JPH04506152A - Formation of triple helix complexes of double-stranded DNA using nucleoside oligomers - 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] Formation of triple helix complexes of double-stranded DNA using nucleoside oligomers Citation of related applications This application is filed under U.S. Patent Application No. 924234, filed October 28, 1986. This is a continuation-in-part application, and the statement is cited in the specification.

Background of the 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 oligodeoxyribonucleosides bound to the purine tract The formation of triple helical structures has been reported (see, eg, (1) and (2)).

Homopyrimidine oligonucleosides 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 The fusion between the rimidine oligonucleoside and the purine strand of Watson-Crick double DNA It was found that this is given by Gusteen base pairing. hoogsteen A triple helix complex containing cytosine and thymidine on the (or third) mirror is They are stable in acidic to neutral solutions, but as the pH increases, separation becomes more difficult. It has been found that

Modified salts of T such as 5-promouracil and C such as 5-methylcytosine The incorporation of groups into the Hoogsteen chain increases the triple concentration over the higher pH range. It has been found to increase the stability of the cell. Cytosine (C) is Hoogsty In order to participate in the pyrimidine-type pairing, a hydrogen is present on the 3-N of the pyrimidine ring for hydrogen bonding. Must be available. Therefore, under certain conditions, C is pro-protonated at N-3. Can be toned.

DNA exhibits a wide range of polymorphic conformations, and these conformations are It can be essential in physical processes. Signal induction by sequence-specific protein-DNA binding The regulation of DNA conformation and the interactions of molecules such as transcription, translation and replication depend on DNA conformation. is considered to be independent in terms of (3) Binds to critical regions of genes and selectively inhibits abnormal cell function, replication, and survival The development of therapeutic agents that inhibit this is shocking. (4) Design of sequence-specific DNA binding molecules and development is being considered in numerous laboratories, and foreign genes (e.g. viruses) or widely used in the diagnosis and treatment of diseases involving genetic DNA degeneration (e.g. cancer). closely related.

Nuclease consisting of methylphosphonate (MP) back bone Resistant nonionic oligodeoxynucleotides in vitro and in vivo It has been investigated as an anticancer, antiviral and antibacterial agent. (5) These analogs The 5°-3° internucleotide linkage is a nucleoside phosphodiester linkage. The conformation is very similar to that of the The phosphonate backbone has one anion Neutralized by methyl substitution of the onphosphoryl oxygen; due to the charged phosphonate group Therefore, inter- and intra-molecular repulsion is reduced. (5) Analog with MP main chain affects living cells and inhibits globin synthesis and vesicular stomatitis virus protein synthesis. conjugation of pre-mRNA in inhibiting H8V replication. It has been shown to inhibit splicing. to nonionic analogs The mechanism of inhibition is the formation of a stable complex with complementary RNA and/or DNA. Contains homeation.

A non-ionic oligonucleoside alkyl complementary to a selected singleton exogenous nucleic acid. - and aryl-phosphonate analogs function in other nucleic acids present in cells. (By binding to or interfering with a specific nucleic acid, the expression or expression of a nucleic acid is or function can be selectively inhibited (for example, U.S. Patent No. 44698). 63 and 4511713). Complementary nuclei taken from mammalian cells ase-resistance, non-ionic oligonucleoside methylphosphonate herpes simplex The use of U, S to inhibit the synthesis of certain viral proteins, including S. virus-1. , disclosed in Japanese Patent No. 4757055.

An anti-sense oligonucleotide or oligonucleotide complementary to a portion of the viral mRNA Interfering with transcription and translation of viral mRNA into proteins of sforothioate analogues. Its use has already been proposed.

The anti-sense construct binds to viral mRNA and binds to cellular ribosomal mRNA. activity, thereby halting the translation of mRNA into protein, or "translation arrest." or may interfere with the process called “ribosome-hybrid synthesis termination.” It will be done. (6) HTLV-I Required for HTLV-I I Replication and/or Expression by administration of oligonucleotides complementary to highly conserved regions of the III gene. Inhibition of cell infection by HTLV-II is disclosed in U.S. Patent No. 4806463. disclosed inside. oligonucleotides as judged by breech expression) known to affect viral replication and/or gene expression. Ru.

