JPH06509945A - Crosslinkable oligonucleotides for enzyme-mediated triplex formation - 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] Cross-linking oligonucleotides for enzyme-mediated duplex formation This invention was developed in September 1988. Application number 07/250.474 filed on May 28th (currently abandoned) It is a partial continuation of the above, and the application number filed on March 18, 1989 This is a partial continuation of No. 07/353.857.

Background of the invention TECHNICAL FIELD The present invention relates to a nucleotide crosslinking agent and in the production of oligonucleotides. Concerning the use of these compounds. The present invention also provides for oligonucleotide production. The present invention relates to derivatives of pyrazolo[3,4-dl pyrimidine] useful as nucleobases.

Oligonucleotides are used to detect target DNA or RNA sequences. useful as a diagnostic probe. Traditionally, such probes consisting of a nucleic acid sequence containing a rimidine, or 7-diazapurine nucleotide base. (U, S, Patent 4°711.955; Robbins et al., J-Canada Ann Journal of Chemistry (J, Can, J, Chew, ), vol. 60, p. 554 (1982): Robbins et al., Journal of Auger Nick Chemistry (J, Org.

Chea+, ), vol. 48, p. 1854, (1983)). chemical part (ch The method for attaching chemical moieties to these bases is to It is mediated by a quinmercurylation reaction, whereby a mercury atom is converted into a pyrimidine ring. covalently bonded to the C-8 position of the purine ring or the C-7 position of the 7-deazapurine ring. (Dahl et al., Proceedings of the National Academy of Sciences) Proc, Natl^cad, Sci, USA ), Volume 70, Page 2238 (1973): Dahl et al. Biochemistry), vol. 14, p. 2447 (1975)), or By reacting an organic mercury compound with a lefin compound in the presence of a balanrum catalyst. (Russ et al., Journal of Organic Chemistry, vol. 43, 2870 pages (1978), Bergestrom et al., Journal of American ・Chemical Bath Society (J, Am, CheIl, Soc,) , Vol. 100, p. 8106 (1978), Binoji et al., Journal of America Kan Chemical Society (J, Am, Chew, Sac,) , vol. 102, p. 2033 (1980).

The sugar composition of oligonucleotide probes is currently ribose or deoxyri or, in some cases, naturally occurring β-arabinose-containing nucleic acids. (Patent Publication EP227.459).

Now, a new group of nucleotide bases, namely 3,4-disubstituted and 3,4 ゜6-Trisubstituted pyrazolo[3,4-dl-pyrimidine was found, which was previously This brings several advantages to the technology. De novo sealing of pyrazolopyrimidines 49) By chemical synthesis and the resulting nucleotide, Incorporating a wide range of functional groups in a variety of different positions and using a variety of sugar moieties make it possible to Also adenine, guanine and hypoxanthine analogs Obtained from a single nucleoside precursor. Furthermore, the synthesis does not require toxic heavy metals or high does not require the use of expensive catalysts. The similar pyrazolo 13.4-d corbyrimidine Known (Kobayan, Chemical and Pharmaceutical Bulletin) Chem, Pharm, Bull, Volume 21, Page 941 (1 (973)), however, differs in substituents from the compounds of the present invention, and Their use is only as xanthine oxidase inhibitors.

Concept of using cross-linkable nucleotide probes in therapeutic and diagnostic applications BR, Baker, “Design of active site-directed irreversible enzyme inhibitors”, Reilly. , New York, (1967), who was involved in the pioneering work of In application, “active-site-directed enzyme inhibitors” cted enzyme II le 1 inhibitors)" was used.

In recent years, the concept of incorporating crosslinks into oligonucleotides has led to better sequence probes. There is sometimes debate over the amount of effort required to develop it. Nore and Vra Soph, Progress in Nuclear Acid Research of Mo. Recular Biology (Prog, Nucl, Ac1d Res, M Ol, Bial, ), vol. 32, p. 291 (1985) N-(2-chloroethyl)-N- attached to the 3'- or 5'-end of otide Sequence-directed cross-linking using methylaniline groups (“complementarity-aware repair”) complementary addressed modification Somerton and Bartlett, Journal ・Of Molecular Biology (JoMol, Biol,), 1 22, p. 145 (1978), a nodonone residue is bonded to the C-4 position and a An 8-atom chain with a highly reactive bromomethylketone group bridges the N-7 of guanosine. We showed that it is possible.

Web and MachiLatch, Nufraink Unrad Research (Nucle) ic^Cid Res, ), vol. 14, p. 7661 (1986), 5-methyl We have prepared oligonucleotides containing Ru-N, N-ethanonetonone bases. , which can be loosely crosslinked with complementary strands. conceptually related The linker arm (1ir+ker　ara) is In alkylation via Yvesson and Delvain, pronoding ・Of National Academy of Sciences U.S.A. (Pr. oc, Natl, Acad, Sci, USA), volume 85, page 4615 ( (1988) showed methylation of the opposite chain, and B of the methylthioether Upon rCN activation, a position two pairs away from the base supporting the linker is caused predominantly on guanidino bases.

Oligonucleotides are sometimes used as chemotherapeutic agents and have been Controlling the expression of gene sequences specific to an organism, e.g. a virus, fungus, parasite or bacterium control In nature, RNA controlled by "antisense" RNA in bacteria There is also expression of A, and complementary target RNA and RNA: RNA hybrid are formed. form and exert their effects by modulating or inactivating their biological activity. It is something that can be demonstrated. Plasmids are used to introduce antisense RNA into eukaryotic cells. Recent various studies using vectors have shown that antisense R introduced into eukaryotic cells Effectively inhibiting NA-targeted rnRNA expression in vivo has been shown (Green et al., Anal Review of Biochemistry ( Ann, Rev, BiocheIl+, ), volume 55, pages 569-597 (1986)). In addition, it is possible to select specific mRNAs from among a large number of mRNAs. protein by hybridization with complementary DNA restriction fragments. The ability to selectively inactivate protein synthesis has been shown to be effective in binding to mRNA. It prevents the translation of mRNA into protein on ribosomes. (Beternon et al., Bronding of the National Academy of Sciences) ・Science (Proc, Natl.^cadSci,), vol. 74, 43 pp. 70-4374 (1977): No. 1 Stay et al., Pronoding of National Academy of Sciences (Proc, Natl, A CAD, Sci.), vol. 75, pp. 1217-1221 (1978).

Sequence-specific antisense oligonucleotides as modulators of gene expression and chemotherapy Tsamecnik and Steph were the first to prove the concept for use as a legal agent. Enson, Proceedings of the National Academy of Sciences USA (Proc, Natl, Acad, Sci, USA), 75 Vol. 280 (1978) describes small antisense oligodeoxynucleotides. Doprobe inhibits Rous sarcoma virus (RSV) replication in cell culture 6, and translation of R3V viral RNA is inhibited under those conditions. (Stephenson et al., Proceedings of None). National Academy of Sciences-USA (Proc, Na tl, Acad, Sci, USA), vol. 75, p. 285 (1978)) . Tzamechnik et al., Bronding of the National Academy of Sciences. Sciences-USA (Proc, Natl, ^cad, sci, 1 JsA), vol. 83, p. 4143 (1986), which is partially compatible with the HIV genome. Complementary oligonucleotides improve protein expression and virus replication in cell culture. It has also been shown that it has the ability to inhibit production. ~95% of inhibition occurs at approximately 70 μM was obtained at an oligonucleotide concentration of . Importantly, they are oligonucleotides Labeled phosphates enter cells intact and are reasonably metabolically stable. Shown with research.

An uncharged methyl sequence complementary to the start codon region of rabbit globin mRNA Phosphonate oligodeoxynucleotides are used in cell-free systems and rabbit reticulocytes. (Blake et al., Biochemistry ( Biochemistry), vol. 24, p. 6139 (1985)). another non Charged methylphosphonate oligonucleotide analog, herpes simplex virus type 1 The 8-nucleotide sequence complementary to the acceptor splice site of the mRNA of Virus replication in intact Vero cells can be inhibited. However, this inhibition required fairly high concentrations (>25mM) of these nonionic probes.

The influence of cross-linking oligonucleotides is of great importance in the field of chemotherapy However, these effects on diagnostic agents based on DNA probes Sound power is also very important. covalently probe-target halide The ability to cross-link hybrids is determined by the background (ba. ckground) and sensitivity limits, as well as novel assays. It has the potential to enable form. Concrete innovations (Gamper, etc., Nucleitsk) ・Acid Research (Nucl, Ac1ds Res,), Volume 14, 994 (previously discussed by p. 3 (1988)) are: (a) incorporating a denaturing wash step to remove background; (b) identifying Using cross-linking as an addition step.

(C) a location that ensures acute specificity and substantially deforms the secondary structure of the target; Crosslinking occurs at or near the melting point temperature of the desired hybrid, thereby increasing the effectiveness of lid formation, and (d) reversing xanbrotocol, e.g. A novel solution hybridization format as shown.

However, the concept of cross-linking suggests potential problems that must be avoided. are doing. For example, an oil containing a crosslinking arm Ligonucleotides very easily bind covalently to target sequences As a result, inappropriate combinations of sequences occur, resulting in host toxic ity) may occur. On the other hand, the crosslinking reaction must be sufficiently rapid. First, a reaction must occur before the correctly assembled sequences can be separated.

To draw this conclusion, it is important to note that during hybridization, as a result, the T Creation of oligonucleotides that produce double strands that are above the physiological temperature of 37°C do it. That way, even a single mismatched base can form a hybrid form. formation and further crosslinking. oligonucleotide length and base composition, Similarly, choosing where to modify bases within a probe can maximize effectiveness. can be achieved. However, the probe should be long enough and specific to only one site. We must ensure that the

European Patent Application No. 86309090.8 is 5-! conversion to lysinyl It describes the formation of chemically modified DNA probes, among which! Substituents are cross-linked However, it contains a chemical or physical reporter group. W○8707611 describes a method for labeling DNA fragments, in which the fragments are chemically modified. This is done by subsequently reacting with a fluorescent dye. Yabusaki et al., U.

S. Patent No. 4,599.303 describes the use of covalently crosslinking oligonucleotides. The mechanism is disclosed and consists of covalently bonding to other nucleotides upon photoexcitation. Due to the formation of furocoumarin-adducts of thymidine, etc. EP 0259186 is Polymers and biotin that can be used as bridging nucleic acid hybridization probes discloses an adduct of W○8503075 is useful as a nucleic acid fragmentation agent. Discloses crosslinkable disulfonic acid esters that can be used. DE3310337 is a tan describes the covalent cross-linking of single-stranded polynucleotides to macromolecules such as proteins. The resulting complex continues to interact with the foreign polynucleotide. used as a marker in hybridization experiments when investigating complementary sequences in be done.

Probe oligonucleotides are used to identify target sequences with high specificity. It must consist of a sufficient base sequence to facilitate covalent bonding with a specific complementary base. provided with one or more crosslinkers to form. Oligonucleos like this used as a highly selective probe in hybridization assays may be done. As an antisense agent for oligonucleotide RNA, e.g. It may also be used in chemotherapy.

Summary of the invention The present invention enables cross-linking between specific sites on multiple oligonucleotide mirrors that are in close proximity to each other. The present invention relates to a crosslinking agent that can be used for crosslinking. The observed cross-linking reaction is also highly specific. It is. The present invention provides an oligonucleotide comprising at least one of these crosslinking agents. and the use of the resulting novel oligonucleotides for diagnostic and therapeutic purposes. Also related to.

More specifically, the crosslinking agent of the present invention is a derivative of a nucleotide base having a crosslinking arm. The following formula (I') is obtained. R, -B-(CH2), -(Y), -(CH2), -A' (1') in the formula, R1 is hydrogen, or optionally at the 3° or 5° position, the sugar moiety is replaced by oxygen. a phosphorus derivative containing the groups Q1, Q2 and Qs, or They may also be substituted with their reactive precursors suitable for nucleotide bond formation. sugar moiety or its analogue.

Q is hydroxy, phosphate or diphosphate: Q2 is =0 Or = S.

Qs is CH2-R', S-R', O-R', or N-R'R''.

Each of Ro and R'' is independently hydrogen or C1-6 alkyl.

B is a nucleobase or an analog thereof, which is a constituent part of an oligonucleotide Yes.

Y is a functional linking group: Each of m and q is independently from 0 to 8.

r is 0 or 1; A′ is a leaving group.

The present invention comprises at least one nucleotide base derivative of the above formula (Io). It also provides novel oligonucleotides.

