EP0302881A4

EP0302881A4 - Polynucleotide probes on solid support through photo-labile linkage

Info

Publication number: EP0302881A4
Application number: EP19870902955
Authority: EP
Inventors: Ashok Kumar Koul
Original assignee: EI Du Pont de Nemours and Co
Current assignee: EIDP Inc
Priority date: 1986-04-02
Filing date: 1987-03-31
Publication date: 1992-01-15
Also published as: EP0302881A1; JPH01500003A; WO1987005942A1

Abstract

Polynucleotide probes bound to solid support through photolabile linkage.

Description

POLYNUCLEOTIDE PROBES ON SOLID SUPPORT THROUGH PHOTO-LABILE LINKAGE FIELD OF THE INVENTION

The subject invention relates to polynucleotide hybridization assays and provides polynucleotide probes attached to solid supports through photo-labile linkages.

BACKGROUND OF THE INVENTION

Polynucleotide hybridization assays are used as research and diagnostic tools for the detection and identification of unique or specific polynucleotide sequences in samples of complete, fragmented, or mixed nucleic acid. Various hybridization formats have been developed.

Southern, J. Mo I. Biol., 3J3:503 (1975), discloses a polynucleo¬ tide hybridization technique employing radiolabeled nucleic acid probes. This procedure permits autoradiographic detection of probe/analyte hybrids and identification of the polynucleotide se¬ quence of the analyte. However, the Southern procedure, as well as the other diagnostic procedures employing radiolabeled nucleic acid probes, are very complex, time consuming, and have the additional problems and expenses generally associated with radioactive materials such as personnel monitoring and disposal. Thus, such assays have remained a tool of basic research and are not generally employed in applied or commercial areas such as clinical diagnosis.

Ward et al., European Patent Application No. 82301804.9, pub¬ lished June 4, 1982, disclose compositions useful as probes in bio- medical research and recombinant DNA technology, wherein said probes comprise purine, 7-deazapurine or pyrimidine covalently coupled to a moiety capable of forming a detectable complex with a polypeptide, said moiety being coupled to purine bases at the 8-posi- tion, to deazapurine bases at the 7-position, or to pyrimidine bases at the 5-position to form a modified nucleotide. The resulting modified nucleotides are incorporated into DNA by nick-translation techniques.

Ranki and Soderland, U.S. Patent 4,486,539, disclose a tech¬ nique for. the sandwich hybridization of nucleic acids, said technique co prising contacting single-stranded nucleic acid from a microor¬ ganism with a pair of different nucleic acid reagents, both reagents of the pair being single-stranded and complementary with the microor^¬ ganism-derived nucleic acid and one of the pair being a nucleic acid fragment attached to a solid carrier , such as a nitrocellulose filter , while the other is a nucleic acid fragment labeled with a radioactive marker , whereby a labeled hybrid is formed attached to the solid carrier , for the identification of a microorganism or group of microor¬ ganisms present in a sample. The correctness of the identification is tested by detection of the extent of formation of a labeled hybrid attached to the solid carrier.

Tchen et al . , PCT Application No. PCT/ FR82/00220 , published July 7 , 1983 , disclose nucleic acid probe compositions which have been chemically modified by the covalent attachment of at least one N-2-acetylaminofIuorene group to one of the bases of the nucleic acid. After hybridization with the target homologous nucleic acid sequence, such hybridization may be detected by the use of enzyme-labeled antibodies .

Kourilsky et al . , PCT Application No. PCT/FR82/00223 , pub¬ lished July 7, 1983 , disclose DNA molecules modified by covalent attachment of an oligomer of modified ribonucleotides, or a single modified ribonucleotide , which provides a means for coupling a chemi¬ cal capable of recognition by another molecule or product.

Co-pending and commonly assigned U . S . Application Serial No. 574,630 , discloses polynucleotide probe compositions which contain a polypeptide moiety capable of enzymatically activating a zymogen to , initiate a detectable enzymatic reaction cascade.

Falkow et al . , U. S. Patent 4,358 ,535 issued November 9 , 1982 , disclose a method for detecting the presence of a pathogen in a clinical sample by depositing and fixing said sample on an inert support and hybridizing the genetic material of the target pathogen to a labeled-nucleic acid probe. The label may be a radioisotope , a ligand , a fluorescer , a chemiluminescer, an enzyme , or an antibody.

