JPH01500003A - Polynucleotide probes on solid supports with photolabile binding - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] On a solid support by photolabile bonding polynucleotide probe of ＾Kaiyu JjIL1 The present invention relates to polynucleotide hybridization assays, and Provides polynucleotide probes attached to solid supports via photolabile linkages do.

Background of the invention Polynucleotide hybridization assay complete or flag unique or unique polynucleotides in a sample of nucleic acids, or mixed nucleic acids. It is used as a research and diagnostic tool to detect and identify code sequences. seed Various hybridization methods have been developed.

5outhern, J, Mo1. Biol, 98: 503 (197 5) Polynucleotide hybridization using radiolabeled nucleic acid probes tion method is disclosed. This technique is useful for probe/analyte hybrid operation. Enables radiographic detection and identification of analyte polynucleotide sequences Make it. However, using the 5outhern method and radiolabeled nucleic acid probes Other diagnostic methods used are extremely complex, time-consuming, and generally involve radioactive materials. related issues and associated costs, such as stakeholder monitoring and disposal. Requires processing, etc. For this reason, such assays have become a basic research tool. It is generally used in applied or commercial fields such as clinical diagnostics. I'm not happy.

European Patent Application No. 82301804 by Ward et al., published June 4, 1982. Specification No. 9 is useful as a probe in biomedical research and recombinant DNA technology. discloses compositions for use in detectable complexes with the polypeptide; A purine, 7-deazapurine or pyrimidine covalently linked to a moiety capable of forming a and the moiety is at position 8 of a purine base, at position 7 of a deazapurine base, or at position 7 of a purine base, or It binds to the 5th position of the rimidine base to form a modified nucleotide. the resulting modification Nucleotides are incorporated into DNA by nick translation.

US Pat. No. 4,486,539 to Ranki and 5oderland Although the specification discloses a method for sandwich hybridization of nucleic acids, this The method involves contacting single-stranded nucleic acids from microorganisms with a pair of different nucleic acid reagents. The reagents in the pair are both single-stranded and complementary to the microbial-derived nucleic acid. and one of the pairs is bound to a solid support such as a nitrocellulose filter. one is a nucleic acid fragment labeled with a radioactive marker, the other is a nucleic acid fragment labeled with a radioactive marker. , thereby forming a labeled hybrid bound to the solid support, which can be detected. Identify microorganisms or groups of microorganisms present in the body. The accuracy of identification depends on the solid support. The test is performed by detecting the degree of formation of combined labeled hybrids.

PCT Application No. PCT/FR821 by Tchen et al., published July 7, 1983 00220 specifies at least one N-2-acetylaminofluorene group. Chemically modified nucleic acid probe compositions by covalent attachment to one of the bases of a nucleic acid disclosing something. After hybridization with target homologous nucleic acid sequences, such hybridization Hybridization can be detected by using enzyme-labeled antibodies .

PCT Application No. PCT/F by Kourilsky et al., published July 7, 1983 R82100223 specifies a chemical substance that can be recognized by another molecule or product. An oligomer or monomer of modified ribonucleotides that provides a means for binding DNA molecules modified by covalently bonding full-strand modified ribonucleotides Disclosed.

Co-pending and common assignee U.S. Patent Application No. 574,630 is detectable The precursor can be enzymatically activated to initiate a cascade of enzymatic reactions. Polynucleotide probe compositions that include a lipeptidic moiety are disclosed.

U.S. Pat. No. 4J58,5 to Falkov et al., issued November 9, 1982. No. 35 discloses that the presence of pathogens in a clinical specimen is determined by depositing the specimen on an inert support. , immobilize and hybridize the genetic material of the target pathogen to a labeled nucleic acid probe. This disclosure discloses a method for detecting by forming.

Labels can be radioisotopes, ligands, fluorogens, chemiluminescent substances, enzymes or may be an antibody.

UK Patent Application No. 79 by Kourilsky et al. published October 31, 1979 No. 13031 discloses a method for detecting the presence of DNA fragments in a specimen However, this method requires fermentation prior to or after the hybridization reaction. Hybridization of the naturally bound RNA probe and the fragment to be searched It consists of causing Presence of target nucleic acid sequence indicates enzymatic labeling of chromogenic substrate revealed by the action of hybridization products.