Nucleotides of the present invention and oligonucleotides incorporating the nucleotides Tido can also be used as a probe. probe vibe υ daizen The reaction is slow but reversible, allowing the probe to align with the precise target sequence. Ensures that the probe irreversibly binds to its sequence each time it hybridizes It is desirable to do so. The covalent cross-linking arm of the nucleotide base of the present invention It appears to permanently modify the chain or cause depurination. The origin of the present invention Gonucleotides can be used for targeted complementation in themselves, cell-free systems, and cellular systems. It is useful for identifying, separating, localizing and/or detecting target nucleic acid sequences. Therefore, The present invention further provides a method for identifying a target nucleic acid sequence; The method comprises preparing an oligonucleotide comprising at least one labeled nucleotide base of the invention. It consists of utilizing a cleotide probe.

The present invention further describes a method for inactivating gene function, which It requires the combination of anti-gene ○DN and recombinant enzyme. Coating ODN with recombinant enzyme The search for similarities within the target gene and the subsequent Encourages main strand formation. The resulting double-stranded complex (triple Cross-linking (nd coa + plexes) inactivates gene function. crosslinking Possible anti-gene nucleoprotein fibers are also described, including (1) within the gene; Covalently attached to an oligonucleotide (ODN) that is complementary to the target DNA sequence and (11) a non-covalent bond with the ODN. Contains a recombinant enzyme that

The present invention also provides novel substituted pyrazolo r3.4-d]pyrimidines. , which is used as a nucleotide base during the preparation of nucleosides and nucleotides. more useful than regular purine or pyrimidine bases or deazapurine analogs .

Brief description of the drawing Figure 1 shows that from the complementary base via the acetamidopropyl sidearm Oligonucleotide cross-linked to deoxyguanononone residues located two sites apart in the 5' direction Represents the modified thioxyuridine residue of leotide.

Figure 2 is! ! p-labeled HPV target and cross-linked product to detect guanonone It shows an autoradiogram cut at the 3' side. Lane 132P- mark Identification 15-ser size marker. Lane 2: React at 20°C for 24 hours. lane 3 ni React at 20°C for 2 hours. Lane 4: Reaction at 30°C for 24 hours. lane 5 near 2 hours React at 30°C. The reaction was stopped with 2-aminoethanothiol and piperidine solution. Processed and effectively cut.

Figure 3 shows the autoradiogram of Up-labeled HPV terquesoto and cross-linked products. /X hybrid fraction by denaturing polyacrylamide gel electrophoresis. It shows distance. 32P-labeled CMV target for lane control. lane 2:2 React at 20°C. Lane 3 Near React at 20°C for 2 hours. Lane 4: 24 hours 30 React at °C. Lane 5 near: React at 30°C for 2 hours. The reaction solution is converted into 2-aminoethanothio treatment with alcohol, which annihilates the iodoacetamide groups.

Detailed description of the invention A, crosslinkable oligonucleotide The present invention provides a novel substituted nucleotide base having a bridging arm, Useful in the preparation of creosides and nucleotides and also as a cross-linking agent . The redeemable base has the following formula (Io): R+B (CH2)-(Y), (CH 2),, A' (1') in the formula, R1 is hydrogen or optionally oxygen at the 3' or 5° position a phosphorus derivative containing the groups Q3, Q2 and Qs, or a nucleus sugars that may be substituted with their reactive precursors suitable for leotide bond formation is a part or an analog thereof; Ql is hydroxy, phosphate or diphosphate; Q2 is = 0 or =S.

Qs is CH2-R', S-R', ○-R', or N-R'R''.

Each of Ro and R'' is independently hydrogen or C1-6 alkyl: B is a nucleobase or an analog thereof, which is a component of an oligonucleotide. can be.

Y is a functional linking group.

m and q are independently from 0 to 8; r is 0 or 1;

8 is a leaving group.

In the practice of this invention, the sugar moiety or analog thereof is a component of a nucleotide. and selected from those that are useful. Such sugar moieties include, for example, ribose, thio xyribose, pen[-su, deoxybentose 1, hexose, deoxybentose xyose, glucose, arabinose, bentofuranose, xylose, lyxo and ncrobentyl. The sugar moiety is preferably , thioxylipose, arabinose or 2°-○-methylribose, Also includes nomer, α or β.

Phosphorus derivatives that bind to sugar moieties include, for example, monophosphates, diphosphates, and triphosphates. Rephosphate, alkyl phosphate, alkane phosphate, phosphorothio It is appropriately selected from ate, phosphoro/thioate, etc.

Reactive precursors suitable for intranucleotide bond formation are used in oligonucleotide synthesis. It is one of the useful substances during nucleotide chain elongation. In the present invention, particularly A useful reactive group is one containing phosphorus. Appropriate linkage for intranucleotide bond formation The ion-containing group is preferably an alkylphosphoramidite, alkylphosphite or or an alkylphosphoramidite. Alternatively, activated phosphatone ether Steal may also be used for this purpose.

Nucleobases or analogs thereof (B) include purines, pyrimidines, deazapurines and and pyrazolopyrimidine. It is preferably uracil -5-yl, nodonon-5-yl, adenin-7-yl, adenin-8-yl, Guanine-7-yl, guanine-8-yl, 4-aminopyrrolo-[2,3-d] Pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidine -5-yl, 4-aminopyrazolo[3,4-d]pyrimidin-3-yl or is from 4-amino-6-oxopyrazolo[3,4-d]pyrimidin-3-yl selected, where the purine is attached to the sugar moiety of the oligonucleotide via the 9-position. , pyrimidine through the 1st position, pyrrolopyrimidine through the 7th position, pyrazolopyrin The midine attaches to the sugar moiety of the oligonucleotide through the 1 position.

The functional linking group Y is ox7, thio, amino or chemically protected thereof. Derivatives such as trifluoroacetamide, phthalimide, C0NR', NR 'CO, and 5O2NR', where R'=H* or Cl-6fluor, Which nuclear group is chosen? Function of those containing aliphatic or aromatic amines -(Cl2), which acts as the point of attachment for the -A' group. They are two people who have the ability to Amino groups and protected derivatives thereof are preferred . The leaving group A' can be, for example, chloro, bromo, iodo, 502R''' or S''R It may be selected from groups such as “Ro”, where each “Ro” and “R” are independently and C6 alkyl or allyl, or Ro and R'' taken together. to form a Cl-6 alkylene bridge. Chloro, bromo and iodo are preferred stomach. The leaving group varies depending on its leaving ability. Nature and reaction of specific leaving groups The groups used are chosen in each case depending on the nature and the desirability of irreversibly binding probes. get specificity.

Ball-and-stick model of double-stranded DNA ck models) and high-resolution computer graphics are , the purine position 7 and the pyrimidine position 5 are major groups in the B-type double-stranded structure of double-stranded nucleic acids. This indicates that it exists in the major groove. child Although these positions can be substituted with significant amounts of side chains, hybridization of the base It is not affected by the version characteristics. These fallen hairs are called dThd or cicy. either derivatization of a or direct total synthesis of the heterocyclic base followed by glycolylation. It may also be introduced depending on the situation. These modified nucleotides can be used in automatic DNA synthesizers. to the appropriate activated nucleotides for incorporation into oligonucleotides. There is also. Cross-linked with pyrazolo[3,4-dl-pyrimidine, an analog of adenine The hands are joined at the 3rd position, but this is also the same for the 7th position of the pudding.

Crosslinkable side chains include purine 7- or 8-position, pyrimidine 5-position, pyrrolopyrimidine For reaching across the major globe from the 5-position or the pyrazolopyrimidine 3-position, be sufficiently long and located on the (3' side of the oligomer) of the base pair containing the modified analog. must react with N-7 of the purine (preferably guanine) . Therefore, the side chain should be at least 3 atoms in length, preferably Preferably, at least 5 atoms, more preferably at least 6 atoms. be. Generally preferred side chain lengths are about 5 to about 9 carbon atoms.

To maximize cross-linking of the strands, modify bases in oligonucleotides containing cross-linking hands. Pair the target strand base being attacked with the first or second base on the 3° side of It seems desirable that the For example, if the target strand base under attack is If the target sequence for a probe containing a modified uranol is ZC (preferably UCC) is dUrd substituted with a bridge at position 5 Complementary GZA (preferably GGA) with probe oligotacleotide containing and Z should contain any base. cross-linked adenine derivatives In the oligonucleotide containing, for example, the triplet adenine modification ΔZ ] C will target GZ'T and 21 is any base. .

When the modified base that develops the crosslinker is uracil and the target sequence is GGA , target sequence of base pairs modified with crosslinker 5° The alkylation of the second guanine on the side is the only one observed (as shown in Figure 1). It is an effect.

The cross-linking reaction is highly specific and results in an “optimal match” of the electrophile to the claimed core molecule. In other words, to find the alkylation site, there are two or more guanine residues may be required in close proximity to the complement of the modified base. Ru.

Two types of modified 2'-thioquine nucleosides are used in the present invention for sequence-directed crosslinking. It exhibits specific properties for incorporation into oligonucleotides as bridging agents. . The first type is 5-substituted-2'-thioxyuridine, whose membrane structure is shown below. show: 5-Substituted-2'-deoxyurin can be prepared by the routes shown in Schemes 1 and 2. can be manufactured.

fXE) For example, the general method of Robin et al. (J Canadian Journal of Chemistry J)-(J, Can, J, Chem, ), 5 Volume Q, page 554 <1 982), Tsuyanal of Orkani, Re-Chemistry (J, Org, C heo, ), vol. 48, p. 1854 (1983)) is shown in Scheme 1. The direct conversion of 1-alkyne (XXI) to 5-iodo-2゛-thioxyuri palladium-mediated coupling with non(XX) to form the acetylene-linked product (XX II) is obtained. The dUrd analogue of acetylene, XXTl, for example, Reduction with Kel gives the saturated compound (XXIII), which can then be used directly to The reactants used in the automatic DNA synthesizer were converted into the same product.

(xxv’r) (XXVT+1 (XX7TI) Starting compound from 5-chloromerk)pho2-deoxyurinone (XX IV) When used as a product, the olefin is directly bonded to the functionalized olefin. Olefin compound (XXVTI) is obtained by palladium catalyzed coupling I can't do that. Alternatively, substituted alkenes (XXV ) and 5-talolomerculio-2'-deoxywacin (XX　TV) Reaction with tanol gives alphamethquin adduct (XXVI), which This was converted into olefin compound XX using trifluoroacetic acid and 1-trifluoroacetic anhydride. Converted to VII. In order to convert the DNA into a synthetic reagent as described below, reduction is performed. to obtain a saturated compound (XXrll).

B Pyrazolo[3,4-dl pyrimidine The second type of modified nucleotide is 2゛-thi A group of oxy-4-aminopyrazolo[3,4,-cj]pyrimidine derivatives.

The above compounds, the general structures of these derivatives are shown below, are 3,4-disubstituted and and 3.4.6-trisubstituted pyrazolo[3,1-d]pyrimidine derivatives derived from the group of new derivatives. be guided. 3,4-disubstituted and 3.4.6-trisubstituted pyrazolo [3,4-dl Pyrimidines and their synthesis are disclosed in applicant's own separate application no. 250.474. and is incorporated herein by reference in its entirety. They are below During the ceremony waiting for the expression (1), R, is hydrogen or a sugar moiety or an analog thereof, optionally the 3' Or, at the 5′ position, it is bonded to the sugar moiety by oxygen, and the groups Q1, C2 and C3 are phosphorus derivatives containing or their reactivity suitable for nucleotide bond formation substituted with a precursor provided that if R3 is hydrogen, R1 cannot be hydrogen: Q, is a human hexaquinone, phosphate or diphosphate.

C2 is 20 or =S.

C3 is CHa R', S-R', O-R', or N-R'R": R" and R'' are independently hydrogen or cl-6 alkyl.

R3 is hydrogen or a group -w-(X), -8; each of W and X is independently This is a chemical linker arm.

A is an intercalator (j, intercalators), metal ion key Rator-1 is an electrophilic crosslinked product, a photoactivatable crosslinked product, or a reporter group and SR , and R6 soreha, independently HlOR, SR, NHOR, NH2 mataha N H(CH2) is 1NH2 and S R is H or C1-6 alkyl.

n is zero or 1.

t is zero or 12.

3.4-disubstituted and 3.4.6-trisubstituted pyrazolo[3,4-dl pyrimidine] synthesis of nucleotides and into nucleic acids either enzymatically or via chemical synthesis Their use as reagents for incorporation offers several advantages over current means. It is something. De novo chemical synthesis of nucleotides is widely used. Functional groups (e.g. NH2, SH, OH, Haloken, C0OH, CN, C0NH2 ) and the use of various sugar moieties. adenine, guanine, and Hypoxanthine analogs are also obtained from single nucleotide precursors. Furthermore, the synthesis is Does not require the use of toxic heavy metals or expensive catalysts.