Kourilsky et al . , U . K . Patent Application No. 7913031 , published October 31 , 1 979 , disclose a method for detecting the possible presence of a DNA fragment in a sample comprising the hybridization of the sought fragment with an RNA probe which is coupled to an en¬ zyme either prior to or subsequent to the hybridization reaction . The possible presence of the target nucleic acid sequence is revealed by the action of the enzyme-labeled hybridization product on a chromogen substrate.

Heller, et al . , European Patent Application No. 82303701 .5 , pub¬ lished January 26 , 1 983 , disclose a heterogeneous hybridization diag¬ nostic method which uses luminescer-labeled , single-stranded poly- nucleotides. After separation of the unhybridized reagent, the sample is exposed to light. Any subsequent light emission is related to the amount of target polynucleotide in the sample. The label may be any of the wel l known luminescent systems.

Heller et al . , European Patent Application No. 82303699. 1 , pub¬ lished July 14 , 1982 , disclose a homogeneous light-emitting hybridization assay wherein luminescer-labeled first and second single-stranded reagent segments are hybridized with a complementary target single-stranded polynucleotide from a physiological sample such that nonradioactive energy transfer occurs between the labels of the two reagent segments . At least one of the labels is of the absorber/emitter type such that energy in the form of a photon absorbed from the other light label is re-emitted as a different wave¬ length . Such secondary emissions can only occur if hybridization has taken place.

Arnold , PCT/US84/01351 published March 14 , 1985 discloses a polymeric support system for the synthesis of oligonucleotides featuring universal oxidatively cleaved primers for oligonucleotide synthesis .

Ohtsuka , et al . disclose 2'-0-(o-nitrobenzyl ) derivatives of uridine , adenosine and cytidine useful in the synthesis of ribo- oligonucleotides . The 2'-ether provides 2' protection and such 2'-ether linkage is photo-labile so that the resulting ribo- oligonucleotide can be collected . SUMMARY OF THE I NVENT I ON

In one aspect, the instant invention provides polynucleotide probes , both RNA and DNA , bound to a solid support through photo-labile linkages . Such support-bound probes may be utilized in conventional sandwich hybridization assays and subsequently cleaved by photolysis for measurement of sandwich pairs . Al ternatively , the unhybridized probes may be cleaved by photolysis for subsequent use in any of the well known hybridization assay formats .

In another aspect of this invention , a support-bound photo-labile primer is provided for attachment of polynucleotide probes thereto for subsequent use in sandwich hybridization assays. Alternatively , such solid-support primers can be used in "building" polynucleotide probes either for use in sandwich hybridization assays or for subsequent photo-cleavage thereof for use in any of the well known hybridization assay formats.

The photo-labile l inkages of this invention have the advantage of being stable to normal hybridization conditions , as well as to con¬ ditions necessary for the synthesis of polynucleotides , e. g. , the conventional "blocking" and "de-blocking" steps associated with the protection of exocyclic a ino groups and 5'-hydroxyl groups during DNA probe synthesis.

DETAI LED DESCRIPTION OF THE I NVENTION

The polynucleotide probe of this invention , bound to a solid support through a photo-labile linkage is of the formula

3' where SS is the solid support, and 0 represents the 3' oxygen of a nucleotide moiety in the polynucleotide probe. Also , the phenyl ring may be naphalene provided that the nitro group is on the ring attached to the 3' oxygen of the nucleotide moiety.

The sol id support can be of a number of polymer supports. Examples of such supports are fluoropolymer resins , polystyrene resins , silica , nylon and graft copolymers; and preferred resins include chloromethylated and bromomethylated resins. These supports are preferably utilized in the form of beads of about 0. 1 to 200 microns in diameter.

The support-bound photo-labile primer of this invention is of the formula

where SS is the solid support bound to the phenyl ring as discussed above, and X is a halogen such as Cl or Br. This primer is useful for the synthesis of polynucleotide probes (as exemplified below) , or for the attachment of previously synthesized (or natural ) polynucleotide probes .

