JPH06510203A - Formation of triple helix complexes of double-stranded DNA using nucleoside oligomers containing purine base analogs - 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] Mie et al. of double-stranded DNA using nucleoside oligomers containing purine base analogs. Formation of shear complex Citation of related applications This application is filed under U.S. Patent Application No. 924234, filed October 28, 1986. U.S. Patent Application No. 368027, filed as a continuation-in-part application of This is a continuation-in-part application filed on June 19, 1989, and the description is cited here. The invention is supported by government grants, including grants from the National Institutes of Health, Contract No. GM-45012. This was done with support. The government has certain rights to this invention. .

The publications and other references cited herein are incorporated by reference into the specification; Numerous citations are given in the following description, and in the bibliography attached immediately before the claims. It is classified as follows.

This application specifically forms a complex with a selected double-stranded DNA structure and a triple helix structure. specific sequences in double-stranded DNA using nucleoside oligomers that can form structures. A novel method of searching and locating.

Polymers in double-stranded DNA are bonded by Hoogsteen hydrogen bonds. by homopyrimidine oligodeoxyribonucleotides bound to the purine tract The formation of triple helical structures has been reported (see, eg, (1) and (2)).

Homopyrimidine oligonucleotides form double helix DNA through triple helix formation. It was found that an extended purine sequence was observed in the main groove. Specificity is homopy Limidine oligonucleotide and Watson-Crick double DNA purine release It was found that it is given by Hoogsteen base pairing. Fugu tea Triple helical complex containing cytonone and thymidine on the single (or third) mirror are stable in acidic to neutral solutions, but as the pH increases, It is known that it will leave. T and 5-methyl such as 5-promouranyl Incorporation of modified bases of C, such as cytosine, into the Hoogsteen chain may result in higher It has been found to increase the stability of the triple helix over a wide pH range. Shito In order for syn (C) to participate in Hoogsteen-type pairing, the piping is required due to hydrogen bonding. Hydrogen must be available on the 3-N of the rimidine ring. Therefore, some kind of Under the conditions, C can be protonated at N-3.

DNA exhibits a wide range of polymorphic conformations, and these conformations are It can be essential in physical processes. Signal by sequence-specific protein-DNA binding Regulation of introduction and molecular interactions such as transcription, translation, and replication depend on DNA conformation. ation is considered to be independent. (3) Important regions of the genome therapeutic agents that selectively inhibit abnormal cell function, replication, and survival. It is very interesting to think about the development possibilities.

(4) The idea and development of sequence-specific DNA-binding molecules is being studied in many laboratories. foreign genetic material (e.g. viruses) or modification of genomic DNA (e.g. It has a wide range of implications for the diagnosis and treatment of diseases, including cancer.

Nuclease-resistant nonionic oligos consisting of a methylphosphonate (MP) backbone Deoxynucleotides (ODNs) have shown potential anticancer potential in vitro and in vivo. It has been investigated as an antiviral and antibacterial agent. (5) 5'- of these analogs The 3' internucleotide bond is a nucleotide-phosphodiester bond. The conformation is very similar. The phosphonate backbone has one anion made neutral by methyl substitution of the phosphoryl oxygen; due to the charged phosphonate group Mirror opening and intrachain repulsion are reduced. (5) Analogs with MP backbone are viable cells mRNA in globin synthesis and vesicular stomatitis virus protein synthesis Pre-mRNA splicing in inhibiting A translation and inhibiting HSV replication. has been shown to inhibit The mechanisms of inhibition by nonionic analogs are complementary contains a stable complex conformation with target RNA and/or DNA.

A nonionic oligonucleoside alkyl compound complementary to a selected single-stranded foreign nucleic acid sequence. Kill- and aryl-phosphonate analogs are highly functional in other nucleic acids present in cells. of a nucleic acid by binding to or interfering with the specific nucleic acid without interfering with its ability or expression. Expression or function can be selectively inhibited (e.g., U.S. Pat. 4469863 and 4511713). harvested from mammalian cells Complementary Nuclease-Resistance, Nonionic Oligonucleoside Methylphosphonate for inhibiting the synthesis of certain viral proteins, including herpes simplex virus-1. Utilization is disclosed in U.S. Pat. No. 4,757,055.

Anti-sense oligonucleotide or oligonucleotide complementary to a portion of virus RNA Because of the interference with the transcription and translation of viral RNA into protein by sholothioate analogues. Proposals have already been made for the use of Antisense constructs are viruses ■ can bind to RNA and block the activity of cellular ribosomes against aRNA, thereby Termination of translation of RNA into protein, ``translation termination'' or ``ribosome hive'' This is thought to interfere with the process called "stopping of lid synthesis." (6)HTLV -Highly conserved regions of the HTLV-111 genome required for 11I replication and/or expression Infection of cells with HTLV-111 by administration of oligonucleotides complementary to the Inhibition is disclosed in US Pat. No. 4,806,463.

The oligonucleotides inhibit reverse transcriptase activity (replication) as well as viral proteins p15 and viral replication and p24 production (gene expression). /or found to affect gene expression.

Certain anti-sense oligonucleotides containing internucleoside methylphosphonate linkages The ability of oxynucleotides to inhibit HIV-induced syncytium formation and expression has been previously investigated. ing. (7) Psoralen-derivatized oligonucleoside methylphosphonates are encoded or Crosslinking of any non-coding heavy stranded DNA is possible; however, double stranded DNA is reported to be not cross-linked. (28) Summary of the invention The present invention provides a method for searching or recognizing a double-stranded nucleic acid or a specific section of a double-stranded nucleic acid sequence. , and a method for inhibiting the expression or function of a specific segment of a double-stranded nucleic acid having a given sequence. Regarding. This destroys the intact duplex structure or base pairing of double-stranded nucleic acids. This is done without interfering with the meeting. The present invention also provides for the production of selected double-stranded nucleic acid sequences. and/or functional inhibition, and may optionally contain nucleic acid modifying groups. Concerning good new modified oligomers. Furthermore, the present invention provides syn-containing compounds for purine bases. New products containing purine nucleoside analogues with chemical modifications that favor their conformation. Regarding oligomers. The present invention also provides specific sections of double-stranded nucleic acids and Because they are complementary, nucleic acid fragments and their base pairing (or hybridization) Concerning the formation of triple helical structures by readable oligomer interactions. this Their double-stranded nucleic acids include double-stranded DNA as well as DNA:RNA hybrids and double-stranded nucleic acids. There is heavy chain RNA.

Therefore, from one aspect, the present invention provides a method for preparing nucleic acid fragments such as the above-mentioned fragments of double-stranded nucleic acids or the like. contact with an oligomer of the present invention that is sufficiently complementary to the sequence of purine bases in a portion of hydrogen bond (or hybridize) with them, thereby forming a triple helix Concerning methods for searching and recognizing specific sections of double-stranded nucleic acids, including forming structures. do.

According to one preferred embodiment, the present invention provides a thin conformation, preferably at position 8. at least one nucleonucleotide containing a purine base that has been modified to favor the Contains double-stranded nucleic acid units and is sufficiently complementary to a specific section or part of a double-stranded nucleic acid. Method for searching or recognizing the specific section using an oligomer forming a triple helix a polypurine in which a portion of the double helix has at least about 7 purine bases; polypurine sequence, and the oligomer is parallel to the strand with the polypurine sequence. It is about the method. In this case, the guanine in the polypurine sequence is Soadenine and cytosine or cytosine analogs (either N −3 is protonated) and the adenine in the polypurine sequence is 8-oxoguanine, thymine and It is read by a base in the oligomer selected from uracil. particularly preferred According to embodiments, the polypurine sequence may include up to about 50% pyrimidine bases. Ru. In that case, the cytosine in the polypurine sequence is 8-fluoroguanine, 8-fluoroguanine, selected from fluoroadenine, 8-methoxyadenine and 8-azaadenine It is read by the base in the oligomer. The above-mentioned 8-modified forms of purine bases are conformation.

According to another aspect, the invention provides for the expression of a specific segment of a double-stranded nucleic acid having a given sequence. or a method for preventing or inhibiting the function of the nucleic acid fragment and the double-stranded nucleic acid. The section is brought into contact with an oligomer of the present invention that is sufficiently complementary to form a hydrogen bond with it. The present invention relates to a method for forming a triple helical structure by

According to a preferred embodiment of the above method, the present invention preferably provides a thin conformation at position 8. at least one nucleotide containing a purine base that has been modified to favor the Using oligomers containing osidic units, a portion of a double-stranded nucleic acid is at least about A specific section of a double-stranded nucleic acid containing a polypurine sequence containing 7 purine bases A method of preventing or inhibiting expression or function, wherein the oligomer is a polypropylene It relates to a method in which the strands are parallel to each other and have a sequence of strands. In this situation, the port Guanine in the liprin sequence is 8-oxoadenine and cytosine or cytosine. selected from N3 analogues (one of which is protonated at N3 at physiological pH). Adenine is read by the base in the oligomer, and adenine is 8-oxoguanine, It is read by a base in the oligomer selected from thymine and uracil.

According to particularly preferred embodiments, the polypurine sequences contain no more than about 50% pyrimidines. May contain bases. In such cases, the cytosine in the polypurine sequence is an ori selected from guanine, 8-methoxyguanine and 8-azaguanine; Read by the bases in the semen, thymine and uracil in the polypurine sequence are , 8-fluoroadenine, 8-methoxyadenine and 8-azaadenine. The base in the selected oligomer is read. The above 8-modified form of purine base is suitable for thin conformation.

In the present invention, the nucleic acid fragment contains genes in living cells, and the formation of a triple helix structure is permanent. The present invention relates to a method for inhibiting or inactivating the above gene.

In a preferred example, the oligomer hydrogen is selected by modifying the oligomer described above. After hydrogen bonding or hybridization with the identified nucleic acid sequence, a chemical reaction is performed with the nucleic acid. a nucleic acid modifying group that irreversibly modifies it. Such a modification is , the formation of a covalent bond, alkylation of a nucleic acid, cleavage of said nucleic acid at a specific position, or It may include cross-linking of oligomers and nucleic acids by degradation or destruction of the nucleic acids.

According to the present invention, purine salts with favorable modifications to the syn conformation Tertiary oligomers are provided that include at least one nucleoside unit having a group. It will be done. These oligomers are linked by any one of a variety of internucleoside linkages. Contains nucleoside units (or nucleoside monomers) that can be attached. these The internucleoside linkages include phosphorus-containing bonds, such as phosphodiester bonds, aryl and aryl-phosphonate linkages, phosphorothioate linkages, phosphoroaminoyl Dart bonds and neutral phosphate ester bonds, such as phosphotriester bonds, and Internucleoside bonds that do not contain phosphorus, such as morpholine bonds, formacecool bonds, sulfamate bonds, and carbamate bonds, but are not limited to It's not like that. Other internucleoside linkages known in the art may also be used in these oligomers. can be used in Also, according to a preferred embodiment, these oligomers moieties, deoxyribosyl moieties and modified ribosyl moieties, such as 2'-0-alcohol moieties. Kyribosyl (alkyl having 1 to 10 carbon atoms), 2°-O-aryl ribosyl and 2'-halogenribosyl (all optionally halogen, alkyl or and aryl), and especially 2°-0-methylribo Nucleoside units having modified sugar moieties including sil moieties may be incorporated. In particular, a set of nucleoside units having a decorative ribosyl, especially a 2'-0-methylribosyl moiety; Incorporation advantageously improves hybridization with double-stranded nucleic acid sequences and Resistance to solutions may also be improved.