In the practice of this invention, the sugar moiety or its analogue is considered as a constituent part of the nucleotide. selected from those that are useful. The moiety may be, for example, a pentose, a deoxypentose, -se, hexose, thioxyhexose, ribose, thioxylyhose, gluco arabinose, bentofuranose, kinloose, lyxose and ncro Bentyl. The sugar moiety is preferably ribose, thioxylipo α, arabinose, or 2′-〇-methylribose, and the anomeric α or Contains either β.

Phosphorus derivatives attached to sugar moieties include, for example, monophosphates, diphosphates, triphosphates, alkyl phosphates, alkane phosphates, phosphorus It is appropriately selected from lotioate, phosphoronethioate, etc.

Reactive precursors suitable for intranucleotide bond formation are used in the synthesis of oligonucleotides. It is one type of substance that is useful for elongation of nucleotide chains. Particularly useful in the present invention The reactive group is one containing phosphorus. Appropriate linkage for intranucleotide bond formation The ion-containing group is preferably an alkylphosphoramidite, alkylphosphite, or or an alkylphosphoramidite. As an alternative, activated phosphatone Es Teru may also be used for this purpose.

In the above formula (■), the chemical linker arm (W alone or together with body, detector proteins, and serves to create a functional group (A) that can more easily interact with chemical reagents. The result When a base pairs with another base in a double-stranded complex, Supports the functional groups that exist. The bond is an alkylene group of 1 to 12 carbon atoms, 2 or an alkenylene group of 12 carbon atoms and one or two olefinic bonds, two an alkynylene group of 12 carbon atoms and one or two acetylene bonds, or or terminally oxy/, thio, amino or chemically protected derivatives thereof ( For example, trifluoroacetamide, phthalimide, C0NR', NR'CO and and 5O2NR', in the formula R"=H or C1-6 alkyl), substituted with a nuclear group such as It may also include groups such as Such functionality can be achieved by aliphatic or aromatic It has the ability to act as a bonding point for the functional group (A,) and exhibits the desired nuclear properties. have power.

The linker arm portion (W alone or together with X) preferably contains at least three more preferably at least 5 atoms. The terminal nuclear group is preferably Preferred are amino or chemically protected derivatives thereof.

Intercalators are planar, aromatic bicyclic, tricyclic, and polycyclic molecules that can be used in nucleic acids. can insert itself between two adjacent base pairs in the double-stranded helical structure of It is something that Intercalators are frame nodes in DNA and RNA. It has been used to induce cut mutations. Through the hands joining the intercalator When appropriately attached (connected with a tether) to the end of a deoxyoligonucleotide, It increases the binding affinity of the oligonucleotide with its target sequence, resulting in Some protect against exonucleases, but not against endonucleases. isn't it. Sun et al., Nucleic Ac 1ds Res, ), vol. 15, pp. 6149-6158 (1987): Shi. Nucleic Ac1ds Research (Nucleic Ac1ds R) es, ), vol. 15, pp. 7749-7760 (1987). connecting Examples of intercalating agents that can xazolopyridocarbazole, acrinone orange, proflavin, acrifla derivatives of vin and proflavin, and 3-azido-6-(3-bromopropylene) 3-amino-6-(3-bromopentylamine)acrydine gin and 3-methquin-6-chloro-9-(5-hydroxypentylamino) There are acridines such as acridine.

oligonucleotides capable of cross-linking to complementary sequences of target nucleic acids. This is because it increases. This means that covalently attached oligonucleotides This can be achieved with a cross-linking agent, which is then chemically activated to form cross-links and Induces a chain break in the complementary sequence of the target and As an example of a claimed electronic bridge moiety that causes irreversible damage to the sequence, Rufer halocarbonyl compounds, 2-chlorostylamines and epoxides Ru.

An oligonucleotide comprising at least one nucleotide base moiety of the present invention When used as a probe in a nucleic acid assay, it should be labeled and labeled. Detecting the presence of a hybrid polynucleotide. Such a label can be used as a reporter group. target nucleotides and their complementary oligonucleotides. It acts as a means of detecting the double-stranded formation between the otide probes.

As used herein, a reporter group is a measurable or detectable physical or chemical group. It is a group with scientific characteristics. Detectability is determined by characteristics such as color change, luminescence, fluorescence, or radioactivity. a reporter group that may be provided by a characteristic or act as a ligand recognition site. It may also be given by the ability of

The pyrazolopyrimidines of formula I of the invention in which R1 is hydrogen are as outlined below. Methods and Kobayano, Chem, Pharm, Bull, Volume 21, 941- 951 (1973). , the disclosure of which is incorporated herein by reference.

Generally, malononitrile (II) is converted into alkyllide (I) in the presence of a base. I) to give anormalononitrile (IV), which is subsequently treated with e.g. Methylated and substituted methoxymethylene with methyl sulfate or diazomethane Lemmalononitrile (V) is obtained. This compound is then combined with hydrazine hydrate. Reacted in boiling alcohol to produce 3-substituted-5-aminopyrazole-4-carbonite. Riru (Vl) was obtained and treated with cold concentrated sulfuric acid to give 3-substituted-5-aminopyrazolo- 4 = carboxamide (Vll) is obtained.

Carboxamide (Vll) can alternatively be replaced with noanoacetamide (Xll) was treated with acid halide (II) to obtain acylinanoacetamide (Xllll). This is then methylated and the resulting methoxylation Compound (XIV) is treated with hydrazine hydrate.

and Vll. Therefore, 3,4-disubstituted pyrazolo[3,4-dl pyrazolo The midines (VIII and It can be obtained by processing with Alternatively, Vl can be heated at or above room temperature. noalcoquinomethyl ester of rubonic acid, then treated with ammonia to form VIII. Also, VII can be prepared by converting the dialkoxy group of carboxylic acids at room temperature or above. Treatment with methyl ester (without subsequent treatment with ammonia) provides compound X Sometimes. 3.4.6-trisubstituted pyrazolo[3,4-dl pyrimidine (IX or and XI), the corresponding VI and Vll are expressed as =R6 (wherein R6 is O or S). alternative as VI and VN with alkyl xanthetes such as potassium ethyl xanthate. salts and alkyl halides such as methyl iodide at temperatures above room temperature. , and then oxidized with a peroxide such as dichloroperbenzoic acid (MCPBA). , followed by treatment with ammonia to give IX and XI, respectively, with the formula R6 in the middle is NH2.

The compound of formula I can be recovered from the reaction mixture, in which it is These compounds are formed by established methods.

In compounds of formula I, where R1 is a sugar moiety, the sugar is addition to the 1-position of sol V or VII, or pyrazolo[3,4-d cobi It can be added to either position 1 of the limidine VIJLJX, X or XI . To add sugar, pyrazole or pyrazolopyridine is treated with sodium hydride, It is then treated with a protected sugar glycolylno\lide.

The uninvented oligonucleotide comprises a substituted pyrazolo[3,4-dl pyrimidine of formula 1] at least one and substantially all of these nucleotides derived from At least one of these nucleotides derived from the substituted nucleotide base of formula 1' and It may consist of almost all of the

To prepare oligonucleotides, protecting groups can be added to nucleosides of formula I or formula The nucleoside is activated and used for oligonucleotide synthesis. protection Conversion to the activated form is a 2'-thioxynucleotide, which has been described in detail in several commentaries. Follow the method about Osido. Sonveaux, BioO -Kani Tsuku Chemistry (Bioorganic Chemistry) , vol. 14, pp. 274-325 (1986); "Synthesis, Practical Method", M. J. Gate Publishing, IRL Press, pp. 23-34 (1 984).

Activated nucleotides can be prepared using methods similar to those for DNA and RNA nucleotides. method into the oligonucleotide, within which the appropriate nucleotides are linked and complementary to the nucleotide sequence of the target DNA or RNA will form a nucleotide chain. Nucleotides can be synthesized enzymatically or chemically It may be incorporated through one of the following steps. The nucleotides have their 5°-〇- 7 Methoxytrityl-3'-(N,N-isopropyl)phosphoramidite It is converted into an anoethyl ether derivative, making it practical for the above-mentioned “oligonucleotide synthesis”. can be incorporated into synthetic oligonucleotides according to the method in ``Methods''. Ru. The N-protecting group is then added to the post-synthesized amino acid along with other oligonucleotide protecting groups. It is removed by thurisis in a manner generally known in the art.

In a preferred embodiment, the activated nucleotides are applied directly to an automated DNA synthesizer. Follow the procedures and instructions for the specific synthesizer used. Ori Gonucleotides are standard industrial scale phosphoramidites or H-phosphonates. It can also be prepared in a synthesizer using chemistry.

In another preferred embodiment, the aminopyrazolopyrimidine nucleotide triphos Langer et al. Bu Sciences U.S.A. (Proc, Natl, Acad, S Ci, USA), Vol. 78, pp. 6633-6637 (1981). It can also be used in place of adenine using the nick translation method. , the disclosure of which is incorporated herein by reference.

Adding a leaving group such as a haloanlu group to the aminoalkyl tail (-CH,), -Y) , which may then be incorporated into oligonucleotides to remove some protecting groups. .

For example, the addition of α-haloacetamides changes the HPLC mobility of the modified compound. to remove the positive charge of the amine group, followed by reaction with 2-aminoethanethiol. , by re-adding a positive charge to the original aminoalkyl oligonucleotide This may be demonstrated by obtaining derivatives with reversed-phase HPLC mobilities similar to .

In certain embodiments, each of the following electronic leaving groups is Aminopropyl group, bromoacetyl, iodoacetyl on virus (HPV) probe It binds the less reactive but conformationally more flexible 4-bromobutyryl. Matched. Bromoacetyl and iodoacetyl have equal reactivity during crosslinking I knew it was something.

The oligonucleotide probe according to the invention comprises a labeled substituted pyrazolo [ At least one 3,4-dl pyrimidine nucleotide moiety and/or the formula and at least one substituted nucleotide base labeled r.

Probes can be prepared by any one of several methods typically used in the art. It is possible to mark the The usual detection method is 3H1+25), 35sS 14 (: or the use of autoradiography using 32p-labeled probes etc. It is. Other reporter groups are labeled with fluorescent reagents, chemiluminescent reagents, and enzymes. contains a ligand that binds to the antibody. Alternatively, probes can be directly fluoresced. Labels and consumables such as photoreagents, chemiluminescent reagents, enzymes and enzyme substrates It is possible to Alternatively, the same composition can be labeled and conjugated with a ligand binding indirectly through a ligand-antiligand complex such as an antibody that is reactive with Sometimes it can be done. The choice of label depends on the required sensitivity and conjugation with the probe. Depends on ease of operation, stability requirements and available instrumentation.

The choice of label determines how the label is incorporated into the probe. radioactive probe Nucleoblasts are typically commercially available and contain the desired radioisotope. Created using leotide. Radioactive nucleotides can be used, for example, using a DNA synthesizer. For use in nick translation and radioactive base transfer with terminal group transferer In the presence of radioactive dNTP's, the DNA polymer is tethered to the 3' side of the probe. Replication of M13 plasmids with specific inserts by the Flenow fragment of or cleave the template using RNA polymerase in the presence of radioactive rNTP’s. By transcribing the RNA, it can be incorporated into the probe.

Non-radioactive probes can be directly probed (e.g., fluorescent reagents, chemiluminescent reagents or enzymes) or indirectly conjugated with ligands. It can be labeled by a nu-yon. For example, link the ligand molecule to the probe as appropriate. Match. This ligand is then essentially a detectable compound or Any suitable combination with an detectable knob such as an enzyme or a photoreactive compound. connects with a receptor molecule. Ligands and antiligands are widely varied . If the ligand is a natural "antiligand", i.e. biotin, tyro If it has ligands such as quinone and cortisol, its labeled It can be used in conjunction with naturally occurring anti-ligands. Alternatively, hapten or or some of the antigenic compounds can be used in combination with appropriately labeled antibodies. come. Preferred labeling methods include (Langer et al., Pronding on National Naru Academy on Science Is tJSA (Proc, Nat Acad, Sci, USA), vol. 78, pp. 6633-6637 (1981 Utilizing biotin-labeled analogs of oligonucleotides (as described in 2013) , which is incorporated herein by reference.

Enzymes of interest as reporter groups are primarily hydrolases, especially phosphatase. -ase, esterase, urease and glycondase, or oxidoreduct enzymes, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, Dannlu, umbelliferone, rare earth acids It is a monster, etc. Chemiluminescence, luciferin, acridinium ester and 2,3-dihydrophthalanedione, such as luminol.