Once the polynucleotide probes are bound to the ^"support through the photo-labile linkage , they can be cleaved by application of UV light having a wavelength of between about 280 nm and 350 nm. As seen in the example below , cleavage takes place between the 3' oxygen and the adjacent benzylic group of the linkage. Of course , when ribonucleotides are utilized , the 2' oxygen is similarly available for such linkage. This cleavage can be carried out simply to collect the polynucleotide probes for later use, or can be carried out follow¬ ing use of the probe as the "capture" probe in a typical sandwich hybridization assay - in which case the "sandwich" is collected for counting . The process for carrying out this sandwich hybridization assay is described in detail in , e .g . , U . S. 4 ,486 ,539. This invention also allows for a diagnostic kit useful in the detection of a target polynucleotide analyte in a physiological sample wherein the kit comprises a first polynucleotide probe bound to a solid support through a photo-labile linkage , and a second polynucleotide probe , both the first and second probes being substantially complementary to substantially mutually exclusive single-stranded regions of the analyte.

^! As used herein , the term "target polynucleotide analyte" refers to a segment of single-stranded polynucleotide having a nucleotide base sequence corresponding to a genetic element whose presence in a -6-

physiological sample is to be detected and/or identified; the term "substantially complementary" refers to sufficient nucleotide base sequence homology between a polynucleotide probe and its target analyte to permit formation of stable probe-analtye hybrids; the term "physiological sample" means a sample of blood, urine or other biolog¬ ical tissue, either unprocessed or processed, containing the DNA or RNA of interest; and the term "substantially mutually exclusive" means that upon hybridization by the first and second probes with each target analyte, the two probes should not compete for the same nucleotide base sequence on the analyte to the extent that hybridization is prevented.

Also as used herein, "polynucleotide" refers to a polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), which can be single- or double-stranded, optionally incorporated or comprising synthetic, non-natural, or altered nucleotides capable of incorporation into DNA or RNA polymers. In the case of natural probe polynucleo- tides, such probes can be conveniently isolated in useful quantities by cloning and amplification of polynucleotide sequences complemen¬ tary to target polynucleotides in plasmid or phage vectors, using techniques that are now conventional to those skilled in the art. A useful reference covering most aspects of DNA manipulation is Maniatis et al., Molecular Cloning, A Laboratory Manual. (Cold Spring Harbor Laboratory, 1982), the disclosure of which is incor¬ porated herein by reference. An exemplary cloning vehicle for production of useful quantities of probe polynucleotides is plasmid pBR322 (ATCC 37017), which is described in detail by Rodriquez, et al., in Scott, ed.. Molecular Cloning of Recombinant DNA, (Aca¬ demic Press, New York, 1977), p. 72. This plasmid contains single Pstl, Baml , EcoRI , Hindlll , and SaU restriction endonuclease recogni¬ tion sites, in addition to genes conferring resistance to the antibiotics tetracycline and ampicilliπ. Plasmid DNA can be amplified by growth in the presence of chloramphenicol (170 ug/ml) according to the method of Clewell, J. Bacteriol. J1_0:667 (1972); and purified by the cleared lysate procedure of Guerry et al., J. Bacteriol. 116:1064 (1973), prior to digestion with an appropriate endonuclease. For example, digestion with Pstl inactivates the ampicillin resistance marker and generates "sticky ends" suitable for ligation to a probe polynucleotide similarly cleaved with Pstl. The resulting recombinant plasmid can then be employed to transform a suitable host bacterium, e.g., E. coli K12 HB101. Upon growth in the presence of chloram- phenicol, high plasmid copy numbers can be attained and the recombinant plasmid DNA isolated and purified as previously de¬ scribed. However, a particularly preferred vector for production of probe polynucleotides is a coiiphage, M13, (ATCC 15669-BI) which, like pBR322, is now commercially available (New England Nuclear Corporation, Boston, Massachusetts, USA). DNA fragments obtained by digestion of phage DNA and DNA complementary to a target DNA of interest can be joined, amplified, and subsequently purified in single-stranded form prior to conjugation with a reporter molecule, e.g., an enzymatic activator polypeptide such as peroxidase. The use of M13 phage as a cloning vehicle has been described by Messing, Recombinant DNA Tech. Bull. 2:43, (1979), the disclosure of which is hereby incorporated by reference.