Certain anti-sense oligonucleotides containing internucleoside methylphosphonate linkages The ability of oxynucleotides to inhibit HIV-induced syncytium formation and expression has been previously investigated. It is. (7) Psoralen-derived oligonucleotide methylphosphonate is Cross-linking of either coded or non-coding heavy chain DNA is possible; It has been reported that double-stranded DNA is not cross-linked. (28) Summary of the invention The present invention searches for and recognizes double-stranded DNA or specific segments of double-stranded DNA sequences. methods and methods for determining the expression or function of a particular piece of double-stranded DNA with a given sequence. Concerning inhibition methods. This is due to the disruption of duplex interactions or the base formation of double-stranded DNA. This is done without interfering with the match. The invention also relates to selected double-stranded DNA sequences. , and optionally contains a DNA modifying group. Regarding regular modified oligomers. Furthermore, the present invention provides novel Regarding oligomers. The present invention also provides specific sections of double-stranded DNA and DNA sections. sufficiently complementary to the DNA section and its base pairing (or hybridization) The interaction of the oligomers involved in forming a triple helix structure.

Therefore, from one point of view, the present invention provides a method for converting a DNA section into the above-mentioned section of double-stranded DNA. or by contacting it with an oligomer that is sufficiently complementary to the sequence of purine bases in its part. , hydrogen bond (or hybridize) with them, thereby forming a triple helix Method for searching and recognizing specific pieces of double-stranded DNA, including forming structures Regarding.

In another aspect, the present invention provides a method for generating specific pieces of double-stranded DNA having a given sequence. A method of inhibiting the DNA fragment or the function of the DNA fragment, the method comprising: is brought into contact with an oligomer that is sufficiently complementary to form a hydrogen bond with it, thereby The present invention relates to a method of forming a triple helix structure.

In the present invention, the DNA section contains genes in living cells, and the formation of a triple helix structure is permanent. The present invention relates to a method of inhibiting and inactivating the above genes.

In a preferred example, the oligomer is modified such that the oligomer is a selected DNA. After hydrogen bonding or nozobrid formation with the sequence, a chemical reaction occurs with the DNA and its These incorporate DNA modifying groups that modify irreversibly. Such a modification is , formation of covalent bonds, alkylation of DNA, cleavage of said DNA at specific positions, or or cross-linking of oligomers and DNA by DNA degradation or destruction. Ru.

In another aspect, the present invention provides methods for detecting nucleotide sequences that are sufficiently complementary to the blue sequence of a particular double-stranded DNA. , novel alkyl- and aryl- which form hydrogen bonds and form a triple helix structure. The present invention provides phosphonate oligomers. Nonionic methylphosphonate Oligomers are preferred.

A preferred type of methylphosphonate oligomer is one containing only purine bases. Ru.

The present invention also provides that a cytosine analog replaces cytosine, and that the cytosine analog has a hydrogen available for hydrogen bonding at the ring position corresponding to N-3 of cytosine, Nucleodis containing a heterocyclic ring capable of forming two hydrogen bonds with a guanine base at neutral pH. The present invention provides an oligomer having a molecule unit.

In another aspect of the invention, 5°-C and 5°-OH and/or 3°-C and 3' Oligomers are provided that include a modified sugar moiety with an extended bond between -OH. this These oligomers containing modified sugar moieties such as As used herein, the following terms are specifically intended to read the purine bases of Unless otherwise specified, it has the following meaning.

The term "oligomer" refers to internucleoside phosphorus groups (internucleosyl phosphorus groups). nucleoside units linked by phosphorus group) and aryl-phosphonate analogs, oligonucleoside phosphors. Holothioate analogs, neutral phosphonate ester oligonucleotides, including analogs, and other oligonucleotides and modified oligonucleotides. .

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". The phosphonate group is a nucleoside It can be used as an internucleoside phosphorus group (or linkage) to link the de units.