Specific hybridization conditions are not definitive and are subject to researcher preference and preference. It will change according to the call and demand. Various hybridization solutions can be used. The polar organic solvent may be added to about 20% to about 60% by volume, preferably about 30% by volume. % capacity included. Typical hybridization solutions have approximately 30-60% V/ V formamide, about 0.5 to 1M sodium chloride, sodium citrate, about 0.05 to 0.05, such as Squirrel HCI, PIPES or HEPES. IMlf approximately 0.05% to 0.5% detergent such as IL solution, sodium dobnol sulfate, and and EDTA between 1 and 10 rnM, 0.01% to 5% Ficoll (approximately 300 -500 kdal), 0.1% to 5% polyvinylpyrrolidone (about 200-5 00 kdal) and 0.01% to 10% bogus serum albumin. Ma Typical hybridization solutions include, for example, partially cleaved fragmented calf thymus or salmon sperm, DNA and/or partially fragmented enzymes About 0.1 to 5111 g of mother RNA, 7 ml of unlabeled carrier nucleic acid, and optionally About 0.5% to 2% wt/vol grits. As another addition, various anionic polyacrylates, such as capacity excluding agents containing polar water-soluble or swellable reagents. Dextra may also contain J-rate or polymethyl acrylate. It may also be filled with sugar polymers such as sulfates.

The specific hybridization technique is not a material element of the invention. hive Redization technology is generally referred to as “nucleic acid hybridization, practical method” , Haymes and Pithins, Publishing, IRL Press, 1985: Girl and Ba Redu, Pronouding on National Academy of Sciences Is USA (Proc, Natl, ^cad, sci, UsA), 63, pp. 378-383 (1969): and John et al. ature), Volume 223, Pages 582-587 (1969) . Improvements have been made in hybridization techniques to make them easier. It can be applied to

The amount of labeled probe present in the hybridization solution can vary widely. be. Generally, a substantial excess of the probe over the stoichiometric amount of the target nucleic acid is used. This is to increase the binding rate between the probe and target DNA. Ru.

Strings with varying degrees of hybridization ncy) can be adopted. Hybridization conditions become more stringent I see, the degree of complementarity between the probe and target to form a stable duplex. must be stronger. The degree of stringency depends on temperature and ionic strength. , 31 by inclusion of a polar organic solvent, etc. For example, the temperature used is Normal temperature ranges from about 20℃ to 800℃, and normal temperature ranges from 25℃ to 75℃. cormorant. Optimal temperature for probes of 15-50 nucleotides in 50% formamide The temperature range can vary between 22-65°C. By performing routine experiments, room temperature It is possible to define one condition for satisfactory hybridization.

The stringency of hybridization also reduces the concentration of formamide to about 20%. % to about 50% to vary the ionic strength and polarity of the reactant solution. It can be changed as appropriate.

Submerging the reaction vessel in a commercially available ultrasonic bath and sonicating it will result in hybridization. The rate of diization often accelerates.

Hybridize for a time and at room temperature appropriate for the specific hybridization solution being used. After ization, the glass with the probe-target hybrid attached , plastic, or filter supports are commonly used in hybridization solutions. containing similar reagents (e.g., sodium chloride, buffers, organic solvents and detergents). into the washing solution. These reagents are similar to hybridization media However, if a higher cleaning condition of 11FW is desired, a lower concentration can be used. is often the case. The time that the support is left in the cleaning solution ranges from a few minutes to a few minutes. It may change over time or more.

Either the hybridization or washing medium is a tight fit (string ent). Accurate hybridization after moderately rigorous cleaning Consistent with the nature of the label, complexes may be readily detected.

Probes may also be directly conjugated with labels. For example, if the label is radioactive In some cases, the support surface with bound hybridization complex substrate is Expose with line film. If the label is fluorescent, the sample is first exposed to specific waves. Detected by long exposure to light. The sample absorbs this light and then the detector (“Physical Biochemistry”, Freifelder) , D0, W, H Freeman & CO0, 1982, pp. 537-542). The sign is If it is an enzyme, incubation with the appropriate substrate for the enzyme The sample is detected by The resulting nobunal is a colored precipitate, colored or fluorescent lysate. , or photons produced by bioluminescence or chemiluminescence .

Preferred labels for dipstick assays are those that produce a colored precipitate and give a positive reading. Point. For example, alkaline phosphatase dephosphorylates indoquine phosphate. oxidizes and then participates in a reduction reaction to form the tetrazolium salt into a highly colored dye. Converted to soluble formazan.

Detection of hybridization complexes involves detecting the signal-generating complex as the target. Requires double strands and probe polynucleotide or nucleic acid binding Sometimes. Typically, such bonds are linked to a ligand-conjugate probe. and between the ligand and the antiligand as an intermediate between the signal and the concomitant antiligand. Occurs through interaction. Binding of signal-generating complexes also results in ultrasonic energy It is easy to accelerate when exposed.

Labels may also allow indirect detection of hybridization complexes . For example, if the label is a hubten or an antigen, antibodies can be used to can be detected. In these systems, the radicals are fluorescent or enzyme molecules. in some cases, by conjugation with an antibody to a radioactive label. (Tijssen, P., 'Biochemistry and Implementation and Theory of Enzyme Immunoassays in Molecular Biology, Laboratory Techniques”, Bull Dong, R.H., Van Nilapenbarb, P.H., Publishing, Elsevier, 1 985L, pages 9-20).

The amount of labeled probe present in the hybridization solution can vary widely. However, depending on the nature of the label and the ability of the labeled probe to bind appropriately to the target nucleic acid in cells, amount and exact stringency of the hybridization medium and/or washing medium. depends on. Generally, a substantial excess of the product over the stoichiometric amount of the target The lobes are used to increase the rate of binding between the probe and target nucleic acid.

The present invention is also directed to methods of identifying target nucleic acid sequences; The method uses a small number of labeled and substituted nucleotide moieties of formula (1) and/or formula (1). It consists of utilizing at least one oligonucleotide probe.

- In embodiments, the method comprises the steps of: (a) modifying the nucleic acid in the sample; (b) converting the target nucleic acid to a labeled substituted nucleic acid of formula I or formula I'; Oligonucleotide probes and hybrids containing at least one leotide moiety The probe in it is derived from a sequence complementary to that of the target nucleic acid. (C) washing the sample to remove unbound probe; (d) treating the sample with a detection reagent; Incubate together (e) Test the samples.

The above methods may be carried out according to procedures well known in the art.

At least one labeled substituted nucleotide moiety of formula I and/or formula ■' A target method that utilizes an oligonucleotide probe containing Assays that identify the nucleic acid sequences of be. Some such assays come in kits. For example, representative A standard kit includes a small number of identified substituted nucleotide moieties of formula I or formula I'. an oligonucleotide containing one or more sequences, i.e., a sequence complementary to the target nucleic acid sequence. oligonucleotide; a denaturing reagent that turns double-stranded nucleic acids into single-stranded nucleic acids: and) 1 hybridase-/con reaction mixture. right. The kit includes a signal generation system such as an enzyme and a base for the system. It may also include quality.

C0 enzyme-mediated triple strand formation It has great potential as a sex drug. For example, chemically synthesized ○DNs are By hybridization with complementary metsenger RNA (mRNA), two Chain formation may also inhibit the expression of specific gene products. Twist Physically, these “antisense” DNs are primarily RNNase-mediated. Message processing by cutting the target mRNA sequence It is believed that it inhibits translation or translation. Because of these inhibitory effects, antiseptic Nance0DNs are useful as antiviral, antiparasitic, and anticancer agents. Sometimes. Moreover, antisense DNs offer a unique opportunity for rational drug development. which gives the gene target a possible target sequence This is because it offers both great specificity and versatility.

However, "antisense" technology suffers from certain fundamental disadvantages.

One of the major challenges is that the development of antisense 0DNs requires in vitro testing. This means that the drug must have sufficient efficacy to be approved. Nuclease resistance, thin rapid uptake into cells, and efficient and stable Antisense ODN formulations that exhibit X hybridization are desired. these Improving one or more of the properties without having a detrimental effect on other properties is , which can be difficult. For example, oligonucleotide methylphosphonate and backbones such as phosphorothioates and phosphorothioates. Antisense 0DNs have superior nuclease resistance in proportion to unmodified 0DNs. However, methylphosphonate 0DNs are refractive with respect to RNNases. Forms an NA-RNA hybrid, and the phosphorothionate 0DNs remain in the cell. Only enough is taken in.

Another way to improve the effectiveness of antisense ○DNs is to connect parts of ○DNs. Together, they enhance their antisense activity. for example,! −Eve The part that directly interacts with the RNA target during redization (ODN-target) A pendant insertion group (pendant inte) that stabilizes the target hybrid calcating grOυps), <RNA cleavage activity based on free radicals 0DNs with or shared with their targets during hybridization 0DNs with the ability to bind associatively) may be helpful in this regard. Ru. Direct cleavage or cross-linking of mRNA targets by ODNs renders the RNA intact. Become active.

Cellular uptake of antisense 0DNs is another area of challenge. charged It is possible that 0DNs are actively taken up into mammalian cells through an energy-dependent pathway. Over time, it is possible to expand this pathway and increase the intracellular concentration of antisense ODN. desirable. °Endocytic approac hes)” enhances cell targeting through receptors, and Contains a cholesteryl group that behaves as a lipophilic anchor for tisense○DN. Transfer: 0DNs into liposomes; and 0DNs into soluble form. It binds one polymer complex.

Applying the above methods alone or in combination, the ability of antisense ODNs to The goal of increasing An alternative to the “antisense” approach to rational drug design is the “antigenetic” approach. Antisense 0DNs are single-stranded mRNAs. Antigene 0DNs targets double-stranded DNA and It has the ability to increase its size and inhibit its functions. More specifically, antigene 0D Nslt forms sequence-specific double-stranded complexes with double-stranded DNA targets and thereby interfering with the replication or transcription of the selected target gene. some kind of RNA DNA contains all genetic information, except for viruses and viroids, which do not have nucleic acids. It is a treasure trove of information such as regulatory control sequences and dormant proviral DNA genomes. Contains unexpressed genes. On the contrary, in the tarp and nt of antisense 0DNs, mRNA1t, a very small subsection of information encoded (encrypted) in DNA. It represents a set. Therefore, anti-gene 0DNs have broader applicability. In addition, it has the ability to simply inhibit mRNA processing and translation. It is more powerful than antisense DNs.

Antigene 0DNs form a sequence-specific complex with chromosomal DNA in the nucleus of living cells. can be formed. The resulting double strand is the IL of the tarp-t double stranded DNA. + restriction and/or transcription can be inhibited. Two target nucleic acids (i.e. Interfering with DNA function based on the known stability of DNA and RNA) Antigenes that inhibit mRNA function have a longer-lasting effect than antisense inhibition of the corresponding mRNA function. have. Mammalian cell DNA is a turnover , rLl; in fact, cells (D, which can arise from environmental disturbances or spontaneous translocations) It has a very complex pathway capable of repairing damage to NA. On the contrary, mR NA is transient and may exist within cells for only a few minutes. One of the mRNA species The potential high copy number of the mR,NA species is This suggests that Sense○DNs may have relatively short-term effects. anti It is necessary to increase the cellular uptake of Gene○DNs to reach a sufficient intracellular concentration. On the other hand, once taken into cells, ○DNs naturally enters the nucleus. It is concentrated in

Anti-gene therapy is based on the ability of certain DNA homopolymers to form double-stranded complexes. This is based on the observation that In these double-stranded complexes, the third strand is It exists in the major glove of the Watson-Crick base-pairing double helix structure, and its Here it forms a hydrogen bond with one of the two sightings. The bond code is the third base (given below). It governs the recognition of base pairs by triplet bases (see triplet bases).

In each case, the bases of the third strand are first, followed by the base pair: the first two bases. Hydrogen bonds between the bases maintain the interaction of the third base.

It is clear from the given base triplet that this base pair recognition code has certain limitations. It is clear. First, there is no ability to recognize the base pairs T-A and C-G, thus , duplex formation consists of homopurine bases on one side and homopyrimidine bases on the other side on the duplex. is limited by the type of run. Second, Nodnon is on the third mirror. (“C”), it adds a proton and can hydrogen bond with the guanine of the base pair G-C. You must do so. The pKa of protonation of cytosine is 46, and the protonation of cytosine is In physical DH, the stability of C-G-C triads is probably compromised. This suggests that there will be. Substitution with 5-methylcytosine or polyvalent cation The use of spermine (such as spermine or spermidine) in the triplet C-G at pH 7.0 - It may also stabilize C. Thirdly, in all cases the triple strand is the third strand is maintained by two hydrogen bonds between the base and the purine residue of the base pair. Therefore , duplex complexes are generally less stable than the original double-stranded DNA and contain purines and pyrimins. Maintained by a combination of two (A-T) or three (G-C) hydrogen bonds between pairs of has been done.