European Patent Application No. 8230 by Eeller et al., published on January 26, 1983 No. 3701.5 discloses a luminescent material label, a heterogeneous method using a single-stranded polynucleotide. A hybridization diagnostic method is disclosed. not hybridized After separating the reagents, the specimen is exposed to light. All subsequent luminescence is caused by the target polynuc in the specimen. It is related to the amount of leotide. The label may be any known luminescent system.

European Patent Application No. 82303 by Heler et al., published July 14, 1982 No. 699.1 discloses a homogeneous luminescent hybridization assay. This allows the bioluminescently labeled first and second single-stranded reagent segments to be Hybridization with complementary target single-stranded polynucleotides obtained from biological specimens and thereby generate non-radioactive energy between the two reagent segment labels. -It causes metastasis. At least one of those marks is an absorber. energy in the form of photons absorbed from the other optical label. This allows the light to be re-emitted at a different wavelength. Such a second emission is the hive This can only occur if redization is performed.

PCT/US8410135 by Arnold, published September 14, 1985 Specification No. 1 discloses a completely oxidatively cleaved protein for oligonucleotide synthesis. Discloses a polymeric support system for oligonucleotide synthesis featuring a primer are doing.

Uridine and adenosylene useful in the synthesis of oligonucleotides 2'-0-(o-nitrobenzyl) derivatives of cytidine and cytidine are disclosed.

The 2'-ether protects the 2', and such a 2'-ether bond Since the temperature is constant, the resulting oligonucleotides can be collected.

Summary of the invention In one aspect, the present invention provides a polymer bonded to a solid support by a precursor stabilizing bond. Provides polynucleotide probes (both RNA and DNA). Such a support Bound probes are used in conventional sandwich hybridization assays. The sandwich pair can then be photolytically degraded for measurement. Ru. Alternatively, unhybridized probes can be photolytically degraded. can then be used in any well-known hybridization assay format. Ru.

In another aspect, after binding the polynucleotide probe, the sandwich Support-bound photolabile probes for use in touch-type hybridization assays Rymer provided. Alternatively, such a solid support primer can be for use in switch-type hybridization assays or for digestion and use in any known hybridization assay format. can be used to "form" polynucleotide probes.

The pioneer stable linkages of the present invention are resistant to conventional hybridization conditions and also Conditions necessary for polynucleotide synthesis, such as exocyclic amines during DNA probe synthesis. Conventional "protection" and "deprotection" associated with the protection of hydroxyl groups and 5'-hydroxyl groups It has the advantage of being stable to solid supports by photolabile bonding. The polynucleotide probe of the present invention has the formula (where SS is the solid support and 03' is the polynucleotide probe (represents the 3' oxygen of the nucleotide moiety in the middle) It is indicated by. In addition, the phenyl ring has a nitro group attached to the 3' oxygen of the nucleotide moiety. Naphthalene may be used as long as it is on the bonded ring.

The solid support can be any number of polymeric supports. Examples of such supports include full Oropolymer resins, polystyrene resins, silica, nylon, and graft copolymers and preferred resins include chloromethylated and bromomethylated resins. Chilled resins are included. These supports are beads with a diameter of 0.1 to 200 microns. It is preferable to use it in the form of a cube.

The support-bound photolabile primers of the invention have the formula (wherein SS is a phenyl group as described above). X is a solid support attached to a ring, and X is a halogen such as Ca or Br. ) It is indicated by. This primer is a polynucleotide primer (such as the one exemplified below). Synthesis of lobes or pre-synthesized (or natural) polynucleotide proteins Useful for joining webs.

When a polynucleotide probe is attached to a support by a pioneering stable linkage, its They can be decomposed by UV light having a wavelength of about 280 nm to 350 nm. Can be done.