The term "phosphodiester" refers to the group o=po-. The phosphodiester group is internucleoside phosphorus linkages (or linkages) to link nucleoside units; It can be used as

The term "alkyl- or aryl-phosphonate oligomer" means at least , one alkyl or arylphosphonate internucleoside bond is Contains an internucleoside phosphorus bond that replaces the ester/internucleoside bond A nucleotide oligomer.

The term “methylphosphonate oligomer” (or rMP~oligomer) At least one methylphosphonate internucleoside bond is a phosphodiester ・Nucleans having internucleoside phosphorus group bonds to replace internucleoside bonds Otide oligomer (or oligonucleotide analog).

The term "third-strand oligomer" refers to a fragment of double-stranded DNA that is read and combined with Mie et al. An oligomer that can form a spiral structure.

The term "nucleoside" refers to nucleoside units, which are interchangeable and These bases include A, G, C, T and U, as well as analogs and bases. Also includes modified forms.

The term "complementary" refers to the third strand of an oligomer. JDNA corresponding (Watson-Crick) base pair purine base and hydrogen bond (and base pairing or hybridize) and form an oligomer with a base sequence that forms a triple helix structure. say.

In the various oligomer sequences listed here, e.g. , "p" indicates a phosphodiester bond, e.g., as in C20, "And" indicates a methylphosphonate bond.

Also, notations such as rTJ refer to nucleic acids linked by methylphosphonate bonds. Indicates an osido group.

The term "purine" or "purine base" refers to the naturally occurring adenine and guanine. It includes not only bases with bases but also base modifications such as bases substituted at the 8-position.

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 to recognize a base sequence. In other words, for reading double-stranded DNA sequences. In this process, the third strand oligomer binds a piece of double-stranded DNA through hydrogen bonding interactions. It can recognize base pairs in doublets, especially purine bases.

The term "triplet" refers to the bases in the third strand of a piece of double-stranded DNA (Watson-Crick). H) Diagrams IA, IB and 2 showing hydrogen bonds with base pairs (i.e. base pairing) It refers to the state that appears from A to 2D.

Brief description of the drawing Figures IA and IB 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 2A 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 30 show that the third strand is [on the reading side, i.e. on the single strand of double-stranded DNA]. Typical polypurine sequence triple helix structure base pairing with purine bases Represents a column.

Figures 4 to 4F show that the third strand "reads", i.e., on the duplex of the duplex DNA. The base sequence of a typical mixed triple helix structure that is base-paired with a purine base is shown. Was.

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 the DC spectrum of the triple helix structure. () is MP-oligomer third strand, () indicates the third strand of the oligonucleotide.

Figures 8A and 8B show (A)-heavy chain DN using psoralen-derived MP-oligomers. A and (B) Cross-linking of double-stranded DNA is shown.

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. contact with an oligomer that can form a hydrogen bond (or nosobridize) with it. Formation of a triple helix structure with the double-stranded DNA sequence by contacting Ru.

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

(I) The first aspect is that without cleavage of base pairs, such as adenine and guanine Extra hydrogen bonding sites of purines in double-stranded DNA and water bonding by bases in the third strand The reading (or recognition) of base pairs in double-stranded DNA sections through elementary bond formation. related. In other words, reading the base pair sequence in a DNA duplex is always The remaining available hydrogen bonding site of the purine in A and the hydrogen bond with the base in the third strand. It is done by reading the purines of base pairs by bond formation. in the third strand purines (e.g. adenine (A) or guanine (G)) or pyrimidines (thi Both min (T) and cytosine (C) form hydrogen bonds with purines in DNA. It can be accomplished. Namely: 1) Adenine (A) in a base pair of DNA reads; That is, it can hydrogen bond with A or T in the third chain.

u) Guanine (G) in the base pair of DNA is read, i.e. C in the third strand or can be paired with G.

In a preferred aspect of the invention, the phosphorus-containing backbone of the third chain comprises methylphosphonate groups and and naturally occurring phosphodiester groups.

(II) The second aspect of this invention is to use purine bases from the third strand to It reads the entire purine (A or G) in the duplex. In other words , this aspect of the invention uses only G to read G in base pairs in a DNA duplex. , only A is used to read A in a DNA double-base pair. chain polarization The stannic antipaste is applied to the strand containing the purine to be read in the duplex. anti-parallel or parallel Both are important.