In another aspect, the present invention provides a method that is sufficiently complementary to a purine sequence of a particular double-stranded nucleic acid piece. Therefore, new nonionic alkyl and and aryl-phosphonate oligomers. nonionic methyl Phosphonate oligomers are preferred.

The present invention also provides that a cytosine analog replaces cytosine, and that the cytosine analog It has hydrogen that can be used for hydrogen bonding at the ring position corresponding to N-3 of cytosine, and is neutral. A nucleozyl monomer containing a heterocycle that can form two hydrogen bonds with a guanine base at pH The present invention provides an oligomer having a position. The cytosine analogs include 5-methyl-cyto Syn, and the analogs shown in Table 5.

Furthermore, the present invention provides for triplet combinations with purine-pyrimidine or pyrimidine-purine base pairs. Concerning oligomers containing purine nucleoside analogs that can form heavy lines. Therefore, the oligomer has both purine and pyrimidine nucleosides. The sequence of one strand of the double helix can be read. In a preferred embodiment, the purine nuclei Oligomers containing only purine nucleosides, including ocide analogs, are provided.

In yet another preferred embodiment, combinations with these purine nucleoside analogs An oligomer containing a pyrimidine nucleoside is provided. these pudding Reoside analogues are preferably modified at the 8-position and therefore have a thin conformation. tion is advantageous. Therefore, the purine analog nucleosides include 8-o-7-iso A.

8-oxo-G, 8-fluoro-A, 8-fluoro-G, 8-methoxy-A, 8 -methokino-G, 8-aza-A and 8-aza-G.

(A) Definition When used herein, the following terms have the following meanings unless specifically stated to the contrary: do.

The term "purine" or "purine base" refers to the natural adenine and guanine bases , as well as modified forms of those bases, such as bases substituted at the 8-position.

The term "nucleoside" refers to a nucleoside unit, which are interchangeable and Also included are subunits of nucleic acids that include carbon sugars and nitrogenous bases. This term is units with A, G, C, T and U as bases, as well as analogs and Also included are modified forms of bases (eg, 8-substituted purines). In the case of RNA, the 5-carbon sugar is It's Bose. In the case of DNA, it is 2°-deoxyribose. this term also includes classes of said subunits containing modified sugars, such as 2°-0-alkyl ribose. Also includes analogues.

The term "phosphonate" refers to the group 0=P-R, where R is an alkyl or aryl group. ). Suitable alkyl or aryl groups are sterically linked to phosphonates. Contains groups that do not interfere with or interact with each other. The phosphonate group is rRJ or "S" configuration. The phosphonate group is It is used as an internucleoside phosphorus bond (or linkage) that connects osidic units. obtain.

The term "phosphodiester" refers to the group o=po, where the phosphodiester group is As an internucleoside phosphorus linkage (or linkage) to link cleoside units It can be used as The term "non-nucleoside monomer unit" refers to Refers to monomer units that do not significantly participate in oligomer hybridization. Before The monomer units listed above do not participate in any important hydrogen bonds with e.g. nucleosides. 5 nucleoside bases or one of its analogues as a constituent. eliminate nomer units.

“Nucleoside/non-nucleoside polymer” refers to nucleoside and non-nucleoside polymers. Includes polymers consisting of side monomer units.

The term "oligonucleoside" or "oligomer" generally refers to about 6 to about 100 nucleoside length, but can be greater than about 100 nucleoside lengths Refers to nucleoside chains connected by internucleoside bonds. They are It is commonly synthesized from nucleoside monomers, but can also be obtained by enzymatic means. vinegar Thus, the term "oligomer" refers to nucleoside monomers that link together nucleoside monomers. Oligonucleotide refers to an oligonucleoside chain with interside bonds. , nonionic oligonucleotide alkyl- and aryl-phosphonates. Analogs, alkyl- and aryl-phosphonothioates, oligonucleotides phosphoramidate analogues, neutral phosphate ester oligonucleotides analogs such as phosphotriesters and other oligonucleoside analogs and modified oligonucleosides, as well as nucleoside/non-nucleoside polymers. Including Rimmer. The term also refers to one or more links between monomer units. If the carbon group bond is a non-phosphorus bond, such as a morpholino bond, a formacecool bond, or a sulfur bond, Nucleosides/non-nucleosides replaced by famate or carbamate bonds The term "alkyl- or aryl-phosphonate oligomer" refers to Both have one alkyl or aryl phosphonate internucleoside bond Refers to oligomer.

The term "methylphosphonate oligomer" (or rMP-oligomer) Oligomers with at least one methylphosphonate internucleoside linkage means.

The term "neutral oligomer" refers to non-ionic nucleases between nucleoside monomers. Orients with interside bonds (i.e., bonds with no pure or negative ionic charge) Refers to polymers, e.g. internucleoside linkages, e.g. alkyl- or aryl groups. sulfonate linkage, alkyl- or aryl-phosphonothioate, neutral phosph E-1-ester bonds, such as phosphotriester bonds, especially neutral ethyl triester bonds. stell linkages, and non-phosphorus-containing nucleoside open linkages, such as sulfamates, Contains oligomers with morpholino, formacecool and carbamate linkages. be caught. Optionally, the neutral oligomer is an oligonucleoside or nucleoside. between the second molecule, including the de/non-nucleoside polymer and the conjugate partner. Conjugates may be included. The conjugate partner is an intercalating agent, an alkyl binding agents for cell surface receptors, lipophilic agents, photocrosslinking agents, e.g. This may include the following: The compound may further incorporate oligomers. modify the interaction between the oligomer and the target sequence, or The drug kinetic distribution of cids can be modified. The essential requirement is that the conjugate contains an oligonucleotide. The cleoside or nucleoside/non-nucleoside polymer must be neutral. Ru.

“The term neutral alkyl- or aryl-phosphonate oligomer2 Both contain one alkyl- or aryl-phosphonate bond. Includes neutral oligomers with interdomain bonds.

“Neutral methylphosphonate oligomer is a term that refers to neutral methylphosphonate oligomers containing at least one Includes neutral oligomers with internucleonid linkages that include phonate linkages.

The term [third-strand oligomerization] refers to reading a fragment of double-stranded nucleic acid and combining it with a triple helix. An oligomer that can form a structure.

The term "complementary" refers to the oligomeric tertiary chain corresponding to the double-stranded DNA ( Watson-Crick) base pairs with purine bases and hydrogen bonds (and base pairing or hybridization) and has a base sequence that forms a triple helix structure. cormorant.

In the various oligomer sequences listed here, e.g. represents a nucleoside group linked by a bond.

The term ``read'' refers to the ability of salts of other nucleic acids to interact through hydrogen bonding interactions. Refers to the ability of a nucleic acid residue to recognize a base sequence. That is, reading the double-stranded DNA sequence In this process, third-strand oligomers cut double-stranded DNA through hydrogen bonding interactions. It can recognize base pairs in the doublet of a piece, especially purine bases.

The term "triblet" refers to the bases in the third strand of double-stranded DNA. Hydrogen bonds (i.e., base pairs) with the (Watson-Crick) base pairs of the section Figure IA.

It refers to the conditions shown in IB and 2A to 2D and 7 to 10.

Brief description of the drawing Figures IA and ID show that the pyrimidine base in the third strand is double DNA (Watson-Climax). (hook) shows a triplet that forms a triplet with a base pair.

Figures 28 to 2D show DNA with double purine bases in the third strand (Watson-Crick). Represents a triplet that forms a triplet with a base pair.

Figures 3A to 3B show that the third chain "reads", i.e., on a single strand of double-stranded DNA. A salt with a typical polypurine sequence triple helix structure that is base-paired with the purine base of Represents the base array.

Figures 4A to 4E show that the third crosshair "reads", i.e., on the duplex of double-stranded DNA. A typical mixed triple helix base sequence that is base-paired with the purine base of represent.

Figure 5 shows the modification with alkyleneoxy bond for extension of internucleoside phosphorus bond. A nucleoside unit having a decorative sugar moiety and a method for synthesizing the same are shown.

Figure 6 shows modified sugar moiety with alkylene bond for extension of internucleoside phosphorus bond. Figure 2 shows a nucleoside unit having a nucleoside structure and a method for its synthesis.

Figure 7 shows that 8-oxo-A in the third strand is a G-C (Watson-Crick) base pair. shows the triple line forming the triple line.

Figure 8 shows that 8-oxo-G in the third strand is an A-T (Watson-Crick) base pair. shows the triple line forming the triple line.

Figure 9 shows that 8-fluoro-G in the third strand is a C-G (Watson-Crick) base pair. A triplet line forming a triplet line with C is shown.

Figure 10 shows that 8-fluoro-A in the third strand is a T-A (Watson-Crick) base pair. The triplet line forming a triplet line with the T of is shown.

FIG. 11 shows the CO spectrum of the triple helix structure. (222) is MP-oligo mer stannic, (222) indicates the oligonucleotide stannic.

Figures 12A and 12B show (A)-backbone using psoralen-derived MP-oligomers. Cross-linking of DNA and (B) double-stranded DNA is shown.

Figure 13A shows 0.5 micromolar I (OA) and 0.5 micromolar II.I Figure 3 shows the absorbance vs. temperature profile of a solution of II (G.C).

Figure 13AA shows I(OA) and II.In at 1.5 micromolar total chain at 7°C. Continuously variable titration of (G.C) is shown.

Figure 13B shows 1,6 micromol I at 10°C (□) and 35°C (...) Observation of solutions containing (OA) and 1.6 micromol II-III (G-C) circular dichroism spectrum and 1.6 micromol I ( OA) and 1°6 micromol II-III (G-C). Showing the spectrum. All were done in standard buffer.

Detailed description of the invention The present invention provides a method for preparing a selected double-stranded DNA sequence that is sufficiently complementary to a purine sequence of double-stranded DNA. oligomers that can form hydrogen bonds (or hybridize) with Regarding the formation of a triple helix structure with the double-stranded DNA sequence by contacting with do.

(B) General overview In addition to the other aspects described herein, the invention includes the following aspects.

(1) The first aspect is that adenine and guanine, such as adenine and guanine, are The hydrogen bond between the remaining hydrogen bonding site of purine in double-stranded DNA and the base in the third strand Concerning the reading (or recognition) of base pairs in a double-stranded DNA section by bond formation Ru.

In other words, reading the base pair sequence in a DNA duplex always Hydrogen bond formation between the remaining available hydrogen bonding sites on phosphorus and the base in the third strand This is done by reading the purines in base pairs. Purines in the third strand (e.g. For example, adenine (A) or guanine (G)) or pyrimidine (thymine (T) or cytosine (C)) can form hydrogen bonds with purines in DNA. . That is, 1) Adenine (A) in a base pair of DNA is read, i.e. i.e., can hydrogen bond with A or T in the third strand, or in a preferred embodiment, A can Read by 8-oxo-G.

1i) Guanine (G) in a base pair of DNA is read, i.e. in the third strand or in a preferred embodiment, G is 8-oxo- Read by A.

In a preferred embodiment of the invention, the phosphorus-containing backbone of the stannic group is a methylphosphonate group or and naturally occurring phosphodiester groups.

(If) The second embodiment of this invention uses purine bases from stannic to form double strands. It is to read all the purines (A or G) in DNA. The polarizability of Kabura is , the antiparallel of stannic against the tin containing the purine to be read in the duplex. either anti-parallel or parallel is also important.