As can be seen from the examples below, decomposition involves the bond between the 3' oxygen and the adjacent benzyl group. I wake up. Of course, when using ribonucleotides, the 2' oxygen can be similarly It can be used as the first part of a combination such as . This split is simply for later use. This can be done to collect polynucleotide probes or ``capture'' in a typical sandwich hybridization assay. capture) can be performed after using it as a J probe (in this case, ” are collected for counting). This sandwich type hybridization Methods for carrying out motion assays are described, for example, in U.S. Pat. No. 4,486,539. detailed in. The present invention also provides a method for targeting polynucleotide analytes in physiological samples. It can also be used in diagnostic kits useful for detecting light, but the kits are a first polynucleotide probe and a second polynucleotide probe bound to a solid support by a nucleotide probe, and the first and second probes are analogs. substantially complementary to a substantially mutually exclusive single-stranded region of the .

As used herein, the term "target polynucleotide analyte" refers to Nucleotide corresponding to a genetic element whose presence in a target sample is detected and/or identified refers to a segment of a single-stranded polynucleotide having a base sequence; "Qualitatively complementary" refers to the null between a polynucleotide probe and its target analyte. Creotide sequence homology facilitates the formation of stable probe-analyte hybrids. the term "physiological specimen" refers to the DN of interest; Unprocessed or processed blood, urine or other biological substances containing A or RN A a sample of physical tissue; and the term “substantially mutually exclusive” refers to and a second probe when hybridized with each target analyte. two probes have the same concentration of analyte to the extent that hybridization is prevented. This means that there is no competition between the nucleotide base sequences.

Furthermore, as used herein, "polynucleotide" refers to ribonucleic acid (RNA) or Represents a polymer of oxyribonucleic acid (DNA'), which can be single-stranded or double-stranded. strands, optionally incorporated into DNA or also RNA polymers. Contains or incorporates synthetic, non-natural, or altered nucleotides that may It may be In the case of natural probe polynucleotides, such probes target in plasmid or phage vectors using methods currently routine to those skilled in the art. Cloning or amplifying a polynucleotide sequence complementary to a polynucleotide By widening the range, useful amounts can be conveniently isolated.

A useful reference document describing most of the DNA manipulations is Maniatiset al. Mo1ecular Cloning, A Laboratory Manu al(Cold Spring Harbor Laboratory, 19 82), the disclosure of which is incorporated herein by reference. a useful amount of An example of a cloning vehicle for producing lobe polynucleotides is lasmid pBR322 (ATCC37017), which was produced by Rodriqu ez at al,, 5Cott, ed.

Mo1ecular Cloning of Recoa+binant DN A (Academic Press, New York, 1977) p. 72. This plasmid contains one Pst Is Baa I, EcoRI, HindI[I, and SalI restriction endonuclease recognition sites and confers resistance to the antibiotics tetracycline and ampicillin. Contains genes that Plasmid DNA was obtained from Clewell, J., Bact. Chloramphenicin according to the method of Eriol, 110:667 (1972). It can be amplified by growing it in the presence of Cole (170μ9/pass); prior to digestion with an appropriate endonuclease, as described by Cuerry et al. ,,J.

Clarified lysate manipulation of Bacteriol, 116:1064 (1973) (cleared 1ysate procedure) can. For example, inactivate the ampicillin resistance marker by digestion with PstI. and probe polynucleotide cut in the same manner using PstI. Forms a “sticky end” suitable for coupling to a bond. Then, the obtained recombinant plastic A suitable host bacterium, such as Escherichia coli (E, coli) K12H BIOI can be transformed. High when grown in the presence of chloramphenicol The plasmid copy number can be obtained and the recombinant plasmid DNA It can be isolated and purified as described above. However, the probe polynucleotide A particularly preferred vector for otide production is coliphage, K13, (ATCC 15689-BI), which, like pBR322, is currently not commercially available. (New England Nuclear Corporation, B oston.

Massachusetts, USA). Complementary to the target DNA of interest DNA fragments obtained by digestion of phage DNA and phage DNA are ligated and amplified. After that, a reporter molecule, e.g. an enzyme activator polypeptide such as peroxidase, is added. It can be purified before conjugation with putido. K as a cloning vehicle 13 Phage is used by Messing.