The base planes of purines and pyrimidines are fixed, and only the furanose ring is slightly Wavy (0.5 people above and below the plane). That is, the three-dimensional structure of the nucleoside The state is more or less about the mutual C'-1 and N-9 or N-1 bond axes. In principle, rotation of these two fixed planes, i.e., the base and the pentose, stipulated in

The sugar-base torsion angle, φCM, is the furano It is formed by the rising edge of the C-1° to O-1° bond of the ring and the line of the base plane. defined as an angle. This angle means that the furanose ring oxygen is a pyrimidine or When it is not planar to C-2 of the purine ring, it is O. , is defined as the positive angle when looking at N from C-1゛ and measured clockwise. Ru. This angle is also referred to as the glycosyl torsion angle.

Using the above definition, for nucleosides, anti-conformation Two ranges of φ, approximately -30° and approximately +150° for thin conformation. It can be seen that CN exists. The range of each conformation is ±45°.

(22,22a) Other researchers have used and proposed a slightly different definition of this angle. are doing. (23, 24, 25, 26, 27) Information about φCN can be obtained by X-ray diffraction , proton magnetic resonance (PMR) and optical rotational dispersion-circular dichroism (ORD-CD). It has been obtained by this method. (22a) One chain (referred to as “Watten chain”) ) to the opposite strand (referred to as the "click strand"). A special conformation of the nucleoside, the group, is used to accommodate changes in the position of the group. 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 Regarding the leoside unit, when reading the purine in the parallel strand in the duplex, the third The conformation of the purine nucleoside units in the chain is called thin conformation. It should be. On the other hand, the corresponding purine in the Ande parallel in the duplex is The conformation of the purine nucleoside unit in the third strand during reading is anti- Must be conformational. i.e. double strands distributed on both strands In reading the purine bases in the same ( The choice is to use stannic with polarity (that is, parallel).

As an example, 5+ 3+ (These two strands are G C I1 Anti-parallel. ) (Watson chain) (Crick chain) The third strand of the three strands with the DNA duplex having the same polarity as Watson strand 5' to 3'. The ditin arrangement is as follows: (parallel to the Watson chain) stannic) The stannic sequence parallel to the click strand is as follows: Gal″t1co1 syn According to one aspect of the invention, the first step for reading purines in double-stranded base pairs is In constructing the tristrand, apply the following guidelines: (i) starting from the 5' end; towards the 3' end, the third strand purine nucleoside unit (A or G) DNA strand ('Watson') reading of purine in base pair (A or G) of strand When cutting, it is necessary to have a thin conformation. of the second base pair In reading the second purine, if the purine is located in the same strand as the first purine. If so, the same conditions apply. However, the second pudding is the opposite anti-parallel. parallel (click) strand, the opposite strand is anti-parallel to the third strand ), the purine nucleoside unit is in the anti-conformation There is a need. In all cases, the adenine in the third strand reads double stranded adenine The guanine in the third strand is used to read the guanine in the double strand. .

(ii) The third aspect of the invention is that the purine base on any of the double-stranded DNA Concerning the length of the third strand phosphorus backbone bond that allows for reading. on any mirror To enable "reading" (or base pairing) with the purine bases of the phosphorus backbone The distance between nucleoside units in the direction must be increased. Knot for phosphorus backbone We present examples of two types of extensions of the congruent form. One type of bonding type of the phosphorus main chain is elongate the entire bond on each nucleoside unit, i.e. all of the third strand This makes the extension connections the same. This type of overall bonding format is only for purine bases. Particularly suitable for third strands containing Therefore, the 5' carbon of "nucleoside unit 1" The length of the bond between the 3° oxygens of the next "nucleoside unit 2" is 2 atoms (-CH 2CH2- etc.) or by 3 atoms (0-CH2-CH2- etc.) Thus, the bonds between individual nucleoside units can be increased by 2 There will be six people longer than before. In order to maintain an appropriate distance between nucleoside units, the distance between units is It is recommended to increase the separation by a number of atoms in the range 1 to 6. Figure 6 shows this A nucleoside unit including a bond extension format and a proposed synthetic route are presented.