Contains tonone/thymine, guanine/adenine and guanine/thymidine. 0DNs are homopurine runs in double-stranded DNA in a sequence-specific manner. It can be combined with These recognition motifs motif) is based on Hoogsteen type base pairing or reverse Hoogsteen type base pairing. It's on. In the C/T recognition motif, the ODN pairs with two homopurine chains. In the G/A recognition motif, the ODN has two homopurines. antiparallel to the strand and depends on the G content of the third strand. These recognition trends Objects may also be sequence dependent. Antigene using C/T recognition motif The sequence specificity of 0DNs allows for homogeneity in plasmid DNA and yeast chromosomes. Hybridization of the 0DNs to purine forms (runs) is possible. Become. Because ODN binding is restricted to homopurine species (runs), the remaining Identify additional heterocycles that can recognize two base pairs, namely C-G and T-A. It is advantageous to Guanosine can also be used in the third strand to recognize the base pair T-A. However, this interaction involves only one hydrogen bond and is relatively unstable. It is fixed.

Analogs of antisense 0DNs, antigene 0DNs, can be used to increase these activities. Modification with various pendant groups designed to It is also possible to do so. Therefore, the inserting group, the cleavage reagent, and the crosslinking moiety are It may also be added to the end of the gene ODN. During duplex formation, these groups are in close proximity It interacts with the double strands that are attached to it, and inserts, cleaves, and crosslinks them, respectively. Furthermore, the C/T recognition model In TIF, cytosine in the third strand ODN is replaced with 5-methylcytosine. In other words, the duplex formed in the 2G”-rich homopurine form (runs) is Always stabilize.

In addition, oligonucleotides of ○DNs with modified backbones are available. Methylphosphonates and phosphorothioates, etc., form double-stranded complexes Sometimes.

Enzyme-mediated duplex formation An important disadvantage of duplex formation discussed above is the relative speed of duplex formation. Be slow. The present invention is particularly characterized by the enzyme catalysis of duplex formation. The aim is to overcome this disadvantage by combining highly desirable enzymes. more meaningful Deeply, enzyme-catalyzed duplex formation is important for overall sequence recognition (non-enzyme-mediated immense advantages (as opposed to A-T and G-C recognition limitations associated with duplex formation) It gives

In summary, the genetic material of all living organisms undergoes occasional homologous rearrangements (hom .

1 Gous rearrangement). similar (or homologous) Recombination that occurs between DNA molecules plays an important role in promoting genetic diversity within a species. catalyzed by enzymes common to both prokaryotic and eukaryotic cells. It will be done. Recombination also plays an important role in DNA repair and immune responses.

Homologous recombination in E. coli occurs in both prokaryotic and eukaryotic cells. It serves as an illustration of the process that takes place. in a defined cell-free system. In research on purified recombinant enzymes derived from Ecoli, homologous recombination was divided into three stages. ing. For the purposes of this example, the “invading” single strand is circular and the tag The target duplex is linear.

In the first stage, “before pairing,” single-stranded circular DNA is exposed to the presence of ATP. Bottom, coated with multifunctional protein recA. the resulting nuclear protein The quality fiber has 18 base pairs per revolution and 3.6 rec per nucleotide, A It has a right-handed helical structure consisting of one protein monomer.

In the second stage, “nonapunsu”, the single-stranded circular nucleoprotein fiber is replaced by the linear double-stranded D A two-dimensional search for homologous sequences is performed along the NA template. The investigation is connected to the spiral. The first complex that does not have a tight network structure (the complex consists of two (quoted as “paranemic joint”) terminating in homologous sequences of the two molecules at A connection between two chains that do not get wrapped around each other. The complex is very unstable and easily separates during membrane proteination. DNA double et al. The fibers are incorporated into the fiber very quickly, and at the same time the two DNA molecules are attached to each other. Wrap into a spiral to form a coil (i.e., intertwine in a spiral). this double strand In this case, the newly incorporated triple strand is connected to one of the parent duplexes (in sequence and polarity). homologous) and complementary to the other parental duplex. The resulting nonapunsu complex In this case, the three strands of DNA actually exist as a hydrogen-bonded double strand, and recA believed to have a close relationship with What is the joining of two chains that do not wrap around each other? In contrast, complexes that spiral around each other into a coiled form are deproteinized. It is stable upon conversion (removal of recA).

The nnapunsu complex, which is coiled by spiraling around each other, is a right-handed helix. Contains 18 triplets per revolution. rec A is a critical part of the complex In other words, sequence specificity lies exclusively in the hydrogen bonds between base pairs. wrapped around each other in a spiral The densely coiled nonabunos complex has two alternative conformations. -Shikon exists. One is that the negative linear strand of the parent DNA is connected to the positive linear strand by Watson Click-type base pairs; the other is the negative linear strand of the parent DNA and the positive It forms a Watson-Crick type base pair with a circular single strand. Which cornho? In Mesicon, the base of the third strand is (according to the code for the binding site above) ) in Hoogsteen type or reverse Hoogsteen type for the other two strands of pudding. Assumed to be hydrogen bonded. The single-stranded circular DNA within the triple-stranded nucleoprotein fiber is , subsequently replaced by a homologous linear strand (“strand exchange”). This replacement is rec Characterized by the energy-dependent release of a linear positive chain, catalyzed by A, a cyclic single chain and a complementary linear strand hybridization complex. nabsis and strand exchange. During both, rec A remains associated with the complex. Finally, recA separates from the newly formed duplex, completing recombination.

During membrane proteination of the nonabsis complex, the protein-free double-stranded DNA complex is It is essentially resistant to single-strand specific nucleases and exhibits a very high T.

This is the same as hydrogen bonding between the so-called “third chain” and the witness (, This is thought to be due to the state in which the coalescence is highly prone to spiral formation. Therefore, it is very The deproteinized sinabun complex, which is highly susceptible to spiral formation, is highly susceptible to spiral formation. It is an analog of rec A-containing complex. Therefore, rec A is It may also catalyze DNA duplex complex formation, which cannot be achieved. In this complex the two positive strands have the same sequence and polarity, and one of the positive strands has the negative It has the ability to form double-stranded hybrids with sight.

Recent evidence suggests that recA plays a role in duplex conformation, including strand swapping. It has been suggested that the current version be changed. In other words, a double-stranded base has two strands of purine salt. This means that it can hydrogen bond with an appropriate purine base regardless of whether it contains a group or not. Ru.

Several factors influence the stability of the Nnabunsu complex, including (1) (2) the shared phase between the invading single strand and the target duplex; Degree of homology (length of homology and especially important mismatches) (3) Shared homology with respect to the end of the target duplex the location of the region, and (4) the double-stranded superhelix character (su), if any. perhelicity).

When selecting and designing an optimal system for stable duplex formation with an antigene, consider the following: The following factors may be important: The oligomer, 50-mar in length, forms a stable presynabunous complex with recA. It should be long enough to allow

Shared homology: recA-stabilized synapunus fiber formation requires a minimum of 13 salts It appears that a shared homology site of the group is required. MT file with one ODN added Photofixation of the recA-stabilized duplex by the run side is performed at minimal or preferred levels. Further restrictions may be placed on the shared homology of Bell. Photofixed and double stranded When the conformational conformation within a complex changes, the invading third strand It becomes possible to switch positions in the double strand. As a result, the bases of the third strand are complementary This results in Watson-Crick base pairing with the chain. Therefore, the preferred embodiment In this case, the ODN and the target share a 50 base homologous site.

Type of mating: shared regions of homology (if any) and target double-stranded DNA Depending on the positional relationship of the ends, the resulting Nnabsis complex can be a proximal junction or an intermediate junction. , or classified as distal junction. Proximal junctions result in recA-stabilized double-stranded complexes The body is located on the left hand side of the linear duplex, and due to strand exchange catalyzed by recA It is unstable. During strand exchange, in the double-stranded complex, the invading third strand Duplexes that are homologous are displaced, and as a result, the incoming strand and the free one This results in a new duplex containing the main strand. Strand exchange requires a free 5′ end; This process easily occurs in close junctions, as it progresses from Ru.

In contrast, the distal and intermediate junctions lack proper termini and are easily accessible to recA. Not subject to catalytic strand exchange. As a result, these joints are highly stable structures. be. Additionally, within the present invention, to form an intermediate or distal junction, re c. Formation of an A-stabilized double-stranded complex is preferred.

Superhelix (SuperheJ, jcity) Varnish-Superhelix targeting of recA may promote the formation of recA-stabilized triple-stranded duplexes.

However, high efficiency duplex formation (DNA target and very small covalent even when using ODNs with homologous regions), Sometimes it happens.

Synthesis and oligomer dispersion for supercoiled targets in model systems The choice of strand target 1-DNA can vary depending on the characteristics of the target double-stranded DNA. It seems that it will.

The present invention focuses on stable double-stranded complex formation catalyzed by recA and synthetic pile gene ODN. It is a combination of s. Enzyme-catalyzed duplex formation has rapid kinetics and a general This shows NA sequence recognition. The antigene ODN coated with recA is as a “guide” to search for homology in double-stranded DNA sequences of During the recognition and binding of this nucleoprotein fiber, recA uses two sequence-specific molecules. Catalyzes the formation of chain complexes. In the present invention, the antigene ODN is small in length. At most 30-40 nucleotides, each of the two strands of the target DNA It has the same base sequence and polarity as Frequency of duplex formation and membrane tongue The stability of the double-stranded complex that follows proteinization is directly related to the length of the antigene○DN. Sometimes they are connected. anti-gene 0DNs (of appropriate polarity) to the endogenous recombination pathway. Used in combination to form sequence-specific double-stranded complexes with chromosomal DNA There is also. As an alternative, double-stranded D targeting antigene 0DNs of various lengths It may also be complexed with a recombinant enzyme before binding to NA.

Catalyzing the formation of hydrogen-bonded double-stranded complexes using the cellular recombination machinery This overcomes several disadvantages associated with current antigene research. Primarily , the enzyme-mediated recognition motif recognizes all four base pairs, thereby allowing Allows targeting of a few double-stranded DNA sequences. Second, rec A-coated - terminal chain antigene ODN (nucleoprotein fiber) is a small uncoated Much more efficient than the target double-stranded DNA (site homologous to the target double-stranded DNA) and thus determine the concentration of antigene ODN required for efficient double-stranded complex formation. reduce Third, new helical strands related to hydrogen bond types and enzyme-mediated recognition. Because of the oxidation, the resulting double-stranded complex is stable at physiological pH. Fourthly , as cellular recombination pathways are utilized, higher-order chromatin structures are DNA that can be used as a target is easy to use as a target. Furthermore, fifthly, the resulting double-stranded complex The body exhibits increased stability with increasing length of the antigene DN.

The ability to conduct effective homology searches is a critical advantage. Preliminary data shows 0 DNs has a very low efficiency in investigating complementary homopurine sequences in double-stranded DNA. It is shown that. For example, non-enzymatically catalyzed duplex formation in vitro CF, M Orson, etc., nuclear acid, analyzed after 60 minutes at 37°C. Research (?1uc1.Ac1ds)7es,), Volume 19, 3435 -41 pages, 1991).

On the contrary, even classical hybridization between two complementary-terminal strands takes several hours. It doesn't take long, but rather it happens within seconds. The human genome is larger than 3x10” base pairs. In particular, if anti-gene 0DNs are used at relatively low concentrations, For example, homology searches can take an prohibitively long time. for double stranded DNA A type of protein formed between -end strand DNA and rec A that exhibits weak non-specific affinity. The use of Unaprennabuns nuclear protein fibers allows homology studies to be carried out from 3D to 2D. Effectively reduce the dimension process. Furthermore, double-stranded and homologous records (homol, og ous registry), the nucleoprotein fibers are divided into corresponding double-stranded and naked It appears to produce more double-stranded complexes than interactions with single-stranded molecules. These factors For the offspring, duplex formation between the recA-covered-end strand ODN and the homologous duplex is Calculation of the rate of spontaneous recovery of complementary-terminal strands under standard hybridization conditions This occurs at a reaction rate that exceeds the value by one or two orders of magnitude.