Recombinant DNA Tech, Bull, 2 + 43. ( (1979), the disclosure of which is incorporated herein by reference. .

In the case of synthetic probe polynucleotides, their synthesis is described in the literature. fruit DNA synthesizers are commercially available. These synthetic probes are approximately 1 It consists of a 2-500 nucleotide portion. Base residues are significantly increased in its ability to hybridize with a target polynucleotide with qualitative complementarity. Can be stably incorporated into single-stranded polynucleotides without adverse effects Can be any purine, modified purine, pyrimidine or modified pyrimidine base . However, the common characteristics of all base residues useful in the present invention are a suitable site for binding (preferably by covalent bond) It is. i.e., the "classical" bases adenine, guanine, cytosine, u In addition to rasyl and thymine, other less common bases, such as 5-methyl lucitosine, 5-hydroxymethylcytosine, orotic acid derivative, methylated base , for example, l-methylguanine, etc., can be incorporated into the probe of the present invention as desired. I can do it. Additionally, the nucleotide can optionally be linked to a base moiety or a sugar moiety. and the hybridization of the resulting polynucleotide with a complementary target polynucleotide. Various substituents can be included that do not adversely affect hybridization ability. Rebo A polymeric “terminal” containing many nucleotides suitable for joining into a binding system. ” to the 3′-hydroxyl end of the single-stranded or double-stranded deoxynucleotide calf thymus terminal deoxynucleotidyl transferer that catalyzes the addition of (TdT) to the probe polynucleotide. (Roychoudhury et al.

Nucleic Ac1d Res, 3: 101 (1976)).

The following example is an application example of the present invention.

Example Nitration of chloromethylated resin: Synthesis of 0-nitro-chloromethyl polymer was carried out by R,B.

Merrifield (Journal of the American Chemical Society, 85: 2149-2154.1968 ).

Dry chloromethyl polymer (copolystyrene-1% divinylbenzene, 5.07 ml) While stirring 59 samples of re-equivalent C (l/9), gradually add 500 fuming nitric acid. (90% HNO, cooled to -10 °C). Add all resin. The mixture is stirred for 1 hour at -5°C and then poured into crushed ice. Glass sintered with its resin Filter through a funnel and strain water (2 x 5001i2), dioxane (2 x 30 Wash the resin with 01C and methanol (2x2001C) for 2 hours. Air dry and dry on Drierite in the freezer. of nitrated resin A nitrogen analysis is performed to confirm proper nitration of the chloromethylated resin.

5′-〇-blocked 2′-deoxynucleoside 〇-nitrochloromethane Bonding to thylated resin: 2’-OH group of ribonucleoside is bonded to 0-nitrobenzyl E, 0htsuka, s, Tanaka for protection by bromide.

M, Ikehara (Synthesis 453-54. July 19 77) 5′-0-blocked nucleoside Coupled to romethylated resin.

Sodium hydride (14,78j!fI/440 micromol) using serum Weigh in a stoppered centrifuge tube (153!12) and keep under argon atmosphere. hold Inject dry dimethylformamide (5IQ) into a syringe (5IQ) through a serum stopper. ) to the sodium hydride. Sodium hydride/dimethylformamide The suspension was drawn off using a separate syringe and placed in dry dimethylform under an argon atmosphere. 5′-〇-blocked 2′-deoxynucleoside in Muamide (2 toge) Transfer to a two-headed flask containing When hydrogen gas generation stops, remove 0-nitrochloromethyl resin. C34x9/440 micromol) is added in small portions. The mixture at room temperature Stir for an hour and then leave at room temperature overnight. Glass filter funnel sintered with its resin Dimethylformamide (2i12X2), 90% dimethylformamide in water, was heated through a furnace. Using chloroformamide (2II2x1) and acetonitrile (2x12x2) and wash sequentially. The resin is then vacuum dried in a desiccator.