The base planes of purines and pyrimidines are fixed, and only the furanose ring is slightly It is wavy (0.5 angstroms above and below the plane). That is, nucleosy The steric structural state of Co-1 to N-9 or N-1 bond axis is as follows: rotation of more or less these two fixed planes, i.e. the base and the pentose In principle, it is determined by The sugar-base torsion angle, φCN, is the binding angle for Co-1. When looking at one of the combinations, the rise of the C-1° to O-1° bond of the furanose ring and the salt It is defined as the angle formed by a line in the ground plane. This angle is - ring oxygen is not planar to C-2 of the pyrimidine or purine ring (ant iplaner) time is 0, and measured clockwise when looking at N from C-1° defined as the positive angle when This angle is also called the glycosyl torsion angle. Ru. Using the above definition, for nucleosides, anti-conformational There are two types of ranges: about -30 @ for N, and about +1501 for N - Conhome Soyon. It was found that there is a φCN of The range of each conformation is approximately ±45 °. (22°22a) Other researchers have used a slightly different definition of this angle. I'm proposing it again.

(23, 24, 25, 26, 27) Information regarding φCN can be obtained from X-ray diffraction, proton methods such as magnetic resonance (PMR) and optical rotational dispersion-circular dichroism (ORO-CD). It has been obtained more. (22a) From one 1ll (referred to as the r-Watson strand) to the opposite strand (referred to as the ``Crick strand'') In order to accommodate changes in the position of the purine bases read up to purine nucleoside in the third strand, defined as the torsional angle of the lycodyl bond A special conformation of the unit is required, whereby the purine in the third strand It turns out that nucleosides can be used to read purines in duplexes . In other words, in order to read purine bases in DNA, purine bases in the third strand are The conformation of the creoside unit is read in DNA with respect to stannic The polarity of the strand containing the purine base (parallel (5° to 3°) or anti- Parallel (3' to 5') direction). purinuk in the third strand For the leoside unit, when reading the purine in the parallel strand in the duplex, The conformation of the purine nucleoside unit in the third strand is a thin conformation. should be a tion. On the other hand, the corresponding protein in the anti-parallel strand in the duplex The conformation of the purine nucleoside unit in the third strand when reading phosphorus is It must be anti-conformist. In other words, it is distributed to both Kabura. When reading purine bases in double strands, either one strand or the opposite strand The choice is to use stannic with the same (i.e. parallel) polarity as the do.

(Watson chain) (Crick chain) The third of the triplet with a DNA duplex with the same polarity as the Watson strand 5° to 3° The ditin array looks like this: The arrangement of stannic rings parallel to the click rings is as follows; parallel stannic) According to one aspect of the invention, the first step for reading purines in double-stranded base pairs is The following guidelines apply when constructing Nitin:

(i) When starting from the 5° end and going towards the 3' end, the stannic purine nucleoside The unit (A or G) is the base pair (A or G) In reading the purine in There is a point.

In reading the second purine of the second base pair, the purine is the same as the first purine. If located in the same strand, the same conditions apply. However, the second pudding is reversed. anti-parallel (click) strand of (where the opposite strand is anti-parallel to stannic) - parallel), the purine nucleoside unit is anti-conformant It must be a message. In all cases, the adenine in the third strand is The guanine in the third strand is used to read the adenine in the second strand. Used for reading.

(ii) "reading" at the purine bases (or salts) on either mirror of the double-stranded nucleic acid; (group pairing) between nucleoside units in the direction of the stannic phosphorus backbone. distance must be increased. We present two examples of types of bond format extensions for the phosphorus backbone. come up with a plan One type of linkage in the phosphorus backbone is the overall linkage on individual nucleoside units. Extensions, ie, all the extensions of stannic are the same. this Such an overall linkage format is particularly suitable for stannic compounds containing only purine bases. Therefore , from the 5° carbon of “nucleoside unit 1” to the 3° acid of the next “nucleoside unit 2” The elementary bond length can be either by two atoms (e.g. -CH2CH2-) or by three atoms. atoms (for example -〇　-CH!　CH2), and its In this case, the bonds between individual nucleoside units can be as long as 2 to 6 angstroms. Ru. In order to maintain an appropriate distance between nucleoside units, the distance between units is set from 1 to 6. It is recommended to increase the number of atoms in the range of . Figure 6 shows this connection extension form. An example of a nucleoside unit included and a proposed synthetic route are shown.

The second type of phosphorus backbone chain extension involves heterogeneous bond extension. stannic The bonds with similar nucleoside unit spacing in the standard phosphodiester backbone are , is adopted when the purines to be read are on the same mirror, while one nucleo The extension on the 3'-carbon of the side unit and on the 5'-carbon of the adjacent nucleoside unit. Extended bonds (15-17 angstroms in length), which may include long bonds, are used to read purine bases located on the mirror of This kind of non-uniform Extended forms of synthesis are particularly useful for pyrimidines (or pyrimidine analogs) and purine bases. Suitable for use with stannic compounds including both.

(III) Reading the base pair sequence in a nucleic acid duplex always salt through hydrogen bond formation with the remaining available hydrogen bonding sites and the stannic base. It has been thought that this is done by reading the purine of the base pair. That is, stannic In order for the oligomer to form a triplet without the need for strand switching, the duplex A homopurine tract in one strand was required. Contains purine/pyrimidine mixtures (double-stranded) sequence reading requires the use of extension linkages in the stannic oligomer. The oligomer can read the purine bases on both mirrors.

However, according to the present invention, both purine and pyrimidine bases are present in double-stranded nucleic acids. Hydrogen bond formation between the available hydrogen bonding sites in the molecule and the base in the third strand and pyrimidine bases are provided. this Depending on the oligomer, e.g. the homopurine tract has one or more pyrimidi Sequences interrupted by tin bases can also be read, and can be read on stannic oligomers. It is also necessary to switch the strand read at the pyrimidine interruption site using an extended linkage. The purines on the other mirror can be read to form a duplex. this The use of their oligomers is advantageous in that only one strand needs to be read. . Also, since only one strand is read, the synthesis of stannic oligomers is simplified. .

These oligomers are parallel to the strands of these double-stranded nucleic acids being read. that is, it has the same polarity as that. These stannic oligomers are used Purine analogues have a syn configuration.

That is, according to the present invention, 8-oxo-adenine forms a complex with G (or 8-oxo-guanosine is used for complex formation with A (or for reading). 8-Fluoro G is used to read C and 8-Fluoro G is used to read C. Oligomers are provided that are used to read A as T (or U?). Sunawa All four bases in the DNA on the same mirror of the double-stranded nucleic acid DNA are these oligomers. can be read using

(B) Formation of triple helix structure (T) Triple (or triple-base pairing) (a) Pyrimidine in the third strand of duplex formation As one aspect of the present invention, the three lines indicate that the pyrimidine base in the third strand forms a hydrogen bond. formation, i.e., when the base pairs with the purine base of a double-stranded DNA sequence It is formed. Such a three-dimensional structure with a pyrimidine base as the base in the third strand Examples of main lines are shown in Figures IA and 1B.

Figure IA shows a second The duplex is shown with T as the tin base. Under these circumstances, the T of stannic Parallel to the A-containing strand of the heavy-strand DNA, the T-containing strand of the double-stranded DNA (also known as the A-containing strand) (anti-parallel) to the containing strands. Therefore, This triple line is expressed as follows.

Figure IB shows the formation of hydrogen bonds, i.e., the G and salt of G-C base pairs in double-stranded DNA. Indicates a duplex with a protonated toson (C+) as the group-pairing stannic base. vinegar. Under these circumstances, the tonton base in the third strand absorbs the hydrogen necessary for a stable duplex. It must be protonated at N3 to form the bond. as desired , notonone is expressed by a tonone analogue with a quaternary nitrogen in the same position as N3. May be replaced. In the triple line shown, C+ of stannic (or its analogue) ) in parallel to the G-containing strand of duplex DNA and in parallel to the C-containing strand of duplex DNA. arranged anti-parallel to the chain (which is also anti-parallel to the G-containing chain) lined up. This duplex sequence is therefore written as follows.

(b) an adenine or guanine in the third strand forming a triplet and other aspects of the invention; Therefore, the three lines indicate that the purine base in the third strand is the same purine base in one strand of double-stranded DNA. Formed when a base forms a hydrogen bond, that is, base pairs with it. Therefore , A pairs with A, and G pairs with G.

In contrast to the pyrimidine third strand triplet, the purine bases in the third strand are Can base pair with purine bases and therefore the chain polarity of purine base containing stannic is parallel to the polarity of the kabura containing purines to be read in the double tJI DNA. or anti-parallel.

For example, Figure 2A shows that the A of stannic is arranged parallel to the A of the double-stranded DNA. , shows triplet lines arranged anti-parallel to the T of double-stranded DNA.

Under these circumstances, the glyconol (C-N) torsion angle of the third chain A becomes conformal. tion, and the glycosyl torsion angles of A and T of double MDNA are both a This conformation is in a straight conformation. This three-line array is expressed as follows.

On the other hand, FIG. 2B shows that A of stannic is anti-parallel to A of double-stranded DNA, Triplet lines arranged parallel to the T of double-stranded DNA are shown. like this Under the circumstances, the glycosyl torsion angles of all three bases are anti-conformal. tion. This triple line arrangement is expressed as follows. G is parallel to G of double-stranded DNA and anti-to C of double-stranded DNA. Shows double strands arranged in parallel. Under these circumstances, the glycosylation of the third chain G The gill torsion angle assumes a thin conformation, and the G-C base group of double-stranded DNA Both lycodyl torsion angles are anti-conformational. This double chain The columns are shown below.

Figure 2D shows that the G of stannic is anti-parallel to the G of the duplex DNA. It shows a double strand arranged in parallel to C of DNA. Under such circumstances , the glycosyl torsion angles of all three bases are anti-conformations . This duplex sequence is represented as follows; (C) the stannic compound forming the duplex. According to a preferred embodiment of the invention, the duplex comprises a thin conformo analogue. Purine analogues with favorable chemical modifications added to the base in which they form (analogs) are formed using stannic oligomers containing nucleosides.

According to this embodiment, the stannic oligomer is attached to the strand of the double-stranded nucleic acid being read. It is parallel.

Some of these purine analogs are capable of hydrogen bonding with purine bases of double-stranded nucleic acids; In other words, the polarity of stannic binds both purine-containing strands to be read in double-stranded nucleic acids. Some can form double strands that are arranged in parallel to polarity. moreover , among others of these purine analogs are the pyrimidine bases and pyrimidine bases of double-stranded nucleic acids. The hydrogen bond, i.e. the pyrimidium, is to be read out in the double-stranded nucleic acid. that can form a double strand that is arranged in parallel to the polarity of the chain containing the There is also. Purine bases to be read are more likely to be hydrogen bonded than pyrimidine bases. Because there are many available sites, the sequence to be read on one value of double-stranded DNA is columns are often purine bases, at least 50% or more Contains bases.

Figure 7 shows that the 8-oxo-A of stannic is parallel to the G of the double-stranded nucleic acid. A duplex is shown that is arranged antiparallel to C of the nucleic acid. This kind of situation Under the circumstances, the glycodyl (C-N) torsion angle of stannic 8-oxo A is syn-conformal. tion, and the glycosyl torsion angles of the G and C bases of the double-stranded nucleic acid are both It is anti-conformation.

Figure 8 shows that 8-oxo-G of stannic is parallel to A of the double-stranded nucleic acid. Shows a duplex that is arranged antiparallel to the T or U of the nucleic acid. this Under these circumstances, the glycodyl (C-N) torsion angle of stannic 8-oxo G is syn- conformation, which is the glycosyl conformation of the A and T or C bases of a double-stranded nucleic acid. Both helix angles are anti-conformational.