In the case of synthetic probe polynucleotides, the synthesis of same is described in the -literature. In fact, DNA synthesizers are commercially available. These synthetic probes consist of between about 12 and 500 nucleotide moieties. The base residue can be any purine, modified purine, pyrimidine, or modified pyrimidine base capable of stable incorporation into a single-stranded polynucleotide without significantly affecting the capacity of the polynucleotide to form hybrids with target polynucleotides having substantial complementarity. However, a common feature of all base residue useful in the present invention is a point or points suitable for attachment, preferably covalent, of any of the well known reporting mechanisms. Thus, apart from the "classic" bases adenine, guanine, cytosine, uracil and thymine, other, less common bases, e.g., 5-methylcytosine, 5-hydroxymethylcytosine, orotic acid derivatives, methylated bases, e.g., 1-methylguanine, etc., can optionally be incorporated into the probes of the present invention. Further, the nucleotides can optionally comprise various substituents, which can be linked to either base or sugar portions, and which do not deleterious- ly affect the capability of the resulting polynucleotide to form hybrids with complementary target polynucleotides. Polymer "tails" comprising a number of nucleotides appropriate for conjugation to a reporting system can be added to probe polynucleotides by use of calf-thymus terminal deoxynucleotidyl transferase (TdT), which catalyzes the addition of deoxynucleotides to the 3'-hydroxyl ends of single- or double-stranded DNA, as disclosed by Roychoudhury et al., Nucleic Acid Res. 3:101 (1976).

The following Example is an illustration of an application of the subject invention.

EXAMPLE Nitration of chloromethylated resin:

Synthesis of the o-nitro-chloromethyl polymer proceeds by the procedure of R. B. Merrifield (Journal of the American Chemical Society, 85:2149-2154, 1968). A 5 gm sample of dry chloromethyl polymer (copolystrene-1% divinyl-benzene 5.07m.eq. of Cl/g) is added slowly with stirring to 500 ml. of fuming nitric acid (90% HNO- which has been cooled to -10°C). After the addition of all resin, the mixture is stirred at -5°C for 1 hour and then poured into crushed ice. The resin is filtered through sintered glass funnel and washed with water (2 x 500 mis.), dioxane (2 x 300 mis.), and methanol (2 x 200 mis.). The resin is dried under vacuum for 2 hours and stored over Drierite in freezer. Nitrogen analysis of the nitrated resin is done to confirm appropriate nitration of the chloromethylated resin.

Attachment of 5'-0-blocked 2'-deoxynucleoside to o-nitro-chlorome- thylated resin:

Attachment of 5'-0-bIocked nucleoside to o-nitro-chloromethyiated resin proceeds by the procedure of E. Ohtsuka, S. Tanaka, M. Ikehara (Synthesis 453-54, July 1977) for the protection of 2'-OH groups of ribonucleosides by o-nitrobenzyi bromide. Sodium hydride ( 14. 78 mg/ 440 unoles ) is weighed in a serum capped centrifuge tube ( 15 mis. ) and held under Argon atmosphere. Dry dimethylformamide (5 mis . ) is added to sodium hydride through the serum cap with a syringe (5 mis . } . The suspension of sodium hydride/dimethylformamide is withdrawn with another syringe and transferred to a three-necked flask containing 5'-0-blocked 2'-deoxy nucleoside in dry dimethylformamide (2 mis . ) under Argon atmosphere. After hydrogen gas generation has ceased , o-nitrochloromethyl resin (84 mg/440 umoles ) is added in small incre¬ ments . The mixture is allowed to stir at room temperature for 3 hours and then is left standing at room temperature overnight. The resin is filtered through a sintered glass filter funnel and washed successively with dimethylformamide (2 mis . x 2) , 90% dimethyl¬ formamide in water (2 mis. x 1 ) , and acetonitri le (2 ml . x 2 ) . The resin is then dried in a desiccator under vacuum.