The second type of phosphorus backbone chain extension involves heterogeneous bond extension. Third strand The bonds with similar internucleoside distances in the standard phosphodiester backbone are , is adopted when the purines to be read are on the same mirror, while one nucleo Extension on the 3-carbon of a side unit and the 5°-carbon of the adjacent nucleoside unit An extended bond (length 15-17 people) that may include bonds is a plate located on the opposite mirror. Used to read phosphorus bases. Such heterogeneous bond extension forms are especially Tertiary containing both pyrimidine (or pyrimidine analog) and purine bases Suitable for use on chains.

(C) Formation of triple helix structure (1) Three-stranded (or triple-stranded) 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 IB.

Figure IA shows the third A duplex is shown with T as the strand base. Under these circumstances, the third strand T is Parallel to the A-containing strand of the heavy-strand DNA, the T-containing strand of the double-stranded DNA (which is also (anti-parallel) to the containing strands. Therefore, This double strand is expressed as follows.

Figure 1B shows the formation of hydrogen bonds, i.e., the G and salt of G-C base pairs in double-stranded DNA. Shows a duplex with protonated cytosine (C) as the group-pairing third strand base. vinegar. Under these circumstances, the cytosine base in the third strand provides the necessary hydrogen for a stable duplex. It must be protonated at N3 to form the bond. as desired , can be substituted with a cytosine analog with a quaternary nitrogen in the equivalent position to N3. can. In the illustrated duplex, the stannic C” (or its analogue) is , parallel to the G-containing strand of the duplex DNA, and parallel to the C-containing strand of the duplex DNA ( This is also anti-parallel to the G-containing chain). It will be done. This duplex sequence is therefore written as follows.

(b) purine in the third strand forming the duplex As another aspect of the present invention, the three lines are Purine bases in the third strand form hydrogen bonds, i.e., the same molecules in the single strand of double-stranded DNA It is formed when base pairs with a dipurine base. Therefore, A is paired with A, and G is 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 or parallel to the strand polarity of the purine-containing strand to be read in duplex DNA. can be arranged either anti-parallel.

For example, Figure 2A shows that the A of stannic is arranged parallel to the A of double-stranded DNA and Arranged anti-parallel to T of stranded DNA. Under these circumstances, the third strand A The glycodyl (C-N) torsion angle is the thin-conformation and double-stranded DNA The glycosyl torsion angles of A and T of are both anti-conformations . This three-line array is expressed as follows.

On the other hand, FIG. 2B shows that the A of stannic is anti-parallel to the A of the duplex DNA. It is arranged in parallel to the T of DNA. Under these circumstances, all three of these The glycosyl torsion angle of all bases is anti-conformation. this double The chain sequence is represented as follows: Figure 20 shows that G of stannic is connected to G of double-stranded DNA, and C of double-stranded DNA is connected to G of double-stranded DNA. Arranged in anti-parallel. Under these circumstances, the glycodyl screw of the third strand G The tilt angle assumes a syn-conformation, and the glycosyl angle of the G-C base of double-stranded DNA Both torsion angles are anti-conformational. This double-stranded sequence is It is shown as follows.

Figure 2D shows that the G of stannic is opposite to the G of double-stranded DNA. A and C are arranged in parallel. Under these circumstances, the glycation of all three bases The codyl torsion angle is the anti-conformation. This double-stranded sequence is as follows. Represented as: (2) Polypurine sequence If one strand of the selected double-stranded DNA sequence contains a polypurine sequence, Oligomers complementary to purine sequences are used, which are pyrimidines or purines, or a mixture thereof. Preferred oligomers are nucleic acids. is resistant to enzymes and produces double-stranded DNA and the triplet structure described above. Contains nonionic MP-oligomers that can be .

The binding of stannic to the polypurine sequence within one strand of a double-stranded DNA sequence results in a double strand. Examples of helical arrangements are shown schematically in Figures 3A-3C. In Figures 3A to 3C, 03 is a Represents rotonized C.