Figure 9 shows that the 8-fluoro G of stannic is arranged in parallel to the C of the double-stranded nucleic acid. The double strands are shown. Under these circumstances, glycosyl of stannic 8-fluoro G ( C-N) torsion angle is thin-conformation, C and C salts of double-stranded nucleic acids The glycosyl torsion angles of both groups are anti-conformity.

Figure 10 shows that 8-fluoro-A of stannic has a large effect on T (or U) of double-stranded nucleic acid. Shows double strands arranged in parallel. Under these circumstances, stannic 8-fluoro -The glycodyl (C-N) torsion angle of A is a thin conformation, and the duplex Both the glycosyl torsion angles of the A and T (or U) bases of a nucleic acid are It is a homeation.

(1) Polypurine sequence One strand of the selected double-stranded nucleic acid sequence contains a polypurine sequence containing all purine bases. If the polypurine sequence is a complementary oligomer, it is or purines, or mixtures thereof. preferred o Ligomers are nuclease-resistant and contain double-stranded nucleic acids and the triblets described above. Contains nonionic NP-oligomers that are capable of producing lattice structures.

A double-stranded nucleic acid sequence is formed by the binding of tin to the polypurine sequence within one strand of the double-stranded nucleic acid sequence. Examples of spiral helical arrangements are shown schematically in Figures 3A-3C. In Figures 3A to 3C, C0 is professional. Represents tonized C.

(2) Mixed array A mixed sequence is one in which the purine bases of the selected nucleic acid sequence are located on both strands of a double-stranded nucleic acid. This is an array that is In one type of this case, on one strand of a double-stranded nucleic acid sequence The polypurine sequence may contain up to about 50% pyrimidine bases.

If only purine bases are read, the stannic oligomer is of each base pair in a double-stranded nucleic acid sequence, regardless of whether one purine base is present in the strand. It must be able to hydrogen bond, or base pair, with the purine base. therefore , the stannic oligomer must be able to “read” across double-stranded nucleic acids. No. Examples of mixed sequences containing complementary stannic compounds are shown in FIGS. 4A-4E.

Figure 4A shows a mixed sequence with pyrimidine-rich stannic.

Figure 4B shows a mixed array with purine-rich stannic.

Figures 4C14D and 4E show a mixed sequence with stannic containing only purine bases. . In Figures 4C to 4E, rSJ represents syn and raJ represents anti. Momo without a superscript represents anti.

The stannic oligomer crosses the duplex from one strand to the reflector and forms a salt with the purine base. If the sugar moieties of the nucleoside units must be “read” in base pair order, By inserting the aforementioned backbone chain format into the connecting phosphorus backbone, elongation can be achieved. It may be advantageous to provide an internucleonid phosphorus linkage.

For example, the internucleoside linkage is the 5°-carbon of the sugar moiety of the nucleoside unit and the 5°-carbon of the sugar moiety of the nucleoside unit. A suitable alkylene (-(CHt)n) or alkylene oxide between the hydroxyl xy(-(CH1)no) extended chain, or 3°-carbon and 3′- can be extended by intervening similar chains between the hydroxyls (Fig. 5 and and Figure 6).

In preferred cases, such extended linkages are present at both the 3'- and 5'-carbons of the sugar moiety. can be intervened.

If consecutive purines of a chosen double-stranded DNA sequence are on the same mirror, such There is no need to employ elongated chains. Nucleosides such as methylphosphonate linkages A phosphorus linkage between consecutive purine salts on one strand of a chosen double-stranded DNA sequence The groups can be read with a suitable base on the third mirror.

Alternatively, the mixed sequence may be present on one strand of a double-stranded nucleic acid sequence at a rate of about 50% or less. If it contains one or more polypurine sequences containing midine bases, Both phosphorus and pyrimidine can be read. Pyrimidine bases are modified by: Read using purine bases. The cytosine in the polypurine sequence is It is read by roG, 8-methquine G, 8-azaG, and thymine or uracil is , 8-fluoroA, 8-methoxyA or 8-azaA. sand That is, the oligomer reads the pyrimidine bases that interrupt the polypurine sequence. Because tin oligomers can read only one strand of a double-stranded nucleic acid sequence, That's fine. Furthermore, in order to make the stannic oligomer read both positions of the two kaburas. No additional modifications, such as extended chains, are required.

(C) Stannic oligomer Preferred oligomers have at least about 7 nucleosides; The length is usually such that the segment of double-stranded nucleic acid can specifically bind to the desired purine sequence. There are enough numbers to More preferably, it has about 8 to about 40 nucleotides. Oligomers, particularly preferred are oligomers having from about 10 to about 25 nucleosides. It's Gomer. Combines synthetic ease with specificity for selected sequences internucleoside interactions such as folding and coiling within the oligomer. Considering the minimization of It is believed that it contains a preferable group.

(1) Preferred oligomers These oligomers have either a ribbonyl moiety or a deoxyribosyl moiety, or modifications thereof, such as a 2'-0-alkylribosyl moiety.

Nucleotide oligomers (i.e. phosphorus present in natural nucleotide oligomers) Phodiester internucleoside linkages and other oligonucleotides and analogs oligos with other internucleoside linkages may be used in this invention. mer, such as oligonucleoside alkyl- and arylphosphonate analogs. phosphorothioate oligonucleoside analogs, phosphoroamida-1 analogues and neutral phosphate ester oligonucleotide analogs may also be used. but However, particularly preferred is the one or more nucleoside units connecting the two nucleoside units. Oligonucleotides in which more than one phosphodiester chain is replaced with a phosphonate chain sid/dr luxy and aryl phosphonate analogs, as well as non-phosphorus linkages. It is an oligomer with Such oligonucleoside alkyl- and alkyl- Preparation of some lylphosphonate analogs and preselected single-stranded nucleic acid sequences Their use in suppressing the expression of No. 4511713, No. 4757055, No. 4507433, and No. 4507433, No. 4591614, and those disclosures are cited as part of the description. Ru.

A particularly preferred group of these phosphonate analogs are methylphosphonate oligonucleotides. It's Gomer.

The method for synthesizing methylphosphonate oligomers (fMP-oligomers) is as follows: B .

L6 Lee et al. [Biochemistry, Vol. 27, pp. 3197-3203 (1988) )], and P, S Miller et al. [Biochemistry, vol. 25, 5092-50 p. 97 (1986), and some of the explanations are based on those disclosures. shall be.

In some cases, at least one phosphodiester internucleoside linkage is An internucleoside methylphosphonyl (MP) group (or oligonucleoside alkyl substituted by and arylphosphonate analogs may be preferred. Methylphosphona The ate linkage is isosteric to the phosphate group of the oligonucleotide. That is, , these methylphosphonate oligomers ("MP-oligomers") were selected It should provide minimal steric constraints on interaction with the DNA sequence. Also like that The alkyl or aryl group does not sterically hinder the phosphonate chain or Other alkyl- or arylphosphonate chains that do not interact significantly are also suitable. Ru. Since methylphosphonyl groups are not found in natural nucleic acid molecules, these M P-oligomers are subject to modification by various nuclease and esterase activities. It should be more resistant to water splitting. Non-ionic methylphosphonate chain Due to their molecular properties, these MP-oligomers are better able to cross cell membranes. and therefore should be better taken up by cells. Certain MP-ori The sesame is more resistant to nuclease hydrolysis and can be used in mammals in culture. It is taken up by cells in intact form and has specific properties for cellular DNA and protein synthesis. It has been found that a different inhibitory effect can be exerted (for example, U.S. Pat. No. 44 69863).

at least about 7 nucleoside units, more preferably at least about 8 nucleoside units Preference is given to MP-oligomers with units whose length is typically that of double-stranded DNA. This is sufficient to specifically recognize the desired segment. Approximately 8 to 40 nucleos More preferred are MP-ligomers having sid, particularly preferred about 10 to 25 It has nuquosides. Ease of manufacture and specificity for selected sequences , and internucleoside interactions such as folding and coiling within oligomers. MP-oligomers of about 14-18 nucleosides to combine minimization of Particularly preferred are the following.

Particularly preferred is a 5′-internucleoside linkage that is a phosphodiester linkage; MP-oligomers in which the remaining internucleoside linkage is a methylphosphonyl linkage be. Having a phosphodiester chain at the 5″-end of the MP-oligomer means that Enables kinase labeling and electrophoresis of oligomers while improving their solubility do.

The selected double-stranded nucleic acid sequence was sequenced and MP-oligomers complementary to the purine sequence were determined. is made by the method reported in the above-identified patent and herein.

From the above, preferred oligomers include those that conform to the thin conformation. Some contain nucleoside units, preferably with modified purine bases.

These bases include those modified at the 8-position, such as 8-oxo-A, 8-oxo -G18-fluoro-A18-fluoro-G, 8-methoxy-A, 8-methoxy -G, 8-Aza-A, 8-Aza-G and other similarly modified There's pudding.

These oligomers can be used in nucleic acid mixtures, or in isolated cells, tissue samples or bodies. It is useful for determining the presence or absence of selected double-stranded nucleic acid sequences in samples containing liquids, etc. be.

These oligomers are useful as hybridization assay probes, Can be used for detection assays. When used as probes, these oligomers It can also be used in diagnostic kits.

If desired, labeling groups such as psoralen, chemiluminescent groups, crosslinking agents, and acridine may be added. .

intercalating agents, or 0-phena:molecules such as notroline copper or EDTA-iron. Inserting a group into the MP-oligomer that can cleave a targeted part of the viral nucleic acid like a pair of scissors. can be done.

These oligomers can be prepared by any of several known methods: 1. can be labeled. helpful Labels include radioisotopes and non-radioactive reporter groups. Isotho The loop labels include 3N], 35S, 32p, +251. Cobalt and 14 There is 0. Many of the methods for isotope labeling involve the use of enzymes, nick transcripts, etc. These include known methods such as thrane, end-labeling, secondary labeling, and reverse transcription. radiation When using functionally labeled probes, hybridization is performed by autoradiography. It can be detected by the Fee method, scintillation counting method, or gamma counting method.

The detection method chosen will depend on the hybridization conditions and the specific labeling used. Varies depending on radioisotope.

Non-radioactive substances can also be used for labeling, and modified nucleosides or Enzymatic insertion of creoside analogues or chemical modification of oligomers, e.g. For example, it can be introduced by the use of non-nucleotide linker groups. As a non-radioactive label may include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, habutens or other Examples include ligands and the like. A preferred method of labeling is the insertion of an acridinium ester. It is in.

Such labeled oligomers can be used as hybridization assay probes. As such, it is particularly suitable for use in hybridization assays.

When used to prevent the function or expression of double-stranded nucleic acid sequences, these By conveniently derivatizing or modifying the oligomer to insert nucleic acid modifying groups, induces a reaction with the nucleic acid, irreversibly modifying its structure and rendering it non-functional. can be made to Our co-pending U.S. Patent Application No. 924,234 (filed October 28, 1986) discloses oligonucleoside alkyl and Derivatization of oligomers containing arylphosphonates and target single-stranded nucleic acid sequences. Oligonucleoside alkyl- and aryl- and oligonucleoside derivatives that are non-functional This disclosure is cited as part of the explanation. do.

A wide variety of nucleic acid modifying groups can be used to derivatize these oligomers.

Nucleic acid modification groups include derivatized oligomers that combine double-stranded nucleic acid segments with triple helices. After generating the structure, covalent bond formation, crosslinking, alkylation, cleavage, and decomposition occur, otherwise result in inactivation or destruction of the nucleic acid segment, or portion of its target sequence. strain, thereby irreversibly suppressing the function and/or expression of the nucleic acid segment. Includes groups that can.