Selective removal of purine/pyrimidine amino-protecting groups, i .e. benzoyl and isobutyryl type :

I n order to test whether the ether linkage binding nucleoside to the resin is stable to alkali while the blocking groups are selectively removed from the purine/ pyrimidine protecting groups , the dimethoxytrityl nucleoside bound to resin is analyzed in the following manner:

Resin ( 1 -2 mg ) is treated with cone, ammonium hydroxide ( 1 ml . ) and allowed to react in a sealed glass ampule in a heating block overnight at 55°C. The resin is centrifuged , and the supernatant (ammonium hydroxide) transferred to another tube. Both the resin and the supernatant are analyzed for the presence of dimethoxytrityl nucleoside based on the liberation of dimethoxytrityl cation when treated without any processing with trichloroacetic in methylene chloride , (supernatant is first evaporated to dryness before treating it with trichloracetic acid in methylene chloride) . The presence of most (90-95%) of the dimethoxytrityl cation in resin compared to the supernatant proves that the 5'-dimethoxytrityl blocked nucleoside is still bound to the nucleoside and thereby confirming that the ether linkage established between the nucleoside and the resin is stable to the alkaline treatment under which the amino blocking groups from purines/pyrimidines are selectively removed . Synthesis of oligodeoxynucleotide on o-nitrochlorpmethylated resin :

O-nitrochloromethylated resin is used to synthesize oligodeoxy- nucleotides either manually or on a DNA synthesizer using phosphora- midite or phosphotriester chemistry. In either case, synthesis begins with the attachment of the first deoxynucleoside (at the 3'-end) to o-nitrochloromethylated resin fol lowing the procedure described earlier. The chain is then extended stepwise to the desired length through the 5'-end of the of the oligomer by coupling subsequent nucleoside phosphodiesters or nucleoside phosphoramidites to the 5'-OH end of the preceding nucleoside. Once the desired oligomer is synthesized , it can be subjected to the alkaline treatment (at 55°C) to deblock amino protecting groups of the purine/pyrimidine bases. Under these conditions , the oligomer stays immobilized to support.

Attachment of completely blocked synthetic oligonucleotide to o-nitrochloromethylated resin :

The procedure to attach completely blocked (protected at 5'-OH group and purine/pyrimidine amino groups) oligonucieotides to the o-nitro-chloromethylated resin proceeds by the procedure of attaching a single nucleoside to the resin as described above.

Removal of the synthetic oligonucleotide from photo-labile support:

The cleavage of the finished oligonucleotide or the hybridized double stranded oligomer from the photo-labile support can be done by irradiating the sample to 320nm lamp for 4 hours in an RPR-100 apparatus ( Ryanote , the Southern Co. , Ha den , Connecticut) .

Claims

C LA I MS WHAT I S CLAIMED I S :

1 . A composition comprising a polynucleotide probe bound to a solid support through a photo-labile linkage .

2. The composition of claim 1 wherein the probe bound to the solid support through a photo-labile linkage is of the formula

3' ^■ where SS is the solid support and 0 represents the 3 oxygen of a nucleotide moiety in the polynucleotide probe.

3. The composition of claim 2 wherein SS is selected from chloromethylated resins , fluoropolymer resins , polystyrene resins , silica , nylon and g raft copolymers .

4. The composition of claim 3 wherein SS is a chloromethylated or bromomethyiated resin .

5. A composition comprising a support-bound photo-labile primer of the formula

⁾ where SS is a solid support and X is a halogen ion .

6. The composition of claim 5 wherein X is Cl or Br.

7. The composition of claim 5 wherein SS is as defined in claim 3.

8. The composition of claim 6 wherein SS is as defined in claim 3.

9. The composition of claim 7 wherein SS is a chloromethylated or bromomethyiated resin .

10. The composition of claim 8 wherein SS is a chloromethylated or bromomethyiated resin .

1 1 . A kit useful in the detection of a target polynucleotide analyte comprising a first polynucleotide probe bound to a solid support through a photo-labi le l in kage , and a second polynucleotide probe , both the first and second probes being substantially comple- mentary to substantially mutually exclusive single-stranded regions of the analyte.

12. The kit of claim 11 wherein the polynucleotide probe bound to a solid support through a photo-labile linkage is of the formula of claim 2.