Purine/pyrimidine amino protecting groups, i.e. benzoyl and isobutyryl Generative removal of purine/pyrimidine protecting groups while the protecting group is selectively removed from the purine/pyrimidine protecting group. , the ether bond that binds the nucleoside to the resin is stable against alkalis. dimethoxytrityl nucleosides bound to the resin to test whether Analyze the code by: The resin (1-2xg) was treated with concentrated ammonium hydroxide (1+0) and heated React overnight at 55°C in a sealed glass ampoule in a container. that resin Centrifuge and transfer the supernatant (ammonium hydroxide) to another tube. resin and supernatant Both were treated with trichloroacetic acid in methylene chloride without any further treatment. (The supernatant was first evaporated to dryness and then diluted with trichloroacetic acid in methylene chloride. dimethoxytrityl cation based on the release of the dimethoxytrityl cation of Analyze for the presence of creosides. Dimethoxytrithi in the resin compared to the supernatant When a large proportion (90-95%) of the cations are present, the 5' becomes dimethoxytrityl. The more protected nucleosides are still bound to the nucleosides, ether bond attached and thereby formed between the nucleoside and the resin is subjected to alkaline treatment, which selectively removes amino protecting groups from purines/pyrimidines. It is confirmed that it is stable.

Synthesis of oligonucleotides using 0-nitrochloromethylated resin: Manually using 0-nitrochloromethylated resin or phosphoramidite or Oligodeoxynucleotide synthesis using a DNA synthesizer using phosphotriester chemistry Synthesize ocide. In either case, using the procedure described above, the synthesis begins with the initial deoxy From binding the nucleoside (3' end) to the 0-nitrochloromethyl resin start. Then the next nucleoside phosphodiester or nucleoside phosphodiester By attaching the lamidite to the 5'-OH end of the nucleoside in front of it The chain is extended stepwise through the 5' end of the oligomer to the desired length. desired o Once the oligomer has been synthesized, it is subjected to alkaline treatment (at 55°C) to The amino protecting group of the rimidine base can be deprotected. Under these conditions, The oligomer remains fixed to the support.

Transfer of fully protected synthetic oligonucleotides to 0-nitrochloromethylated resins Join: fully protected (5'-OH group and Procedure for conjugation of oligonucleotides (with protected purine/pyrimidine amino groups) As described above, the single nucleoside attachment procedure to the resin is used.

Removal of synthetic oligonucleotides from precursor stable supports: of the completed oligonucleotide or hybridized double-stranded oligomer. Decomposition from the forerunner stable support was performed using an RPR-100 apparatus (Ryano te, the 5outhernCo,, Hamden, Connecti This can be carried out by exposing a 320 nm lamp (cut) for 4 hours.

international search report +a′″′a″−”−’PCTノ’: JS87/nnAE0

Claims (12)

[Claims] 1. From a polynucleotide probe attached to a solid support by a photolabile bond composition. 2. The probe attached to the solid support by a photolabile bond is expressed ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (where SS is the solid support and 03' is the polynucleotide probe) (represents the 3' oxygen of the nucleotide moiety) The composition according to claim 1, which is represented by: 3. SS is chloromethylated resin, fluoropolymer resin, polystyrene resin, silicon according to claim 2, which is selected from silica, nylon and graft copolymers. Composition. 4. Claim 3, wherein SS is a chloromethylated or bromomethylated resin composition. 5. formula ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ (wherein SS is a solid support and X is a halogen ion) A composition comprising a photolabile primer attached to a support shown in . 6. 6. The composition according to claim 5, wherein X is Cl or Br. 7. A set according to claim 5, wherein SS is defined in claim 3. A product. 8. A set according to claim 6, wherein SS is as defined in claim 3. A product. 9. Claim 7, wherein SS is a chloromethylated or bromomethylated resin composition. 10. Claim 8, wherein SS is a chloromethylated or bromomethylated resin Compositions as described. 11. a first polynucleotide protein attached to a solid support by a photolabile linkage; a polynucleotide probe and a second polynucleotide probe; Both probes are directed to substantially mutually exclusive single-stranded regions of the analyte; A kit useful for detecting target polynucleotide analytes that are complementary to each other. 12. Polynucleotide probe attached to solid support by photolabile linkage 12. The kit according to claim 11, wherein has the formula according to claim 2.