The position of the nucleic acid modifying group in the oligomer can vary and depends on the particular nucleus employed. This may vary depending on the acid modifying group and the double-stranded nucleic acid segment targeted. However, Thus, the nucleic acid modifying group may be located at the end of the oligomer or may mediate the end. plural Nucleic acid modifying groups may be included.

In a preferred embodiment, a reaction is induced, i.e. a bridge between the oligomer and the double-stranded nucleic acid. For bridging purposes, nucleic acid modifying groups are photoreactive (e.g., specific wavelengths of light, or wavelength ranges). ).

A representative example of a nucleic acid modifying group that can induce crosslinking is the aminomethyltrimethylpsoralen group ( psoralen such as AMT). AMT is conveniently photoreactive; That is, before crosslinking can occur, it must be activated by exposure to light of a specific wavelength. Must be. Other bridging groups, which may or may not be photoreactive, are also included in these can be used for the derivatization of oligomers.

Alternatively, the DNA modifying group can be detached from the oligomer by reaction and covalently may contain an alkylating agent group that binds to and inactivates the DNA segment.

Suitable alkylating agent groups are those known in the chemical art and include alkyl halides, halide Includes groups derived from rogenated acetamides, phosphotriesters, etc.

DNA modifying groups that can induce cleavage of DNA segments include ethylenediamine Transition metal chelate complexes such as tetraacetic acid (EDTA) or its derivatives may be mentioned. Ru. Other groups that can be used to cleave are phenanthroline, porphyrin or pleomycin etc. When using EDTA, iron is conveniently mer and assist in the generation of the cleavage radical. EDTA is the preferred DNA opener. azo compounds, or other nitrogen-containing substances such as nitrenes, or or other transition metal chelate complexes may also be used.

Third-strand oligomer nuclei that read purine bases on both strands of a double-stranded DNA sequence Rheoside units can be a mixture of purine and pyrimidine bases, or just purine bases. may include.

When reading purine bases on both mirrors of a double-stranded DNA sequence, only the purine bases are read. Preference is given to using oligomers having . Such pudding-only oligomers (a) Purines have higher stacking properties than pyrimidines, thereby (b) The use of purines alone tends to increase the stability of the triple helical structure; It eliminates the need to protonate cytosine (therefore, water is added to the N-3 position at neutral pH). (with hydrogen available for elementary bond) or at the position corresponding to N3 on the pyrimidine ring , eliminating the need to use cytosine analogs with such available hydrogen. , which is convenient for several reasons, such as allowing the use of universal elongated chains. I am convinced that

Purine bases (as well as pyrimidine bases) are usually anti-conformational. It is. However, the barrier to rotation of the base to the thin conformation is low. stomach. In the formation of the third triple helix, the purines on the third strand are It can be estimated that it adopts a thin conformation. If desired, normal or thin Purines can be modified to be conformational. For example, methyl , bromo, isopropyl, or other bulky groups. It can be assumed that by modifying the 8th position of , a thin power distribution is obtained under normal conditions. That is, that Nucleoside units containing purines substituted in can take a lot of action. Therefore, the purine base in the thin conformation is shown. If so, this invention optionally substitutes such a substitute for unsubstituted A or G. Consider the insertion of a purine substituted at position 8. Inventor's (Kendrew) model From a study by It turned out that it was not supposed to be.

For the use of purine nucleoside units in anti and syn conformations More preferably (according to the rules for reading double-stranded DNA described herein) two The purine on both mirrors of the double strand is read by the third strand of stranded DNA and purine only. can generate a triple helical structure.

Double-stranded DNA is a third-strand oligomer containing both pyrimidines and purines. When used to read purines on both strands of Use the mat as described herein to cross from one strand of a duplex to the other. It can be cut and read by the second strand.

(2) Oligomers containing cytosine analogs As another aspect of the present invention, A cytosine containing a heterocycle having an available hydrogen at a ring position similar to the 3-N of the cytosine ring. Syn analogues replace cytosine with guanine base and two waters at neutral pH. can form elementary bonds, i.e., Hoogsteen-type base pairing, or Cytosine does not require protonation for riblet formation. New oligomers containing rheoside units are provided.

A preferred nucleoside unit has hydrogen available for hydrogen bonding to N-3 of cytosine. a 6-membered compound that can form two hydrogen bonds with a guanine base at neutral pH. including analogs with a heterocycle of 2'-deoxy-5,6-hydro 5-azadeoxycytidine (1), pseudoisocytidine (II), 6-amino -3-(β-D-ribofuranosyl)pyrimidine-2,4-dione (III), and 1-amino-1,2,4-(β-D-deoxyribofuranosyl)triazine -3-[4H]-one (IV) and the like. Their structures are shown in Table 5. Also 5 -Methylcytosine is also included.

(3) Oligomers containing purine analogs According to yet another aspect of the invention, the 8 Novel oligonucleotides containing nucleoside units containing modified purine bases or analogs Sesame is provided.

In particular, these purine analogs have the ability to form triplets with G-C base pairs. There is 8-oxo-A, which has been demonstrated (see Example 4). Purine analogue 8-oxo -G can be used to read the A of the AT base pair.

(Watson-Crick) salts by using certain 8-modified purine analogs. The base pair pyrimidine base can be read. In particular, 8-fluoro-A has a reading of T 8-Fluoro G can be used to read C. A and G 8-methoxy and 8-aza analogs of were used to read T and C, respectively. obtain.

(D) Production of MP-oligomers (1) General discussion As mentioned earlier, methylphosphonate oligomers are manufactured in the United States. Patent No. 4469863, Patent No. 4507433, Patent No. 4511713, Patent No. No. 4591614 and No. 4757055.

A preferred method of synthesizing methylphosphonate oligomers is described by S., Lin et al. Mistry, Vol. 28, pp. 1054-1061 (1989)], B. L. Lee et al. [Biochemistry, Vol. 27, pp. 3197-3203 (1988)], and P, S Miller et al., vol. 25, pp. 5092-5097 (1986). and their disclosures are cited and incorporated into this description. Modified with extended chain Oligomers containing nucleoside units containing sugar moieties (see Figures 5 and 6) - can be conveniently produced by these methods.

Oligomers containing phosphodiester internucleoside phosphorus linkages are including automated solid-state chemical synthesis using ethyl phosphoramidite precursors (29). can be synthesized using any of several conventional methods.

Nucleoside units optionally containing the above-mentioned cytosine analogs (Table 5) are suitable By substituting a cytidine analogue (see Table 5) in the reaction mixture, MP-O Can be inserted into oligomers.

(2) Production of MP-oligomers having extended chains in the phosphorus main chain (a) 5°-(E) (tyreneoxy) substituted sugar intermediate MP-oligomer is a 5'-carbon and 5 Either between '-hydroxyl or between 3'-carbon and 3'-hydroxyl Modified nuclei in which the bond is replaced with an alkyleneoxy group such as an ethyleneoxy group It can be produced using osside.

Figure 5 shows either a 3゛-(ethyleneoxy) or a 5゛-(ethyleneoxy) chain. The reaction scheme presented for the production of modified nucleoside intermediates having . In Figure 5 , B represents a base, Tr and R represent a protecting group, and Tr represents a base such as dimethoxytrityl. protecting group, R is t-butyldimethylsilyl or tetrahydropyranyl Indicates a protecting group.

optionally with such extensions at both the 3′- and 5′-positions of the sugar moiety. Nucleoside units can be produced.

(b) 5°-β-hydroxyethyl substituted sugar intermediate with purine bases on both chains When reading double-stranded DNA sequences, slightly elongated internucleoside chains are It may also be preferable to use MP-oligomers having on the main chain.

Such MP-oligomers contain sugar (deoxyribosyl or ribosyl) moieties. modified to produce 5'-β-hydroxyethyl substituted nucleosides as shown in Figure 6. In the synthesis reaction formula for the production of 5-hydroxy, β-hydroxyethyl (OH-C H! can be prepared using nucleosides replaced by CH2) groups.

Figure 6 shows the reaction equation proposed for the 5°-β-hydroxyethyl substituted saccharide. . In Figure 6, DCC is dicyclohexylcarbodiimide and DMSO is dimethylsulfate. sulfoxide, B represents a base. A suitable protecting group R is t-butyldimethylsilyl, or and tetrahydropyranyl.

(c) Production of MP-oligomers having extended internucleoside chains in the phosphorus main chain The MP-oligomer incorporating the modified nucleoside unit described above was prepared according to the above procedure. Produced by substituting modified nucleoside units.

In the production of oligomers containing only purine bases, oligomers with the same extended chain are used. Utilization of creoside units may be employed. However, pyrimidine (or pyrimidine-like In the production of oligomers containing both bases and purine bases, A mixture of non-containing nucleoside units and extended chains is used. 3゜- of the sugar part Nucleoside units with extensions on both the carbon and the 5′-carbon are advantageous. obtain.

(3) Production of derivatized MP-O oligomers The derivatized oligomers have the desired D It can be easily produced by adding NA modification groups to oligomers. mentioned earlier As described above, the number of nucleoside units within the oligomer and the DNA modifications within the oligomer The position of the decorative group(s) may vary.

The DNA modifying group(s) is an origin that can most effectively modify the target sequence of DNA. It can be placed in a gomer. Therefore, such optimal location is known in the art. Although readily determined by conventional techniques, the location of DNA modifying groups is often altered by the DNA segment being expressed and its primary target site(s). It can be changed.

(a) Preparation of psoralen-derivatized MP-oligomers 8-Methquin psoralen and M with psoralen such as 4″-aminomethyltrimethylpsoralen (AMT) Derivatization of P-oligomers is described by J. M. Keene et al. [Biochemistry, 27 Vol. 9113-9121 (1988)] and B. L. Lee et al. Tory, Vol. 27, pp. 3197-3203 (1988). This disclosure is incorporated herein by reference.

(b) Production of EDTA-derivatized MP-oligomers MP-oligos with EDTA The derivatization of mers is described by S. B. Lin et al. [Biochemistry, Vol. 28, 105] 4-1061 (1989)], and the disclosure is cited in the explanation. Part of it.

(E) Usefulness According to this invention, the specific segments of double-stranded DNA reported herein are Purine bases in the double helix of double-stranded nucleic acids according to the guidelines of triplet base pairing. can be detected or recognized using the MP-oligomer third strand to read. M.P. - The third strand of the oligomer has the base of each nucleoside unit corresponding to that of the double-stranded nucleic acid DNA. The sequence was chosen to form a triplet with the base pair and obtain a triple helical structure. It has columns. Hybridization of detectably labeled oligomers The presence of a specific double-stranded nucleic acid sequence, e.g. can be detected.

This invention also provides for reading nucleic acid sequences and using MP-oligos that form triple helical structures. Method of preventing expression or function of selected sequences of double-stranded nucleic acids by use of mer I will provide a. The generation of triple helices prevents the opening of the double helix for transcription, Aspects such as prevention of binding of vector molecules (e.g. proteins), and/or function. Using derivatized oligomers to detect and locates and then cross-links (psoralen) the target site within the nucleic acid duplex. , or irreversibly repaired by cleaving one or both strands (EDTA). It can be decorated. The selected nucleic acid sequence by carefully choosing the target site for cleavage Oligomers can be used as molecular scissors to specifically cut out.

Reacts with the fragment or its target sequence to irreversibly modify, degrade, or degrade the nucleic acid. or a nucleic acid reactive group or modification that can be destroyed and thus irreversibly inhibit its function. Groups may be inserted to derivatize the oligomer.

That is, these oligomers can be traced to specific genes or within living cells. irreversibly inactivates or suppresses the target sequence and selectively inactivates or suppresses It can be used to These oligomers are then deficient or undesirable. genes that produce undesirable products or induce undesirable effects when activated can be used to permanently inactivate, eliminate, or destroy.