(3) Mixed array A mixed sequence is one in which the purine bases of the selected DNA sequence are present on both strands of the double-stranded DNA sequence. This is an array located in . Therefore, the stannic oligomer cannot hydrogen bond. That is, each base pair of a double-stranded DNA sequence must have two base pairs with a purine base. Purine bases are located regardless of which strand of heavy chain DNA. therefore , third-strand oligomers must be able to “read” across double-stranded DNA. No. Examples of mixed sequences containing complementary third strands are shown in Figures 4A-4E.

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

Figure 4B shows a mixed sequence with a purine-rich third strand.

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

The third strand oligomer traverses the duplex from one strand to the reflector to form a salt with a 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 phosphorus linkages between nucleosides.

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 ((CHz)n=) or alkylene oxy between the hydroxyl ((CHz)no'-) elongated chain, or with the 3°-carbon of the sugar moiety. It can be extended by intervening a similar linkage between the 3°-hydroxyls ( (See Figures 5 and 6). In suitable cases, such extended linkages can be added to the 3'- or and 5°-carbon.

If consecutive purines of a selected double-stranded DNA sequence occur on the same mirror, such There is no need to adopt a straight elongation chain. Nucleosides such as methylphosphonate linkages In interdomain phosphorus linkages, consecutive phosphorus linkages on one mirror of a selected double-stranded DNA sequence The base can be read with a suitable base on the third mirror.

Therefore, in suitable cases, by employing such elongated chains, the selected Oligomers that can be read with purine bases on both mirrors of a double-stranded DNA sequence can be created.

(D) Third strand oligomer Preferred oligomers are those having few (about 7 nucleosides); The length is usually determined by the length of the double-stranded DNA segment to specifically bind to the desired purine sequence. There are enough numbers to get it. More preferably, the particularly preferred are oligomers having from about 10 to about 25 nucleosides. It is an oligomer. Combines synthetic ease with specificity for selected sequences. internucleoside phases such as folding and coiling within the oligomer. Considering the minimization of interactions, oligomers having about 14 to about 18 nucleosides - is believed to include a particularly preferred group.

(1) Preferred oligomers These oligomers have either ribosyl or deoxyribosyl moieties. , or modifications thereof. However, their ease of synthesis and increased stability In order to combine deoxyribosyl moieties or modified deoxyribosyl moieties with Oligomers containing a double moiety are preferred.

Nucleotide oligomers (i.e. phosphorus present in natural nucleotide oligomers) acid diester internucleoside linkages, and other oligonucleotide analogs. can be used in this invention, but preferred oligomers include oligonucleotides. Side alkyl- and arylphosphonate analogs, phosphorothioate oligonucleotides Gonucleoside analogs, phosphoramidate analogs, and neutral phosphate esters Contains oligonucleotide analogs. However, it is particularly preferred that two nucleotides Phosphonate one or more phosphodiester chains linking the de units. Oligonucleoside alkyl- and arylphosphonates replaced in a chain It is an analogue. Such oligonucleoside alkyl- and arylphos The production of some Honat analogues and the inhibition of expression of preselected single terminal strand nucleic acid sequences. No. 446, incorporated herein by reference. No. 9863, No. 4511713, No. 4757055, No. 450743 No. 3, and No. 4591614. These phosphonates A particularly preferred group of analogs are methylphosphonate oligomers.

Preferred method for synthesizing methylphosphonate oligomers (rMP-oligomers) B, L., incorporated herein by reference.

Lee et al. [Biochemistry, Vol. 27, pp. 3197-3203 (1988)] , and p,s, Miller et al. [Biochemistry, vol. 25, 5092-5097 (1986)].

Preferred oligonucleoside alkyl- and arylphosphonate analogs are , at least one phosphodiester internucleoside linkage connects 3'-5' internucleoside methylphosphonyl (MP) groups (or “methylphosphoners”) It has been replaced by The methylphosphonate chain is an oligonucleotide It is isosteric to the phosphate group of cleotide. That is, these methylphos Sphonate oligomer CrMP-oligomer) is a reciprocal polymer with a selected DNA sequence. It should provide minimal steric constraints for action. Also, such an alkyl group or a The lyl groups do not sterically hinder the phosphonate chains or interact with each other Other alkyl- or arylphosphonate chains are likewise suitable. Methi Because phosphonyl groups are not found in naturally occurring 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 is used for cellular DNA and protein synthesis. It has been found that specific inhibitory effects can be exerted (for example, in US Pat. No. 44,698). (See No. 63).