The present invention provides (1) homologous to a part of one of the target DNA duplexes, and (2) an ODN that is complementary to a similar portion, and (2) an inactivation of the target DNA sequence. It is a combination of enzyme-catalyzed duplex formation that is achieved. In a preferred embodiment In some cases, the ODN has a bridging moiety covalently linked to it. target Upon hybridization to the target DNA sequence, a third HODN (antigenetic Cross-linking to both parent terminal strands (child 0 DN) inactivates the Targetera 14) NA sequence do. ODN, crosslinking part, target DNA sequence and target DNA sequence Depending on the environment and characteristics of the column, inactivation of the parental duplex may be permanent. be. Therefore, a single administration of one or more antigene 0DNs can completely retrograde Virus cetum, evisome helbesvirus genome or mutant tumorigenesis It may also block gene expression.

Following duplex formation and covalent cross-linking, the modified target DNA is no longer Does not constitute an endorsement of reproduction or transcription. Unlike all other lesions in DNA However, this modification is not reparable. usually, Cross-linked DNA is repaired by a combination of excision repair and homologous recombination.

However, in cross-linked double-stranded complexes, target genes that enter recombination There is probably no copy of this that is not damaged. By analogy with the prokaryotic cell model Instead, try using the misrepair (or 5OS) route to remove crosslinks. However, eukaryotic cells induce mutations as a sacrifice. In such a case, Gene function will be irreversibly silenced by the resulting mutation.

Significantly, when recombinant enzymes are used in combination with antigene○DNs, This increases the efficiency with which this MODN “finds” its complementary target DNA sequence. When the anti-gene ODN (e.g. in a nucleoprotein complex) associates with a recombinant enzyme, When combined, the efficiency of duplex formation is greatly increased.

Within the present invention, suitable target DNA sequences include structural genes and both upstream and downstream sequences. regulatory control sequences. These regulatory sequences are involved in either transcription or replication. Sometimes they are connected. Anti-gene ODNs are tags selected for the purpose of altering function. -Gera 1-Determined and designed by DNA sequence and selected Targera l-D N will have a complementary sequence to one of the two strands of A.

Crosslinking agents suitable for use in the present invention include photochemical reagents such as psoralen and It is a chemical crosslinking agent. Preferred chemical crosslinkers are those described in Section A above; ○Electronic moieties attached to the 3′ and/or 5° ends of DN: and ○ Contains an attached masked electronic moiety. Photocrosslinkers can be applied topically or extracorporeally , a light-sensitive target, also useful for in vitro use. Sometimes. Chemical crosslinks may be used without limitation and are not covered by the claimed invention. It is particularly preferable for use within the range of .

Preferred recombinase enzymes include recA, human recombinase and N. melanogaster. Prokaryotic and eukaryotic recombinants such as Drosophila recombinases Human recombinase is particularly preferred.

In particularly preferred embodiments, the antigene ODN is administered to a cell or host to When the child DN enters the target cell nucleus, it binds to the recombinant enzyme in the nucleus.

In an alternative embodiment, the anti-gene ODN and recombinant enzyme are combined ex-vipo. in vivo) and then administered to cells or hosts as nucleoprotein fibers do.

In this embodiment, it is advantageous to administer the nucleoprotein fibers in liposomes. Sometimes it is.

The following examples illustrate the invention without limiting it. “RT” is room It means warmth.

mistress Thin layer chromatography on silica gel 60F254 plate (Anarte Tsuku) using the following solvents: A-90% methylene chloride, 10% methanol: B -50% ethyl acetate 50% hexane; C-70% ethyl acetate 10% methanol Le: 10% water: 10% acetone + D-50% ether, 50% hexane. Hula Chromatography was performed using 60F254 silica (Merck). O Ligonucleotides were produced on an Applied Biosystems Model 380B synthesizer. Synthesized. Oligonucleotides are linked to T4 polynucleotide kinase (BRL) and and γ-”P-ATP using the New England nucleus). Essentially labeled.

Example 1 6-(tritylamino)caproic acid 6-aminocaproic acid (26 g, 0.2 mol) was dissolved in triethylamine (100 ml). ) was added and dissolved in dichloromethane (200 Ill). Trityl chloride (120 g, 0°45 mol) was added and the solution was stirred for 36 hours. The resulting solution was diluted with 1N HCl. The organic layer was evaporated to dryness. Residue 2-Propertool/IN It was suspended in NaOH (300@t/100 ml) and refluxed for 3 hours. Steam the solution It was allowed to evaporate to a thick syrup and dichloromethane (500ml) was added. add water , acidified. Separate the layers and dry the organic layer over sodium sulfate and evaporate to dryness. Ta. The residue was suspended in hot 2-propanol, cooled and filtered to remove the intermediate compound. 6-(tritylamino)5-(tritylamino)pentylhydroxymethylene 6-(tritylamino)-caproic acid (20,0 g, 5 3 mmol) and triethylamine (20 ml) in dichloromethane. Isobutyl chloroformate (8.3 ml, 64 mmol) was added dropwise over 0 min. I put it down. The mixture was stirred in a water bath for 2 h and freshly distilled malonitrile (4 , 2 g, 64 mmol) were added in one portion. The solution was placed in a water bath for 2 hours and then at room temperature for 2 hours. Stir for a while. The dichloromethane solution was washed with water-cooled 2N HCl (300ml) and diluted with dichloromethane. The phase mixture was filtered to remove the precipitated product (13.2 g). separate phases The organic phase was dried and further evaporated to a thick syrup. add syrup Lolomethane was applied to deposit fine crystals of the product. filter the crystals and dried to obtain 6.3 g of product for a total yield of 19.5 g (87%). It is useful as an intermediate.

Example 3: 5-(Tritylamino)pentylmethquinomethylene malononitrile ether/di Malononitrile of Example 2 in chloromethane (900Ial/1001ml) A suspension of (13 g, 31 mmol) cooled in a water bath was added to a freshly prepared diluted Azomethane (Diazard (trademark) (Aldrich Chemical Kanno Sakuranie) 5 (from 0 mmol) in ether. Stir the solution for 6 hours, then add vinegar Neutralized with acid (10ml). The solution was evaporated to dryness and the residue was dissolved in /chloromethane/acetate. Norikakel's chromatography using ton (4/1) as eluent Ta. Both portions containing the product were collected and evaporated to Noronobu. Shiro Npu wo Ji It was triturated with chloromethane and crystallized. Filter the crystals, dry and chromatograph 8.3 g (61%) of a product which is pure and useful as an intermediate compound was obtained. Two.

Example 4・ 5-amino-3-[(5-tritylamino)pentyl] bira-nor-4-ka Rubonitrile In a water bath, a methanol solution (1 00 ml) of hydrazine monohydrate (7.8 ml, 160 mmol). I dripped it every minute. After stirring in a water bath for 30 minutes, the solution was evaporated to dryness. Cool the residue After suspension in methanol, filtration and drying, the 5-amino-3- [(5-tritylamino)pentyl]pyrazole-4-carbonitrile 7.1 ( 100%) was obtained. Analytical samples were prepared by recrystallization from water.

Example 5: 5-amino-1-(2-thioxy-3,5-)-〇-toluoyl-β-p-technique 1 Notrobentofuranonor)-3-[(5-tritylamino)pentyl]virano 4-carbonitrile A water-cooled solution of carbonitrile (3.5 g, 8 mmol) from Example 4 was heated with hydrogen. Treated with sodium and stirred at 0-4°C for 30 minutes. 1-chloro-1,2-' ) Add thioxy-3,5-di-oh fluoroyl lipofuranose and dilute the solution to 0-4 Stirred at °C for 1 hour. Pour the solution into a saturated solution of sodium bicarbonate and dichloromethane. Extracted with tan. The organic layer was dried over sodium sulfate and evaporated to dryness. residue of Norica gel using toluene/ethyl acetate (5/1) as eluent. The sample was subjected to microchromatography. Two main products were isolated, N-1 and N- Identified as two isomers, the respective yields are 57% (3.6 g) for N-1 and N-2it was 20% (1.2 g). Approximately 1 g of N-1 and N-2 isomer mixture I also collected compound items.

The total yield of glycosylated raw material was 5.8 g (92%). N-1 isomer , 5-amino-1-(2-thioxy-3,5-di-toluoyl-β once-2 1-notropentofuranonor)-3-[(5-tritylamino)pentyl]pyra /-ru-4-carbonitrile was used in Example 6 without further purification.

Example 6 l-(2-thioxy-β-and-monoerythropentofuranosyl)-3-[(5-( h'notylamino)pentyl]pyrazolo[3,4-d]pyrimidine-4-ami Example 5 (3.5 g, 4.4 mmol) of pyrazole-4-carbonide I J) To the toluene (100ff11) solution, add ethoxymethyl acetate ( 1,111,67 mmol) was added.The solution was kept at 80-9.0°C for 5 hours. It was then evaporated into white whelk. Add syrup to dichloromethane (10ml) and added to water-cooled methanolic ammonia (100 ml) in a glass pressure bottle. Ta. After two days at room temperature, the contents of the bottle were evaporated to dryness. Dissolve the residue in methanol and The pH was adjusted to 8 with freshly prepared sodium methoxide to completely deprotect. -evening After stirring, the solution was treated with Dowex™-50H+ resin. filtered and evaporated to dryness. Dissolve the residue in acetone/hexane (3/2). (used as a releasing agent) and chromatographed on silica gel to obtain an analytically pure 2.0 g (77%) of product was obtained.

Example 7゜ 1-(2-deoxy-β-erythropentofuranosyl)-3-[5-(tri- ethylamine) pentylcopyrazolo[3,4-d]pyrimidin-4-amine 5 -monophosphate Pyrazolobi 1 nomidine-4- of Example 6 in trimethyl phosphate (5 ml) Phosphochloride lJ7 was added to a water-cooled solution of amine (250 mg, 0.43 mmol). 50 μm) was added and the solution was kept at 0-4°C. The reaction starts from 100% O in water. Followed over 25 minutes by reverse phase HPLC using a linear gradient of acetonitrile. . After stirring for 5 hours, an additional portion of phosphoryl chloride (25 μm) was added and the solution was poured into another portion. Stirred for 30 minutes.

Purification was performed by reverse phase HPLC using a 2 mm x 5 Q cm C1,8 column. column was equilibrated with water and eluted with a gradient of 0 to 100% acetonitrile over 20 minutes. Ta. Fractions containing the desired substance are collected, lyophilized, and chromatographed. 160 mg (56%) of essentially pure nucleotide was obtained.

Example 8: 1-(2-thioxy-β-D-erythrobentofuranonor)-3-(5-[( 6-biotinamido)hexaneamidopentyl) pyrazolo[3,4-CI] Nucleotide salts of pyrimidine-4-amine 5'-monophosphate Example 7 solution (10 ml), balanoum hydroxide in carbon (50 mg L) and cyclo The xadiene (1 ml) was refluxed for 3 days, filtered and evaporated to dryness. Clean the residue DMF (1, 5 ml) of N-hydroxysuccinimine rubiotinylaminocabroe. (50 mg). - After stirring overnight, N-hydroxysuccinimidyl 6 - Add an additional amount of biotinamide cabroate (50 mg) and stir the solution for 18 hours. Stirred. The reaction mixture was evaporated to dryness and chromatographed according to the method of Example 7. Attached. Fractions were collected and lyophilized to yield chromatographically pure biotinylate. 80 mg of mid-substituted nucleotide was obtained.

Example 9゜ 1-(2-deoxy-β-D-erythropentofuranonor)-3-[5-(6- biotinamide) hexaneamide pentyl] pyrazolo [3,4-dl virimi Syn-4-amine 5'-h rephosphate The monophosphate of Example 8 (80 ffig, approximately 0.1 mmol) was added to triethyl alcohol. Min (14 μm) was added and dissolved in DMF. Carbonyldiimidazole (8 1mg.

0.5 mmol) was added and the solution was stirred at room temperature for 18 hours. Pour the solution into methanol ( After stirring for 30 minutes, tributylammonium pyrophosphate (0.5 g in 0.5 ml DMF) was added. After stirring for 24 hours, add another portion of the trib. Tylammonium pyrophosphate was added and the solution was stirred overnight. reaction mixture was evaporated to dryness and chromatographed according to the method of Example 8. two generation The materials were collected and treated separately with concentrated ammonium hydroxide (IJI) for 18 hours at 55°C. Processed. UV and HPLC analysis showed that after ammonia treatment both products were shown to be identical, which were collected and lyophilized to produce nucleotide trinucleotides. Phosphate 35°Nisocoustranslanecon reaction The triphosphate of Example 9 was prepared using the two-translane copolymer of Langer et al. (supra). was incorporated into pHPV-16 using the following protocol. Triphosphene of Example 9 Bio-11-dUTP (Sig. The probe was compared with a probe prepared using a commercially available chemical company (Ma Chemical Company). filter Hyperdigestion and in 5 itu smear No significant differences were observed in any of s).