Another aspect of this invention is to read the purine sequence and generate a triple helix structure. selected in that it is sufficiently complementary to the purine sequence to be able to form a detectable A specific double-stranded nucleic acid sequence containing a purine MP-oligomer third strand labeled as Provide a kit for detection.

To further understand this invention, we conducted a computer simulation. Examples include reporting of the results of a series of experiments. Of course, this regarding this invention These examples are not intended to specifically limit the invention, and those skilled in the art Changes in the invention, now known or which may later occur, to the best of the knowledge of It is considered to be within the scope of the invention.

Example Example 1 Triple helix structure computer noyumi lane these convenience turns; The primary purpose of milaneyon is to have a methylphosphonate ffM P J) backbone. Two nonionic nucleotides/analogs are formed by Hoogstay-type base pairing. As the third strand of the strand helical DNA, unmodified oligodeoxynucleotide ff0DN The purpose was to see whether it binds with higher affinity compared to J. fruit Experimental results indicate that ODN binding to double-stranded DNA and transcription inhibition may undergo duplex formation. (8) suggested that there is a triple linkage between analogs with MP backbones versus intact ODNs. No experimental comparison of DNA helix formation was made. Non-ionic with MP backbone It was not known in the past whether sexual analogues fit into the major groove of triple helical DNA. Not yet. Additionally, fully elucidated proteins with native or MP backbones in the third chain The conformation of the double-stranded helical DNA was not determined.

Site-specific oligonucleotide binding via duplex and double-stranded DNA complex formation , was recently shown to suppress the transcription of human cancer genes in vitro (8.9 ).

The purpose of this example is to construct a second strand helical complex using molecular dynamics experiments. We considered nonionic oligonucleosides with an MP backbone in the process and included them in this process. The aim is to elucidate the molecular mechanism(s) that cause

(A) Simulation method Double-stranded poly(dT+o)-poly(dA+o)-poly(dT　Io)[T　IAT z] The ligand is the A-DNA X-ray structure of Arnott and Selsing (10) Obtained from. The same ligand is poly(dT,.)-poly(dA+o)-poly(d T. ) was used as the starting geometry for methylphosphonate [T, AT, MP].

Geometry optimization and analysis of dimethyl ester methylphosphonate fragments and partial atomic charge assignments are 3-21G* and 5TOG* from the beginning, respectively. Quantum mechanical method using QUEST (version 1.1) using Pysissets (11). The latter basis set is the AMBER database used to maintain uniform charge assignment with those previously calculated for nucleic acids in I've been bored. From the final monopolar atomic charge assignment for the MP fragment, the first The total neutral charge for each base of the three DNA strands, the furanose and the MP backbone is obtained. It was. On the other hand, the Rp and Sp methyl substitutions of the main chain phosphoryl oxygens of the TAMP chain stand This was done using physical computer images. Since synthesis cannot be controlled, MP diastereo The mer substitutions were carried out in a manner that approximately approximates the experimental yield.

A complete base of AMBER for molecular mechanics and molecular dynamics calculations using total atomic force fields. (1.2.13) using the vectorized version (version 31). Everything All calculations were performed on CRAY X-MP/24 and VAX8600 computers. It was.

The negative charge on the DNA phosphate backbone is due to the phosphorus atom that divides the phosphate oxygen into three halves. Neutralized by positive antagonist configuration in 4A: the antagonist is MP-substituted mirror Not placed on top. Triple helices and counterions have periodic boundary conditions It was surrounded by an IOA shell of TIP3P water (14) molecules. T, AT2 and T, A72MP system has 9283 and 10824 atoms, respectively. Ru.

The box dimension is 101,686.8A3, T, AT, per TIAT2. It was 1.24.321.1 A per MP. First, DNA and opposing ionic atoms is sufficiently constrained, but the surrounding water molecules have a convergence value (effective value of gradient [rms] , <0.1 kg force 1 mole/A)8. OA non-binding cutoff (cut The energy was minimized using (toff). DNA, opposing ions and water are followed by an additional 1500 cycles of minimization with activated shaking (SHAKE); The energy was then minimized for 220 cycles without any geometric constraints ( 15). 5 without restraint under steady temperature and pressure (300 and 1 bar) Molecular dynamics using HAKE is applied to 4Qpsec trajectory for each of two molecules. I went.

(B) Results of simulation research The third DNA strand with the MP backbone is the reinforcement of the ODN with the MP backbone in the triple helix. brought about some changes that are consistent with the proposed combination. Average hydrogen bond distance and average Atomic fluctuations are successful in the T, AT2MP) libretto. consistently smaller (Table 1). The mirror-opening phosphorus atom distance is A-T, Nikki 9.6A (+/-0,91) and 8.3 (+/-0,58) for A-T2MP. Ta. The reduced mirror-opening phosphorus atomic distance and the smaller average atomic swing between the second and third chains The movement is due to interring electrostatic repulsion associated with MP substitution in the main chain.

Both triple helix DNA systems exhibit strand-specific polymorphic conformational features during molecular dynamics. Ta. Critical conformations in the furanose relative to the starting geometries of both systems (Table 2). In the T, AT2 helix, the strands of TI and T2 The lanose ring occupancy is mostly in the A-DNA conformation (C3' end). The remaining, largest portion of adenine sugar is in the B-DNA conformation (C2 “end”) Take. A significant percentage of the adenine furanose conformation is T , the 01° endoconformation in AT2 and TIAT2MP. M Sugar puckering pattern of P-substituted helices ) has a large proportion of 01° and C2’ endocolons as opposed to unsubstituted helices. It has an accommodation. Analysis of other conformational parameters supports the mixed structural nature of the triple helix. Helical twist angle (TI and A Kagami Kai) has an average of 39°4 degrees (+\-2,86) for T, AT, and structure, and B- A better match to the DNA conformation (range 36-45). T, AT2M The average P helix twist angle is 32.0 degrees (+\-2,1 9), and depending on the A-DNA conformation (range 30-32.7) close. The average repeat unit (between Tl and A chain) for the entire structure is 10.2 TlAT x and 11.2 degrees for T, AT, and MP. 4Qpsec orbit (traj Table 3 shows the average mirror-opening phosphorus atomic distances of in both helices , the optical distance of the T1 strand is determined by the A-DNA conformation (7, OA) (coincidence). do. The optical distance of the TIMP chain is better aligned with the B-DNA conformation. and differs greatly from values more consistent with A-DNA for the T2 strand.

In order to measure the changes associated with MP substitution, the inventors analyzed opposite ion The coordination of ions and water was analyzed. Average coordination distance and atomic rock of the opposing ion with a phosphorus atom Fluctuation is 3.8A (+\-0,6) per T and AT2. And T, AT.

It was 4.6 A (10\-0,9) A per MP helix. Flat in T, AT, MP The increase in coordination distance and atomic motion (0,8A) is due to the increase in coordination distance and atomic motion (0,8A). This is most likely due to the proximity of the Rp (axial rising) methyl group to the ion.

The average number of water molecules coordinated to the phosphanate group does not change significantly in the MP-substituted helix. do not have.

Table 4 shows the DNA main chain and CI'-N (base) dihedral changes of the two types of helical systems. .

Comparison of α-diface of adenine chain is the average position of dihedral with MP substitution. 4Qpsec There is a large overlap during the transient motion of both planes in orbit. Average Sp-MP di In the astereomer, there is a significant change (27,0 degrees) in the β dihedral angle from the baseline. there were. The fluctuation of the β diplane containing the Sp-MP diastereomer is remarkable compared to that of Rp-MP. There were fewer

(C) Interpretation of simulation results The results of these molecular dynamics calculations indicate that the collinear second strand with Hoogsteen pairing The MP-substituted ODN incorporated as ), using a triple helix complex as the second strand, which is more stable than the original ODN. Suggests formation. Enhanced binding of MP binder reduces electrostatic repulsion It depends. The MP-substituted helix reduces the hydrogen bond distance and opens the mirror A-T! Li This reduces the chain distance and causes less fluctuation due to the atomic position relative to the original triple helix. minutes Closer fitting and reduced atomic motion during molecular dynamics result in MP-substituted triple helices Quantitatively consistent with the larger enthalpy of binding and stability of the complex. These knowledge It appears that the MP-substitution in the second strand reduces the mirror-opening phosphate repulsion, resulting in more intimate contact. The formation of a triple helix structure was investigated secondarily by Hoogsteen and Watson. This indicates close proximity to click hydrogen bond interactions. Fugus Although a significant effect on teen pairing can be expected, in these calculations, MP replacement There is an unexpected enhancement of Watson-Crick hydrogen bonding with . The latter finding is T 1 and T, MP strands reduce electrostatic repulsion and interference (due to second strands). This is most likely due to

The conformations of these DNA structures differ from experimental data based on fiber shapes. Ru. The structure of poly(dT) poly(dA)-poly(dT) is Determined by line-diffraction measurements, this is a loosely resolved structure. (10). Molecular dynamics simulation , has a completely resolved DNA structure under periodic boundary conditions with opposing ions. Ru. Generally, B-type DNA is predominant in solution, and under low humidity conditions, A-DNA The A conformation is thought to be predominant (16). Several double-stranded DNs The A helical structure was determined by X-ray diffraction test, and under low humidity and high salt concentration conditions, Uniformly observed in A-DNA conformation (10.17.18). these Computer simulations of different DNA conformations showed that Mie et al. coexist within the helix, with the individual strands of the helix having a dominant conformational occupancy; -gusteen pairing/MP-substituted strands predict that the B-form is predominant. both Most of the 01' endosaccharides in the triplet of are in this furanose conformation. C2' end and C3, endo DNA sugar backers (16) 0.6 kg It is interesting because 1 mole of lolly is expensive. The DNA-N date crystal structure (19) is clearly It has a remarkable 01' end occupancy, and the molecular dynamics simulation of dsDNA by Siebel et al. In the simulation, a prominent part of the 01° endosugar pucker was observed (20) . Both helical structures are commonly associated with purine nucleos that adopt a C2' end geometry. This follows the classical observation of pyrimidines adopting a C3' end geometry with a tide.

Large perturbation of β-diplane in three lines and Rp and Sp, MP diastereoeye The various structural fluctuations of somers are due to non-bonding and hydrophobic interactions.

The Sp-MP group is very close to the thymine methyl group (in the main groove) on the same DNA mirror. are in contact with each other and interact with each other due to van der Waals forces, which is , a partial helix is formed by interlocking thymine to the Sp-MP main chain. Can be destabilizing. There is a large deflection in the β-biface of the Rp-MP group. That's this These groups protrude into the solvent water and are located far away from the thymine and methyl groups. This is because it is in Orientation of methyl substituents on phosphorus atoms and DNA duplex stability It is known that there is a correlation between the two, and the stability of the Sp-MP diastereomer It has been proposed that the mechanism of sex reduction is due to local steric interactions ( 21). The inventor's calculations show that steric interactions are sensitive to local helical destabilization. The dominant mechanism is the methane of the sp-Mp backbone on the same mirror. It is transmitted by a non-bonding interaction between the group and thymine, which locally binds the DNA. This suggests that it is becoming unstable.

Example 2 Detection of Triple Helix Formation Using Circular Dichroism Spectroscopy Circular dichroism spectroscopy is performed using the following nucleic acid This was carried out using a double-stranded helical structure formed using a combination of side oligomers.