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-oligomers with sid, particularly preferred between about 10 and 25 It has nuquosides. Ease of manufacture and specificity for selected sequences , and interactions between nucleosides such as folding and coiling within oligomers. MP-oligomers of about 14-18 nucleosides to combine minimization of effects. - are particularly preferred.

Particularly preferred is that the 5°-internucleoside linkage 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 DNA sequence was sequenced and an MP-origin complementary to the purine sequence was sequenced. The sesame is made by the method reported in the above-identified patent and herein.

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

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

If desired, labeling groups such as psoralen, chemiluminescent groups, cross-linking agents, acridine, etc. intercalating agents, or molecular scissors such as 0-fetentroline copper or EDTA-iron. MP-oligo has a group capable of cleaving the target protein of the viral nucleic acid. can be inserted into the market.

These oligomers can be labeled by any of several known methods. useful sign Examples include radioactive isotopes and non-radioactive reporter groups. Isotho Labels include 3H, 35S, 32p, 1251° cobalt, and 14C. . Many methods of isotope labeling involve the use of enzymes, including nick translation. These include known methods such as transcription, end labeling, secondary strand synthesis, and reverse transcription. radioactive label When using lobes, hybridization can be performed using autoradiographic methods. , scintillation counting, or gamma counting. To be elected Detection methods depend on the hybridization conditions and the individual radioisotopes used for labeling. It can change depending on the position.

Non-radioactive substances can also be used for labeling, such as modified nucleosides, or enzymatic insertion of nucleoside analogs or chemical modification of oligomers; For example, it can be introduced by the use of non-nucleotide linker groups. As a non-radioactive label Examples include fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, habutens, or the like. Examples include other ligands such as . A preferred method of labeling is acridinium ester This is the insertion of a file.

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 DNA sequences, these Advantageously derivatizing or modifying oligomers to insert DNA modifying groups causes a reaction with the DNA, irreversibly modifying its structure and rendering it non-functional. It can be made to stay. The inventors' co-pending publications are incorporated herein by reference. Pending U.S. Patent Application No. 924,234 (filed October 28, 1986) includes Oligomers containing oligonucleoside alkyl- and arylphosphonates derivatization of target single-stranded nucleic acid sequences, and oligos derivatized to make the target single-stranded nucleic acid sequence nonfunctional The use of nucleoside alkyl- and arylphosphonates is reported.

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

The DNA modifying group includes derivatized oligomers that combine double-stranded DNA segments and triple et al. After the formation of a helical structure, covalent bond formation, crosslinking, alkylation, cleavage, and decomposition occur. strain, otherwise inactivate or otherwise inactivate the DNA segment, or portion of its target sequence. causes disruption, thereby disabling the function and/or expression of the DNA segment. Included are groups that can be reversibly suppressed.

The position of the DNA modifying group in the oligomer can vary widely, depending on the particular It can vary depending on the DNA modifying group and the double-stranded DNA segment targeted.

Thus, the DNA modifying group may be located at the end of the oligomer or mediate the end. . May contain multiple DNA modifying groups.

In one preferred embodiment, a reaction occurs, i.e., a reaction occurs between the oligomer and the double-stranded DNA. For cross-linking, the DNA-modifying group is photoreactive (e.g., specific wavelengths of light, or range).

A typical example of a DNA modifying group that can cause a crosslinking reaction is aminomethyltrimethyl psorale. psoralens, such as AMT (AMT). AMT is advantageously photoreactive; Therefore, before cross-linking is carried out, it is activated by exposure to light of a specific wavelength. . Other bridging groups, which may or may not be photoreactive, can be added to these oligos. can be used to derivatize molecules.

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. Ru. Suitable alkylating agent groups are those known in the chemical art and include alkyl halides. , halogenated acetamides, phosphotriesters, and the like.