More specifically, the procedure includes the following materials and steps.

material.

DNase (ICN Biomedicals) - 4 μg/ml DNA Polymerase 1 (U, S, Biochemicals) -8U/ml pHPV-16-2, 1611g /ml, which contains the human papillomavirus type 16 genome sequence. It's Mido.

Nucleotide-mix A-dGTP, dCTPSTTP 2 and 4 each (Pharmano a) Mix U-dGTP, dCTP, dATP 2 liters each Bio-11-dUTP-1 ,0mg/ml (BRL)Bio-12-dAPFTP-1,0mg/ml Moderate.

10X-DP (4ml), pHPV-16 (2ml), nucleotide mix Xu A (6 ml), Bio-12-dAPPTP (2 ml) and H2O (2 0 ml) 0) DNase (], ml) and DNA polymerase 1. (2.4ml) was added. The reaction mixture was incubated at 16°C for 1 hour.

Bio-12-dAPPTP (consisting of the triphosphate of Example 9) and Bio-11-dUTP and nucleotides instead of nucleotide mix A This procedure was repeated using Mix U.

Nucleic acids were separated by ethanol precipitation and slotted onto nitrocellulose (sl otted) was hybridized with pHP V-16. hybridized Streptavian conjugate of biotinylated probe with BCIPlNB It became clearer with alkaline phosphatase. biotinylated nucleotides Probes prepared with either gave the same /ethyl. probe in the neck Tested also in in-note format in Cervical smears However, it was shown that there was no qualitative difference in signal and background.

Example IJ 5-amino-3-r(5-tritylamino)pentylcobyrazole-4-carbo Quinoamite The method of Example 2, except that ananoacetamide is used in place of malonitrile. According to is prepared from 6-(tritylamino)caproic acid. After this, Example 3 treatment with azomethane to obtain the methokino derivative, which was then carried out 5-Amino-3-[(5 -tritylamino)pentyl]pyrazole-4-carboxydiamide is obtained.

Example 12・ 4-Hydroxy-6-methylthio-3-(5-tritylamino)pen-tylco Virazoro C3,4-d″Jpi) Chisel Machine The carboxamide from Example 11 was combined with potassium ethylxanthate and ethylxanthate. 4-hydroxypyrazolo[3,4-dl pyrimidine] = Obtain the potassium salt of 6-thiol. This salt is then reacted with iodomethane 4-hydroxy-6-methylthio-3[(5-tritylamino)pentyl]pi Lazolo[3,4-dl pyrimidine is obtained.

1-(2-thioxy-β-p-erythrobentofuranonor)-4-hydroxy- 3-[5-0 lythylamino)pentyl] pyrazolo[3,4-dl] pyrimidine -6-amine Following the method of Example 5, the birazolopyrimidine of Example 12 was prepared with sodium hydride. treated with l-chloro-1,2-sibuoxy-3,5-cyo-toluoylrivof React with lanose. The resulting compound was combined with MCPBA and methanolic ammonia. The product is obtained by removing the toluoyl protecting group.

Example 14 1-(2-thioxy-β-ri-erythrobentofurano/l)-4-hydroxo= 3-C5(6-biotinamido)hexaneamidopentyl]pyrazolo "3,4- d] Pyrimidine-6-amine 5"-monophosphate Practical according to the method of Example 7. The birazolopyrimidine of Example 13 was reacted with phosphoryl chloride to form the corresponding 5-monomer. Obtain nophosphate.

Following the method of Example 8, the above 5°-monophosphate was treated with palladium/chlorine and React with chlorohexanoene and convert the residue to N-hydroxysuccinimine rubio. 1-(2-thioxy-β-p-erytho) by reacting with thinylaminocabroate. Bentofuranosyl)-4-hydroxy-3-[5-(6-biotinamide) xaneamidopentyl]pyrazolo[3,4-fi] birimicin-6-amine 5 °-monophosphate is obtained.

Example 15゜ ■-(2-thioxy-β-D-enotropentofuranosyl)-4-hydroxy -3-E5-(6-biotinamido)hexaneamidopentyl]pyrazolo[3, 461 Pyrimidine-6-amine 5'-triphosphate Following the method of Example 9, The 5°-monophosphate of Example 14 was treated with carbonyldiimidazole; After that, the corresponding 5゛- Obtain triphosphate.

Example 16・ 1-(2-thioxy-β-and-erythropentofuranosyl)-3-[5-(tri thylamino)-pentyl]pyrazolo[3,4-d]pyrimidine-4-benzoy Ruamine 1-(2-thioxy-β-Kiichi erythropentofuranosyl)-3 from Example 6 -[5-(tritylamino)benthylcopyrazolo[3,4-d]pyrimidine -4-amine is reacted with benzoyl chloride and pyrimidine to produce a C-3,5-DΩ-benzoyl-β-2-erythrobentofuranonor)-3- C5-(tritylamino)-pentyl]pyrazolo[3,4-d]pyrimicin- 4-benzoylamine is obtained. This is treated with aqueous sodium hydroxide to partially The compound was deprotected and the 1-(2-thioxy-β-erythropentofuranosyl )-3-[5-(tritylamino)-pentyl]pyrazolo[3,4-c+3 Pyrimidine-4-benzoylamine is obtained.

Following the method of Example 8, the benzoylamine of Example 16 was hydroxylated in carbon. treatment with radium followed by trifluoroacetic anhydride to obtain 1-(2-thio xy-β-bentfuranosyl)-3-[5-(trifluoroacetate) (amido)pentyl]pyrazolo[3,4-d]pyrimidine-4-benzoylamide 1-(2-thiokine/-5-0-dimethoxy7trityl-β-and-erythropent furanonor)-3-[5-(trifluoroacetamido)pentyl]pyrazolo[ 3゜4,-d]]pyrimicin-4-benzoylamine 3゜0-(N,N- Diisopropyl) phosphoramidite noanoethyl ester Compound of Example 17 was reacted with cymethoquinotrityl chloride and pyrimidine to form the corresponding 5'-dimetrityl chloride. A toquinotrityl compound is obtained. This compound was then dianoethylchloro-N. N - React with diisopropyl phosphoramidite (such as Shinno, nuclear Nucleic Ac1ds Research (Nucleic Ac1ds Res), Volume 12, 4 5395-(4-phthalimidobutodo-1-yn-1-yl)-2'-deoxyu Lysine 5-iodo-2°-deoxyuridine (354 mg, 1 mmol) was added to Dissolved in 10 ml of chillformamide. Copper iodide (76 mg, 0.4 mmol) ), tetrakis(triphenylphosphine)palladium(0) (230mg, 0.2 mmol) and triethylamine (200 mg, 2.0 mmol) added. 4-phthalimidobudo-1-yne (300 mg, 15 mmol) once In addition, the reaction was kept at 60°C for 3 hours. Then evaporate the clear yellow reactant and salt Methylene chloride was added. Crystallize almost all of the product by scraping the inner wall of the flask. This was filtered and recrystallized from 95% ethanol to give 335 mg of the title compound. (78%) was obtained as fine, feathery needles.

Example 20: 5-(4-phthalimidobudol-1-yl)-2'-deoxyuridine Example 19 Dissolve 100 grams of deoxyuridine in 95% EtOH and Approximately 3 g of the liquid was added. After 48 hours, the catalyst was carefully filtered off and the filtrate was evaporated to a solid. and recrystallized from methanol-water to give the title compound 960a+g (97%) 5-( 3-iodoacetamidopropyl)-2°-deoxyuridine 5-(3-trif fluoroacetamidoprob-1-yl)-2-deoxy-uridine (0,3 m mol) of ammonia, then N-hydroxysuccinimidyl α-iodoacetate. (0.5 mmol). The reaction mixture was evaporated to dryness and chromatographed. 5-(3-iodoacetamidopropyl)-2'-deoxy Obtained uridine.

Example 22 5-(4-(4-bromobutyramido)butyl)-2′-deoxyuridine implementation 5-(4-phthalimidobutol-1-yl from Example 20) according to the method of Example 21 )-2'-deoxyuridine with ammonia and then with N-hydroxysphnonimide. 5-(4-(4-bromobutyramide) ) butyl)-2°thioxyuridine is obtained.

Phosphoramidite preparation and DNA synthesis nucleotides were prepared according to known methods. was obtained in approximately 85% yield by 5'-dimethoxytritylation, and 3'-phosphor amidite with diisopropylaminoper/anoethylchlorophosphite (upper) (as in “Oligonucleotide Synthesis: A Practical Method”) in methylene chloride. Made with diisopropylethylamine. Acetate phosphoramidite The solution was made into a 0.2N solution with nitrile and applied to an automatic DNA synthesizer. These new and Incorporation of modified phosphoramidites is similar to regular phosphoramidites. (for trityl assay, which develops color with UV) 97-99%).

Oligonucleotides were removed from the DNA synthesizer in tritylated form and treated with 30% ammonia. Deprotection was performed at 55° C. for 6 hours using A. 0.5M sodium bicarbonate 10μm In addition, acidification during concentration was prevented. The oligonucleotide was evaporated to dryness under vacuum; Redissolved in 10 ml of water. Add oligonucleotide to 0. IN triethylane acetate 20 by HPLC using 15-55% acetonitrile in monium acetate. Purified for minutes. The unsubstituted oligonucleotide was diamino-derived after 10 minutes. The conductor took 11-12 minutes. Collect the desired oligonucleotide and evaporate to dryness. and then redissolved in 80% aqueous acetic acid for 90 minutes to remove the trityl group. Desalination is , on a G25 Sephadex column and appropriate fractions were collected. Concentrate the fraction Make to specific volume, dilute to check total yield, and perform analytical HPLC to check purity. Ta.

Oligonucleotides were kept frozen at -20°C until use.

According to the above method, the nucleoside 5-(3-trifluoroacetamidoprobu-1 -yl)-2゛thioxyuridine to 5'-〇-dimethoxytrityl-3'-(N .

N-diisopropyl) phosphoramiditone anoethyl ester derivative Ta. Add this to a DNA synthesizer and add the following 14-mer oligonucleotide sequence. Prepared.

In the 3'-CT TCCU'TG TAG GTC-5' sequence, Ul is 5-(3- Aminoprop-1-yl)-2'-deoxyuridine (oligo A).

In the same manner, 5-(4-phthalimidobutol-1-yl)-2°-deoxyurid 5゛-〇-dimethoxytrityl-3゛-(N,N-diisopropyl)phosphorus Convert it into a foramidite cyanoethyl ester derivative and add it to a DNA synthesizer. In the above 14-mer oligonucleotide sequence, Ul is 5-(4-aminobudo-1- yl)-2'-deoxyuridine (oligo C) was prepared.

In the corresponding 14-IIIer oligonucleotide, Ul is unmodified deoxyuridine. We also prepared .

Example 24: Derivatization of oligonucleotides Generally, to add a crosslinker to an aminoalkylioligonucleotide, an aminoalkylated A solution of 10 μg of kill oligonucleotide and 0.1 M borate buffer, pH 100 such as α-Nakaguchi-acetate or 4-halobutyrate in 8, 5, 10μm X molar excess of n-hydroxysuccinimide! 10 acylate at ambient temperature 30 Incubated in the dark for minutes. The entire reaction was equilibrated with sterile water. The mixture was passed through a ram and eluted with sterile water. Collect appropriate fractions based on UV absorption, Concentrations were measured spectrophotometrically.

Introduction of the haloacyl moiety was investigated by HPLC. Solpax (trademark) ( Zorbax) oligonucleotide column (DuPont) from 60% of the composition Eluted with an 80% gradient for 20 minutes: A (20% acetonitrile = 80% 0.02N NaH2PO4) and B (1.2N NaCl in 20% acetonitrile: gQ%0.02NNa82POt). A reactive α-haloanol moiety is present That is, the corresponding aminoalkyl oligonucleotide after exposure to IN nosteamine Retention time of α-haloanolamide alkyl oligonucleotide for otide Indicated by the response. When nosteamine is introduced, aminoalkyl oligonucleotides The charge pattern between the otide and α-7X loanolamide oligonucleotides is equal. It becomes.

According to the following method, 14-mer oligonucleotide 3°-CT TCCU’T In the G TAG GTC-5' sequence, U' is 5-(3-aminoprop-1-yl) -2'-deoxyuridine (Oligo A1 Example 23) By reacting with cisphnonimide α-iodoacetate, the 1.4-mer ori In the sequence of the oligonucleotide, U' is 5-(3-iodoacetamidoprob-1-yl). )-2'-deoxyuridine (oligo B) was obtained.