I: d(CTCTCTCTCTCTCTCT) Abbreviation d(CT)s E”’=9.2X 10’M-’CI-’II: d(AGAGAGAGAGAG GAGAG) Abbreviation symbol d(AG)* E”’=　1.45　X　105M-’c11-’m: d(CpTpCpTp CpTpCpTpCpTpCpTpCpTpCpTp) Abbreviation d (CpTp ).

E”=8.5X I Q’M-’cm-’(a)2:1d(CT)s-d(AG )s and (b) 1:1:1d(CT)@・d(AG)m・d(CpTp)s The circular dichroism (CD) spectrum of the double-stranded helical structure formed by 0 at pH. Measurements were made using a CD spectropolarimeter in IM phosphate buffer.

CD specs for 1:1d(CT)s・d(AG)s・d(CT)s()] Toll is shown.

Example 3 Crosslinked psoralen derivation of triple helix structure using psoralen derivatized MP-oligomers dTp(T)s oligomer Hariichi B, L, et al. Biochemistry 27: 3197-3203 (1988).

The T7 oligomer has the following sequence containing a 15-mer polyA subsequence; 5' 10 2 0 30 40)' It was hybridized with DNA having .

4'-(aminoethyl)aminomethyl-4,5',8-trianethylsorale 4゛-(aminobutyl)aminomethyl-4,5°,8 -trimethylpsoralenbi(ab)AMT"co and 4'-(amino-hexyl) Aminomethyl-4,5°,8-trimethylpsoralen ["(ah)AMT"] MP-oligomers derivatized with (a) single-stranded DNA of the above DNA sequence; and (b) hybridized with the double-stranded DNA of the above sequence at 4°C, as described by Lee et al. Crosslinking was induced by irradiation as described.

The results are shown in Figures 12A and 12B. Figure 12A shows psoralen-derivatized T7 oligo Figure 3 shows cross-linking by a mer and a single-stranded (polyA-containing) DNA sequence.

Figure 1.2B shows crosslinking of double MDNA and double stranded DNA sequences.

Example 4 Formation of double-stranded complexes using stannic oligomers Formation of triple antitussive acid complexes As shown in the table, the Watson-Crick base pairing "target" duplex, II -Tested with III and third strand oligomer ■. pyrimidine, purine, When the pyrimidine triple helix is formed, the sugar phosphate base backbone of the oligomer ■ It has the same polarity as the purine chain of the heavy helix.

Oligomer ■ (X is 8-oxo-adenine), I (OA ) and target duplex (where base pair y-z is G'C), II・I When II(G-C) is mixed, (m~( A), maximum hypochromia at 26On- was obtained with a stoichiometry of 1:1. This characteristic is This was consistent with the formation of a helix I-II/III (OA-GC). When heated , two cooperative transitions were observed. The first tran, whose midpoint occurs at 29°C Jishon is the third chain! It was identified as corresponding to the melting point of (OA). The midpoint is The second transition that stops at 45°C is the double helix II/III (G-C) It was identified as corresponding to the melting point of .

Similar melting characteristics were observed when using Oligomer I (C) (see Table VI). (see).

Furthermore, the formation of triple helices I, II, and III (OA-G-C) is shown in Figure 13B. This was confirmed by the circular dichroism (CD) spectrum. At 10°C, I(OA )・AE・■工■ (GC) CD spectrum is 22on11 and 28o stop delta E, a property identified as characteristic of triple helix formation. (Pilch , D., Sl. et al., Proceedings of the National Academy of Sciences. Biosciences, 87:1942-1946 (199 0)). 3rd tfJ [(7) 35℃ temperature, CD speed The spectrum is a combination of the spectra of I (OA) Oyohi II and III (G-C). It was essentially the same as the total. Triple Helix I, II, III (C-G-C) CD The spectra show similar behavior at 10°C and 40°C, and the triple helix stroke The spectrum was virtually identical to that of I.A.III (OA-G-C).

As shown in Figure 7, 8-oxoadenosine has two hydrogen bonds with G (G's with N-7 and 0-6 atoms). 8-Oxoadenosine is syn-co- It was shown that it exists in the keto form with a base in the conformation. 8-Oxoa If the keto form of Dennin is in the syn conformation, then 1(OA) and E. Triple helix formation with III(G-C) can be performed. Proposed hydrogen bond outline Abbreviations are tt tail shown in Table 4 +, - I, II-III (OA-G-C ) evidenced by the similarity of the melting properties of Oyohi I, II-III (C-G-C). It was.

According to molecular model experiments, 8-oxoadenine in the syn configuration is Although it can be compatible with the C-G base pair of triple helix ■, II, and III (OA-C-G) , 8-oxoadenine and C-G are completely unexpected. It has been suggested that 1:1 mixture of I (OA) and II/III (C-G) The melting curve actually showed two transitions, but the concentration of the first transition was The color was only about 7% of the total, and the melting temperature of stannic was about 9°C. Triblet For stannic compounds (OA-U-A), the melting temperature is higher than that. (13°C) was observed.

The increase in Tra is due to the N-5 exocyclic amino group of 8-oxoadenine and the U-A base pair. This may be due to the single hydrogen bond formation between 0 and 4 of uracil in . Also single Hydrogen bonds can be formed by 0-4 of 8-oxoadenine and thymine, but Double helix formation occurs in a 1=1 mixture of I (OA) and II/III (T-A). This was not observed at all. In that case, the triple helix formation is due to the 5- It can be prevented by interaction of the methyl group with the 5-membered ring of 8-oxo-adenine.

The molecular model shows that non-oxoadenine is located opposite the A-T base pair of the triple helix. Although applicable, triple helix formation is limited to I (OA) and II/III (A - T) It was shown that this was not observed at all for the 1.1 mixture. This finding is based on 11 A target double helix that contains adjacent A bases and has a curved structure that can prevent triple helix formation. This may be due to the properties of II and III (A and T). Engineering・II・III(T-A, -T) stannic is 10℃ lower than stannic of I, II, III (C, G-C) The finding that it melts at

Table 1 Average hydrogen bond distance (RMS) Watson-Crick Without MP With MP ^DEllIN6B-TIIYO42 , 33 (+/-0, 31) 1.98 (+/- 0, 15) ADE Nl -TR Y I’! 3 2.10 (+/-0,17) 1.95 (+/-0,13) Fu Gusteen ADE BN6^-T [IYO42,12(+/-0,22) 2.09(+/ -0,19) Steen hydrogen bond distance (Angstrom). These distances Calculations were made for triple helix DNA complexes. Oscillation due to atomic position (effective value [r+ ms]) (expressed in angstroms).

Table 3 Average intrachain phosphate atomic distance (RMS) chain without MP with MP TI 6.5 (±10.28) 6.2 (±10.31) A 7.0 (±10. 26) 7.0±10.24) 72 6.3 (±10.29) 6.8 (±10 ．． 26) TIAT2 and T, intra-end phosphate distances of the AT2MP helix. 4Q Calculated intrachain phosphate distances averaged over psec orbitals (Angstroms) ) is shown for a complete triple helix system. The standard mirror-opening phosphorus atomic distance is for A-DNA. 6. 7. for OA and B-DNA. It is OA (15).

Table 4 71 280.5 (18,5) 288.4 (11,3) A 252.9 (28 , 7) 233.2 (28, 5) 72 2851 (11, 6) 21 + 8.5 ( 1t, 3) βP-05'-C5°-C4゛ T 1 161.2 (11,5) 168.7 (8,7) A 151.5 (1 0,7) 159.3 (21,5) T 2 140.8 (8,8) FOR1 58,8 (21,9) PO3167, 8 (8,7) YO5°-C5°-C4°-C3 T 1 70.6 (14,6) 62.6 (9,3) A 104.8 (27,9 ) 112.3 (25,5) T 2 67, 2 (10,5) 64.0 (10, 4) δC5'-C4''-C3°-03° T190.3 (12,8) 82.5 (11,1) Table 6 Melting temperature of the oligodeoxyribonucleotide complex second strand (al　d−c+’r T-c+TT-'rTT-Tx"r-T"rT (I) Target double helix: d- GAA-GAA-AAA-AYA-AAA (II) CTT-CTT-TTT- TZT-TTT-d (III) 8-oxo A G C2945 8-OxoACG941 8-Oxo A IJ A 13 42 (a) C+ is 5-methyldeoxyditidine.

(b) M solution experiment consists of 0.5 micromolar NaCl, 20 mmol C12 and and an oligomer concentration of 50 mmol Tris-HCI (pH 7.0). .

(C) Transition, triple helix → double helix + 1° (d) Transition: Double helix → single strand.

literature 1 Moscou, H.E., et al., Science Vol. 238, pp. 645-650 (198 7 years) 2. Povitt, TJ et al., Journal of American Chemical Society Etty Vol. 111, pp. 3059-3061 (1989) 3, Wells, R. , D. et al., FASEB Journal Vol. 2, pp. 2939-2949 (1988) ) 4 Durban, P1 Science Vol. 232, pp. 464-471 (1988) 5 , Miller, P, S et al., Anticancer Drug Design Volume 2, Volume 1 pp. 17-128 (1987) 6 Yachon et al., “AIDS Treatment J, Scientific American No. 11 Pages 0-119 (October 1988) 7. Sarin et al., Pronoding of the National Academy of Sciences Jenswiss (USA) Vol. 85, pp. 7448-7451 (1988) 8 Ni, M. et al., Science Vol. 241, pp. 456-459 (1988) 9 Inkström, E., et al., Proseding of the National Academy of Sciences. ・Of Sciences (USA) Vol. 85, pp. 1028-1032 (1988 Year) 10, Arnoff, S., et al., Journal of Morcucous Biology No. 8 Volume 8, pages 509-521 (1974) 11. Schimpf, UC et al., Journal of Computer Science - Vol. 5, No. 129-1.45 (1,984) 12 Nomph, UC et al., AMB ER (UC3F) version 3.1 (1988) 13 Uni Iner, S, J et al., Journal of Combinational Chemistry, Volume 7, Tube 230- 252 pages (1986) 14 Njorgenson, W. J., et al., Journal of ・Chemical Physics Volume 79, pages 926-935 (1983) 15 Lendsen, H. J., C. et al., Journal of Chemical Physics No. 81 Volume 3684-3690 (1984) 16 Sanger, W., Brisimpal. Of Nucreek Acid Structur - York, 1984) 17 Arnosoto, S. et al., Nature Vol. 244, No. 99 -101 pages (1973) 18, Arnott, S. et al., Science Vol. 181 Bundle pp. 68-69 (1973) 19 Toulu, H.R., et al., Prosuedi. National Academy of Sciences (USA) No. 78 Volume No. 2179-2183 (1981) 20 Sabel, G. L., et al., Professional Succession of National Academies Me of Science (USA) Vol. 82, pp. 6537-6540 (198 5 years) 21, Bower 1M, et al. Nucleitsk Acid Research Vol. 1 No. 4915 -4930 pages (1987) 22 Tonohiyu, 3. et al., Journal of Molecule Biology, Vol. Volume 2, page 363 (1960) 22a, Ts'o, P, 0. P., Basic Principals in Nucle Itsuku Acid Chemistry, pp. 453-584 (P, O, P, Ts’o, 7 Kademik Press, New York, 1974) 23, Sanglalingam 1M1 Biopolymers Vol. 7, p. 821 (1969) ) 24 Arnott, S. et al., Progress in Nuclear Acid Reduction. Search Molecule Biology Volume 10, Page 183 (1970) 25 Sashisekharan, ■, et al., Conformation of biopolymers, paper ・International Symposium 1967, Volume 2, Page 641 (1967 ) 26, Rakunyuminarayanan, A, V et al., Biochemical Biophysics. Le Acta Volume 204, Page 49 (1,970) 27, Lakshminarayanan , A., V. et al., Piopolymers Vol. 8, pp. 475 and 489 (1970 Year) 28 Lee, B. L. et al. Nucleitsk Acid Research Vol. 16 No. 106 pp. 81-10697 (1988) 29 Barone, A, D et al., Nucleitsk Acid Research Vol. 12, No. 4 pp. 051-4060 (1984).

