preferentially performed at 55 °C as incomplete reaction was indicated for some oligomers when using milder desilylation conditions as revealed by MALDI-MS analysis.
  • the ⁇ -L-LNA phosphoramidite was prepared as out-lined in WO 00/66604 and the ⁇ -D-LNA phosphoramidite was prepared as outlined in WO 99/14226.
  • the various natural nucleobases in-corporated into the oligomers was obtained as phosphoramidites from commercial suppliers.
  • the reference DNA oligomers 8 and 13, the LNA oligomers 9 and 14, and the ⁇ -L-LNA oligomers 10 and 15 have been prepared and studied previously (Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem.
  • Hybridization experiments i.e., determination of T m values toward single-stranded DNA and RNA complements were conducted as described by Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; Kumar, R.;
  • oligomers 8-12 different variations of a 9-mer mixed-base sequence are studied.
  • Both 9 and 10 display a weak RNA selectivity as witnessed by the slightly lower thermal stabilities of the duplexes involving the DNA complement.
  • oligomer 12 When three ⁇ -L-RNA monomers are incorporated (oligomer 12), hybridization towards both DNA and RNA is adversely influenced. However, hybridization is more adversely influenced towards DNA.
  • the results for oligomers 11 and 12 indicate that the incorporation of one or more ⁇ -L-RNA monomers into an oligomer may impart selectivity towards hybridization with RNA targets into an oligomer.
  • T depicts a ⁇ -D-LNA monomer bearing a thymine nucleobase (thymin-1-yl)
  • ⁇ L T depicts a ⁇ -L-LNA monomer bearing a thymine nucleobase
  • L T depicts ⁇ -L- LNA bearing a thymin-1-yl group.
  • T m values Melting temperatures (T m values) towards complementary single-stranded DNA and RNA targets were obtained as the maximum of the first derivative of the melting curve (A 26 o vs temperature) in a salt buffer (10 mM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0) using 1.5 ⁇ M concentrations of the two complements.
  • a comparable RNA selectivity has not been observed for longer homothymine sequences (e.g., 14) nor for partly or fully modified LNAs with mixed base compositions (see Oeram, H.; Jacobsen, M. H., Koch, T.; Vuust, J.; Borre, M. B. Clin. Chem. 1999, 45, 1898 and Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V K.; Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54
  • results for 14-mer DNA, LNA/DNA, ⁇ -L-LNA DNA, and ⁇ - L-RNA/DNA chimera as homothymine oligomers are depicted showing similar results as summarized in Table 1 above for the 9-mer series.
  • DNA-R DNA reference
  • DNA-T DNA target
  • RNA-T RNA target oligomers
  • Tm [33:DNA-T] no Tm detectable above 5 °C
  • Tm [33:RNA-T] 16 °C
  • ⁇ -L-RNA/ ⁇ -L-LNA chimera toward 3'-exonucleolytic degradation in vitro was evaluated using snake venom phosphodiesterase (SVPDE) using a procedure previously described by Rosemeyer, H.; Seela, F. Helv. Chim. Acta 1991, 74, 748 and Svendsen, M. L.; Wengel, J.; Dahl, O.; Kirpekar, F.; Roepstorff, P. Tetrahedron 1993, 49, 11341.
  • SVPDE snake venom phosphodiesterase
  • ⁇ -L-RNA/ ⁇ -L-LNA chimera 19 and 20 are both very significantly stabilized toward degradation by SVPDE.
  • ⁇ -L-RNA/ ⁇ -L-LNA chimera like ⁇ -L-DNA (see Asseline, U.; Hau, J.-E; Czernecki, S.; Le Diguarher, T.; Perlat, M.-C; Valery, J.-M.; Thuong, N. T. Nucleic Acids Res. 1991, 19, 4067), are significantly protected toward 3'-exonucleolytic degradation.
  • T TL depicts a ⁇ -D-LNA monomer bearing a thymine nucleobase (thymin- 1-yl)
  • ⁇ Lr TpL depicts a ⁇ -L-LNA monomer bearing a thymine nucleobase
  • ⁇ L T depicts a ⁇ -L- LNA bearing a thymin- 1-yl group.
  • T m values Melting temperatures (T m values) towards complementary single-stranded DNA and RNA targets were obtained as the maximum of the first derivative of the melting curve (A 26 o vs temperature) in medium salt buffer (10 mM sodium phosphate, 100 mM sodium chloride, 0.1 mM EDTA, pH 7.0) using 1.5 ⁇ M concentrations of the two complements.
  • the complementary RNA oligonucleotide target was 5'-[ 32 P]-labelled with polynucleotide kinase and mixed with unlabelled RNA oligo.
  • RNA/DNA oligomer to be studied (34-36) was hybridized to the complementary RNA (4:1 ratio) in 20 mM Tris-HCl (pH 7.5), 100 mM KCl at
  • the RNase H digest was performed in 20 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 , 100 mM KCl, 1 mM DTT by mixing the E.coli RNase H enzyme and the mixture of hybridized oligonucleotides and incubating at 37 °C. Aliquots of the reaction mixture were removed and stopped at 5 and 60 minutes after the initiation of the reaction and the reaction products were analyzed by electrophoresis on 20% acrylamide/urea gels followed by autoradiography.
  • RNA target sequence 5'-r(AGG UCC AUA GAG AC)
  • Control (Co) is the complementary RNA oligonucleotide target that was 5'- [32P]-labelled with polynucleotide kinase.
  • ⁇ L U ⁇ -L-RNA uracil monomer
  • oligomer 34 supports RNase H cleavage when hybridized to the complementary RNA sequence, but oligomer 34 is less efficiently cleaved than oligomers 35 and 36.
  • RNA complement is cleaved most efficiently, but not exclusively, 3 'to the ⁇ -L-RNA residue in the corresponding oligonucleotide. This suggests that
  • RNAse H cleavage can take place in close proximity to an ⁇ -L-RNA monomer.

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Abstract

La présente invention concerne de nouveaux monomères d'ARN-L-α, qui, lorsqu'ils sont incorporés dans un oligonucléotide altèrent une tendance plus élevée à l'hybridation avec un complément d'ARN, comparé à un complément d'ADN. L'invention concerne également un procédé de préparation d'un oligonucléotide modifié d'ARN-L-α et un intermédiaire permettant sa fabrication. Lesdits nouveaux oligonucléotides sont utiles dans une variété d'applications thérapeutiques, diagnostiques et de biologie moléculaire générales.
PCT/IB2002/005080 2001-11-05 2002-11-05 Oligonucleotides modifies a l'aide de nouveaux analogues d'arn-l-alpha WO2003039523A2 (fr)

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