Oligo A and oligo B were synthesized with the above 1.4-mer '[Jl is unmodified deoxyuran] Analyzed on a Solpax column, all identical sequences, similar to those in lysine, are It had a lower retention time. Unmodified 14-mar, 931 min; aminopropyl 14- mar (oligo A), 7.36 min, and iodoacetamidopropyl 14- mer (oligo B), 10.09 minutes.

In the same way, aminopropyl 14-mer (oligo A) was converted to n-hydroxysuccinimide. reaction with niimide 4-bromobutyrate to form a 14-mer with Ul as 5-(3-( 4-bromobutyramido)prop-1-yl)-2'-deoxyuridine I got something.

Aminobutyl 1.4-mer (Oligo C1 Example 23) Cinimide α-iodoacetate or N-hydroxysphnonimide 4-pro 14-mer with Ul is 5-(4-iodo) acetamidobut-1-yl)-2゛-deoxyuridine or 5-(4-( 4-bromobutyramido)budol-1-yl)-2'-deoxyuridine I got each of them.

Example 25 Questions about cross-linking reactions The reaction for cross-linking the DNA probe with the target nucleic acid sequence was performed using 1 μg haloacyl amino acid. Doorkyl probe and 0,1 lvf Tris, pH 8,0, and 0.9 MN 32 p-labeled target with cordycepin at the end in 200 μm of aC1. The cells were incubated at 20°C or 30°C. some part Removed at 24 hour or 72 hour intervals, 1Qrr + M Nosteamine 20μ The 710 acylamide group was extinguished by diluting to m.m. Store these solutions at room temperature, 1 μm was used for denaturing polyacrylamide gel electrophoresis (PAGE) analysis.

According to the above method, two models of oligonucleotide sequences are used to The potential of the probe to crosslink with its complement was evaluated. Human papillomavirus (H PV) or human cytomegalovirus (CMV). -propyl]-2-deoxyurin, or U=5-[3-(α -・iodoacetamide)- or 4-(4-bromobutyamide)-butyl]- 2'-deoxyuridine.

The target for HPV is 30-mer, and the target for CMV is 30-mer. is a 24-mer. The crosslinking probe is a 14-mer, C It was a 15-mer with respect to MV. Each probe once in the above sequence Contained the indicated single modified deoxyuridine.

Cross-linking HPV targets with 5-(3-iodoacetamidopropyl) The results of the reaction with a limited amount of probe are shown in FIG. On denaturing PAGE gel Analysis of the cleavage pattern of the cross-linked It was shown that the hybrid will lose. The intensity of this band is It depends on the degree of crosslinking. Two separate /X-band knobs on the gel There is a non-sequence-directed alkyl in either the target or probe strand. strongly asserts that oxidation is occurring (including intramolecular probe alkylation) It is.

Compared to the actual 15-mar run in the adjacent lane, the main cut It is suggested that the truncated fragment is a 9-mer. Unique autoradiolog Precise testing with a ram shows bands that move more slowly and have very low strength. was recognized. This pattern is consistent with predominant alkylation at G-21 and G-20 This would be consistent with a small amount of alkylation at . Cross-linkable HPV hybrids The Dreien model test is based on the 5-(3-yo) By slightly distorting the helical structure of the side hand, the target It shows that it can contact the G-21 residue of the chain.

The alkylation is located 2 units away from the 5° side of the modified deoxyuridine base pair. If it occurs predominantly in the target town of Guanon, the CMV sequence will not react. Dew. This result was actually observed. Not reacting with CMV is a further feature of the present invention. This supports the specificity of the cross-linking mechanism.

Example 26 Time and temperature dependence Time and temperature dependence studies were performed on the HPNr system of Example 25. It was carried out with acetamidoprop-1-yl)-2'-deoxyuridine. .

32 by cordycepin with a terminal group transferase attached to the target end. p-labeled Maniatis et al., “Molecular Cloning - Experimental Manual”, Cole. Spring Harbor Laboratory, 11982, p. 239), up to 20°C. Incubate with excess probe in pH 80 Tris buffer either at 30°C or at 30°C. Cubated. Aliquots were removed after 0.24 or 72 hours of incubation. and 10 mM mercaptoethylamine (reacted with iodoacetamide). Stop the reaction with equal volumes and leave at room temperature for subsequent analysis by denaturing or non-denaturing PAGE. Saved with.

Cross-linking of the hybrid was followed with modified PAGE, which was This was evident at both temperatures and after 72 hours (see Figure 3). cross-linked The amount of Ibrid increased at all temperatures and times. /1 Ibrid's approx. Cross-linking was carried out on a 20% wall and incubated at 30° C. for 72 hours.

Experiments at separate temperature ranges showed that the half-life of cross-linking at 37°C was approximately 2 days, and the reaction showed completeness after 24 hours at 58°C. This time-dependent reaction is , the iodoacetamide moiety does not hydrolyze or react with the buffer solution. It's implied. The increase in reaction rate at higher temperatures is due to the fact that / and subsequently indicate that the alkylation rate shows the desired increase with temperature. te 0ru.

Example 27・ Site specificity of alkylation In order to elucidate the site specificity of alkylation, the cross-linked HPV/- Brid (U is 5-(3-iodoacetamidoprob-1-yl)-2'-de Oxyuridine) was added to a 10% piperidine solution at 90° C. for 60 minutes. Ma Kisimam et al. (Proceedings of the National Academy of Yves) NNIS-USA (Proc, Natl^cad, sci, UsA) , Vol. 74, p. 560 (1977), this process This method quantitatively cleaves the Tarp-Sotho chain 3° with respect to the binding site. As a result The data obtained by , alkylation of the terp-but base (guanine) was the only effect observed. The cross-linking reaction in the HPV model system is highly specific. It showed that.

Chemically modified crosslinkable 0DNs Psoralen-modified photo-crosslinkable ○DNs were prepared using a photochemical method.

Briefly, an ODN is hybridized with a complementary DNA sequence and a 4'-hydroxymer Chill-4,5°, illuminated at 360 nm in the presence of 8-trimethylpsoralen (HMT). injection, thereby forming a sequence-specific cross-link with a unique 5'-TpA-3' sequence. did. Partial reversal of crosslinking by exposing to 25Qnm light for a short time Modify HMT furan side-adduct ○DN according to photoreversal) Separated by PAGE.

As an alternative, psoralen derivatives can be coupled to ODNs by chemical synthesis. Sometimes I let them do it. Using these synthetic mechanisms, we synthesized the 5° end of ODN and psoralen. 4,5°, 8-trimethylpsoralen (TMP) through the bond spanning the 4′ position. Combined ○DNs may also be obtained. One method is to use modified TMP as appropriate. React with 5′-activated and deprotected ODN (B, L, Ri et al., Biochem. Story (Bioche, ), vol. 27, pp. 3197-3202, 1988 ). As a second method, TMP phosphoramidite-conjugated compounds can be added at the end of solid-phase synthesis. Add to (U Virus and U, Ingrinoe, Nucleitsk Acids) Research (Nucl, Ac1ds Res,), Volume 17, Pages 285-99, (1989). A similar adduct is 4'-aminomethyl-4,5',8-trimethyl It may also be prepared using thiolated derivatives of psoralen (AMT) (J, Thi. Ale and P. Warrenzien, Nukleitsk Ands Research (N uclJcids Res, ), volume 17, pages 3359-72, 1989) . As a further alternative, TMP-modified deoxyadenosine phosphoramidites can be used on a solid phase. It may be inserted at any position in the ODN during synthesis (U Virus et al. Itsuku Ansos Research (Nucl, ^cids Res, ), Volume 17 , pp. 8967-78, 1989). In this synthon, TMP is The 4th position is modified with a linker that binds to the 08th position of the nonn.

Example 29゜ Prototype using chemically modified 0DNs for Turke Soto double-stranded DNA, i.e. rec When complexed with homologous NA in the A-stabilized duplex, both Watson and A psoralenized ODN that can be efficiently crosslinked with both the strand and the click strand is used. the end A psoralenized 5゛ modified ODN was synthesized, and the side of the rec A-stabilized duplex was Maximum photocrosslinking occurs within the 5'-TpA-3' sequence in contact with the surface. In summary, so Larenylated ODNs are used in the presence of recombinant enzymes to create sequence-specific cross-links between double-stranded D into a complementary site in the NA target. The ODN has a DNA target and It has a Hoogstein type base pairing, which is connected to the 5-hydroxypsoralen moiety or 4. Modify its 5' end with a 5', 8-trimethylnlalene moiety. solare By linking the furocoumarin to the 5' position, the furocoumarin can be attached to the duplex immediately adjacent to the duplex. It can be easily inserted into the 5'-TpA-3' sequence. Furan ring and pyrone Both rings are precisely positioned to photoreact with thymidine. psoralenized tail The length of the psoralen may be varied to maximize insertion of the psoralen into the duplex-duplex junction.

Figure 1 ・mgd　’’P-1mHPV target @32P　mm15-mer marker ---ψ　Crosslinked product ...Mockery-32p-labeled HPV target Figure 3 Continuation of front page (51) Int, C1,5 identification symbol Internal reference number Cl2N 15/11 //Cl2Q 1/68 Z 7823-4B (72) Inventor Mayer, Ripp Chi Bee Jr. Woodinville, Washington, USA 98072 N.E. One Hundred and Seventy Six Brace 1541 Number 1 I (72) Inventor: Tipone, John See, Washington, USA 98011 Pourcel, N.E. One Hundred and Sixty Six ・Brace No. 12117 (72) Inventor: Burst, Gerald Dee, United States of America 77380 Text 8th Floor, West High Ouse, The Woodlands, Sass. (72) Inventor: Gamber, Howard Bee, Washington, USA 98072 Hundred and Twelve, N.E., Woodinville, Tong. Drive 14048

Claims (6)

[Claims] 1. Structural genes, corresponding to regulatory sequences or parts thereof that influence gene function homologous to one strand of the target double-stranded DNA and homologous to the other strand. A complementary oligonucleotide (ODN) is attached with a crosslinking agent to form a crosslinked ODN. , contact the crosslinkable ODN with a recombinant enzyme to form a nuclear protein fiber, and To form a triple-stranded complex between the nuclear protein fiber and the target double-stranded DNA. It takes enough time to combine, The gene consists of cross-linking the three-stranded complex to change the gene function. How to change functionality. 2. 2. The method of claim 1, wherein the crosslinking agent is a nucleoside crosslinking agent. 3. The nucleoside crosslinker has the following formula: R1-B-(CH2)q-(Y)r-(CH2)m-A'(I') In the formula, R1 is hydrogen, or is bonded to the sugar moiety through oxygen at its 3' or 5' position and a phosphorus derivative containing groups Q1, Q2 and Q3, or a nucleotide linkage. A sugar moiety or the like, optionally substituted with a reactive precursor thereof suitable for synthesis. It is a likeness; Q1 is hydroxy, phosphate or diphosphate; Q2 is =O or = S; Q3 is CH2-R', S-R', O-R', or N-R'R''; R ' and R'' are each independently hydrogen or C1-6 alkyl; B is a nucleobase or an analog thereof that is a constituent part of an oligonucleotide. , ; Y is a functional linking group; m and q are independently 0 to 8; r is 0 or 1; A′ is a leaving group The method according to claim 2, which is represented by: 4. Structural genes, corresponding to regulatory sequences or parts thereof that influence gene function homologous to one strand of the target double-stranded DNA and complementary to the other strand a chemical cross-linker covalently bound to an oligonucleotide; A chemically cross-linked antigene core consisting of a recombinant enzyme non-covalently linked to a cleotide protein fiber. 5. The chemical according to claim 4, wherein the chemical crosslinker is a nucleoside crosslinker. Cross-linked anti-gene nucleoprotein fibers. 6. The nucleoside crosslinker has the following formula: R1-B-(CH2)q-(Y)r-(CH2)m-A' (I') In the formula, R1 is hydrogen, or is bonded to the sugar moiety through oxygen at its 3' or 5' position and a phosphorus derivative containing groups Q1, Q2 and Q3, or a nucleotide linkage. A sugar moiety or the like, optionally substituted with a reactive precursor thereof suitable for synthesis. It is a likeness; Q1 is hydroxy, phosphate or diphosphate; Q2 is =O or = S; Q3 is CH2-R', S-R', O-R', or N-R'R''; R ' and R'' are each independently hydrogen or C1-6 alkyl; B is a nucleobase or an analog thereof that is a constituent part of an oligonucleotide. ; Y is a functional linking group; m and q are independently 0 to 8; r is 0 or 1; A′ is a leaving group The chemically crosslinkable anti-gene nuclear protein fiber according to claim 5, represented by Wisdom.