/Zri itZ- C'G 3 501 9 G) /: Kuj /Fugu j- Crosslinking kinetics Time (minutes) ^260

Claims (38)

[Claims] (1) Detects specific sections of double-stranded nucleic acids without interrupting base pairing of the double helix. or recognition method in which favorable modifications are made to the thin conformation. at least one nucleoside unit containing an 8-modified purine base; contacting the nucleic acid fragment with an oligomer that is sufficiently complementary to the acid fragment or a portion thereof; This method involves forming a triple helix structure, with one strand of the double helix contains a polypurine sequence having at least about 7 purine bases, and the oligomer contains a polypurine sequence having at least about 7 purine bases; parallel to the strand with the polypurine sequence, and further Guanine is 8-oxo-adenine and cytosine or cytosine analogs (this Either N-3 is protonated at physiological pH and forms a hydrogen bond with guanine. in the polypurine sequence. an oligo whose adenine is selected from 8-oxo-guanine, thymine and uracil. A method that is read by the bases in the mer. (2) Claim 1, wherein the polypurine sequence contains at least about 12 purine bases. Method described. (3) Oligomeric nuclei containing 8-oxoadenine or 8-oxoguanine 2. The method according to claim 1, wherein the osid has a syn conformation. (4) The polypurine sequence contains pyrimidine bases at a rate of 50% or less, and The cytosines in the sequence are 8-fluoroguanine, 8-methoxyguanine and 8-fluoroguanine. It is read by the base in the oligomer selected from azaguanine, and the polypurine Thymine in the column is 8-fluoroadenine, 8-methoxyadenine and 8-aza according to claim 1, which is read by a base in the oligomer selected from adenine. Method. (5) 8-oxoadenine, 8-oxoguanine, 8-fluoroadenine, 8- Fluoroguanine, 8-methoxyadenine, 8-methoxyguanine, 8-azaa Oligomeric nucleosides containing denine or 8-azaguanine are 5. The method according to claim 4, wherein (6) The oligomer is an oligonucleotide, an alkyl- or aryl-phosphorus Honothioate oligomer, phosphorothioate oligomer, alkyl- or are aryl-phosphonate oligomers, phosphotriester oligomers, and phosphonate oligomers. Loamidate oligomer, carbamate oligomer, sulfamate oligomer , containing morpholino oligomers or formacetal oligomers, claims The method according to any one of Items 1 and 4. (7) The nucleoside unit of the oligomer is ribose, deoxyribose, 2'- O-alkyl ribose, 2'-O-aryl ribose and 2'-halogen ribose (all of which are optionally substituted with halogen, alkyl or aryl) 8. The method according to claim 7, comprising a sugar moiety selected from the following. (8) The nucleoside units of the oligomer are ribose, deoxyribose and 8. The method of claim 7, comprising a sugar moiety selected from '-O-methylribose. (9) a double-stranded nucleic acid having a given sequence of each of the two strands in a complementary nucleic acid duplex; A method for inhibiting the expression or function of a specific section of at least one nuclease containing an 8-modified purine base with a favorable modification added to the An oligomer containing an osidic unit and sufficiently complementary to the nucleic acid fragment or a portion thereof The method includes contacting the nucleic acid fragment with the nucleic acid fragment to form a triple helical structure. polypurine in which one strand of the double helix has at least about 7 purine bases the oligomer is parallel to the strand having the polypurine sequence; Furthermore, guanine in the polypurine sequence is 8-oxo-adenine and cytosine. or cytosine analogues (either of which are N-3 protonated at physiological pH). can form hydrogen bonds with guanine) The adenine in the polypurine sequence is replaced by 8-oxo-guanine, thymine and u A method of reading based on bases in oligomers selected from rasyl. (10) The polypurine sequence contains at least about 12 purine bases. 9. The method described in 9. (11) Oligomeric nuclei containing 8-oxoadenine or 8-oxoguanine 10. The method according to claim 9, wherein the leoside is in a thin conformation. (12) The polypurine sequence contains approximately 50% or less of pyrimidine bases, Cytosine in the phosphorus sequence is linked to 8-fluoroguanine, 8-methoxyguanine and It is read by the base in the oligomer selected from 8-azaguanine, and Thymine in the sequence is 8-fluoroadenine, 8-methoxyadenine and 8-fluoroadenine. Claim 9, which is read by a base in an oligomer selected from azaadenine. How to put it on. (13) 8-oxoguanine, 8-oxoadenine, 8-fluoroadenine, 8 -Fluoroguanine, 8-methoxyadenine, 8-methoxyguanine, 8-aza Oligomeric nucleosides containing adenine or 8-azaguanine are 13. The method of claim 12, wherein the method is in homeation. (14) The oligomer undergoes a reaction with the nucleic acid double helix, changing the structure of the nucleic acid. 10. The nucleic acid according to claim 9, wherein the nucleic acid modification group is modified to include a nucleic acid modification group capable of irreversibly modifying the nucleic acid. Method. (15) the oligomer is induced to react with a nucleic acid fragment or a target sequence therein; irreversibly modifying the nucleic acid, thereby irreversibly altering the expression or function of the nucleic acid fragment. 10. The method according to claim 9, which comprises a nucleic acid modifying group capable of inhibiting oxidation. (16) The modification is shared between one strand of the nucleic acid double strand of the nucleic acid double helix and the oligomer. 16. The method of claim 15, comprising binding or cross-linking the nucleic acid. (17) The modification according to claim 15, wherein the modification includes cleavage of one or both strands of the nucleic acid fragment. Method. (18) When a nucleic acid fragment contains a gene in a living cell, the formation of a triple helix structure 16. The method of claim 15, which permanently inhibits or inactivates. (19) The oligomer is an oligonucleotide, an alkyl- or aryl-phosphate Sulfonothioate oligomer, phosphorothioate oligomer, alkyl-moshi or aryl-phosphonate oligomers, phosphotriester oligomers, Holamidate oligomer, carbamate oligomer, sulfamate oligomer - Contains morpholino oligomers or formacetal oligomers. 13. The method according to claim 9 or 12. (20) The nucleoside unit of the oligomer is ribose, deoxyribose, 2' -O-alkyl ribose, 2'-O-aryl ribose and 2'-halogen ribose Bose (all optionally substituted with halogen, alkyl or aryl) 20. The method according to claim 19, comprising a sugar moiety selected from the following. (21) The nucleoside units of the oligomer are ribose, deoxyribose and 21. The method of claim 20, comprising a sugar moiety selected from 2'-O-methylribose. (22) Selected double-stranded nucleic acid sequences and modifications favoring thin conformation at least one nucleoside unit containing a decorated 8-modified purine base; contains and is sufficiently complementary to read the polypurine sequence in one strand of the nucleic acid sequence. In other words, it has a triple helical structure consisting of oligomers that can form triplets. The oligomer is parallel to the chain with the polypurine sequence, and The guanine in the sequence is 8-oxo-adenine and cytosine or cytosines. analogues (either of which are protonated at N-3 to form guanine and hydrogen at physiological pH) are read by bases in the oligomer (which can form bonds) and Adenine in the phosphorus sequence is selected from 8-oxo-guanine, thymine and uracil. The structure read by the bases in the oligomer. (23) Oligomeric nuclei containing 8-oxoadenine or 8-oxoguanine 23. The structure of claim 22, wherein the leoside is in the syn conformation. (24) The polypurine sequence contains approximately 50% or less of pyrimidine bases, and Cytosine in the phosphorus sequence is linked to 8-fluoroguanine, 8-methoxyguanine and It is roughened by the base in the oligomer selected from 8-azaguanine, and the polypropylene is Thymine in the sequence is 8-fluoroadenine, 8-methoxyadenine and 8-fluoroadenine. Claim 22, which is read by a base in an oligomer selected from azaadenine. Structure described. (25) 8-oxoadenine, 8-oxoguanine, 8-fluoroadenine, 8 -Fluoroguanine, 8-methoxyadenine, 8-methoxyguanine, 8-aza Oligomeric nucleosides containing adenine or 8-azaguanine are 25. The structure of claim 24, wherein the structure is in homeation. (26) The oligomer is an oligonucleotide, an alkyl- or aryl-phosphate Sulfonothioate oligomer, phosphorothioate oligomer, phosphotrieth oligomers, phosphoramidate oligomers, carbamate oligomers, Rufamate oligomer, morpholino oligomer, alkyl or formaceta 25. A structure according to any one of claims 22 or 24, comprising a polymer oligomer. (27) The nucleoside unit of the oligomer is ribose, deoxyribose, 2' -O-alkyl ribose, 2'-O-aryl ribose or 2'-halogen ribose Bose (all optionally substituted with halogen, alkyl or aryl) 27. The structure of claim 26, comprising a sugar moiety selected from the following. (28) The nucleoside units of the oligomer are ribose, deoxyribose and 28. The structure of claim 27, comprising a sugar moiety selected from 2'-O-methylribose. (29) Claim 27, wherein the oligomer comprises about 12 to about 16 nucleoside units. structure. (30) the oligomer is an alkyl or aryl phosphonate oligomer; 25. A structure according to any one of claims 22 or 24. (31) 8-modified purine salt with favorable modification of syn conformation contains at least one nucleoside unit containing a guaniline group in the polypurine sequence. 8-oxoadenine and cytosine or cytosine analogs (either can be protonated at physiological pH to form hydrogen bonds with guanine) The adenine in the polypurine sequence is read by the base in the oligomer selected from in an oligomer in which the ion is selected from 8-oxo-guanine, thymine and uracil. It is possible to read the polypurine sequence of one strand of the double-base nucleic acid sequence, which is read by the base. oligomer. (32) The polypurine sequence contains approximately 50% or less of pyrimidine bases, and Cytosine in the phosphorus sequence is linked to 8-fluoroguanine, 8-methoxyguanine and It is read by the base in the oligomer selected from 8-azaguanine, and Thymine in the sequence is 8-fluoroadenine, 8-methoxyadenine and 8-fluoroadenine. Claim 31, which is read by a base in an oligomer selected from azaadenine. Oligomers listed. (33) Neutral oligomers are oligonucleotides, alkyl- or aryl -Phosphonothioate oligomer, phosphorothioate oligomer, alkyl- or arylphosphonate oligomers, phosphotriester oligomers, Sphoramidate oligomer, carbamate oligomer, sulfamate oligomer mer, morpholino oligomer or formacetal oligomer. 33. The oligomer according to any one of 31 and 32. (34) The nucleoside unit of the oligomer is ribose, deoxyribose, 2' -O-alkyl ribose, 2'-O-aryl ribose or 2'-halogen ribose Bose (all optionally substituted with halogen, alkyl or aryl) 34. The oligo according to claim 33, comprising a sugar moiety selected from Mar. (35) The nucleoside units of the oligomer are ribose, deoxyribose and 35. The oligo according to claim 34, comprising a sugar moiety selected from 2'-O-methylribose. Mar. (36) The oligomer of claim 34, comprising about 12 to about 16 nucleoside units. -. (37) Claim 3 comprising an alkyl- or aryl-phosphonate oligomer. 33. The oligomer according to any one of Items 1 and 32. (38) The oligomer according to claim 37, comprising a methylphosphonate oligomer.