Examples of DNA modifying groups that can cause DNA segment cleavage include ethylenediamine. Examples include transition metal chelate complexes such as tetraacetic acid (EDTA), or its derivatives. It will be done. Other groups that can be used for cleavage include phenanthroline, porphyri or pleomycin. When using EDTA, it is convenient to open the iron. It binds to the oligomer and helps generate cleavage radicals. EDTA is preferred DNA-cleaving groups, but not azo compounds or other nitrogen-containing groups such as nitrenes or other transition metal chelate complexes may also be used.

A third strand oligomer that reads purine bases on both mirrors 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; eliminating the need to protonate cytosine (thus at neutral pH at the N-3 position) with hydrogen available for hydrogen bonding), or N-3 on the pyrimidine ring Using a cytosine analog with such an available hydrogen at the position corresponding to Several features, such as eliminating the need for It is believed to be convenient for a reason.

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. Third In the formation of the triple helix, the purine on the third mirror undergoes a syn-covalent process during the hydrogen bonding process. It can be presumed that the government will take reformation. If desired, Shin Kong Ho can be used even if it is normal. pudding can be modified to be a mation. For example, methyl, bromo , isopropyl, or other bulky groups at the 8-position of the purine. When modified, it can be assumed that the thin configuration is assumed in the normal state. That is, if placed like that Nucleoside units containing converted purines usually adopt a thin conformation. can be obtained. Therefore, if a purine base is shown to be in the thin conformation , in this invention, in place of unsubstituted A or G, such 8-position is optionally substituted. Consider insertion of substituted purines. Research using the model of the inventors (Kendrew) From our research, such substitutions should have no effect on the formation of the triple helix structure. It turns out that there is something.

Use of purine nucleoside units in anti and syn conformations , preferably (according to the rules for reading double-stranded DNA described herein) The purines on both mirrors of the double strand are read by the third strand of double stranded DNA and purine only. can be used to generate a triple helix structure.

Double-stranded DNA is a third-strand oligomer containing both pyrimidines and purines. When used to read the pudding on both mirrors, - non-regular chain form 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 third strand.

(2) Preferred purine oligomers This invention is based on the following formula (wherein Bp is a purine base, R does not sterically hinder the phosphonate chain, or R' groups independently selected from alkyl or aryl groups that do not interact with each other; is hydrogen, hydroxy, or methoxy, alk is alkylene of 2 to 6 carbon atoms , or alkylene of 2 to 6 carbon atoms) A new type of purine oligomer containing nucleoside units selected from

Preferred oligomers are those containing at least about 7 nucleoside units.

Preferred nucleoside units are those in which R is methyl. Also, R″ is hydrogen Certain nucleoside units are also preferred. The preferred base Bp is both optionally at position 8. including substituted adenine and guanine, with preferred substituents being methyl, bromo, Isopropyl etc.

Preferred alk groups are those having about 2 to about 3 carbon atoms.

Particularly preferred alk groups are those having 2 carbon atoms and include ethylene. .

(3) 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 disjoint bonds, thus forming Hoogsteen-type base pairs, or tribs. The nucleosomes do not require protonation, which is necessary for cytosine, for the generation of let. New oligomers containing side 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 (I), butoidisocytidine (II), 6-ami No-3-(β-D-monoribofuranosyl)pyrimidine-2,4-dione (III) , and 1-amino-1,2,4-(β-D-deoxyribofuranosyl)tri Azin-3-[4H]-one (IV) and the like. Their structures are shown in Table 5.

(E) Production of MP-oligomers (1) General discussion As previously mentioned, the preparation of methylphosphonate oligomers is described in U.S. Pat. No. 69863, No. 4507433, No. 4511713, No. 45916 No. 14, and No. 4757055.

Preferred methods of synthesis of methylphosphonate oligomers are described herein by reference. [Biochemistry, Vol. 28, 1054-106] 1 page (1989) Ko, B. L., Lee et al. [Biochemistry, Vol. 27, 319 7-3203 (1988)] and P. S. Miller et al. -, Vol. 25, pp. 5092-5097 (1986). extended series A nucleoside unit containing a modified sugar moiety with a chain (see Figures 5 and 6) The containing oligomers can be conveniently prepared by these methods.

Oligomers containing phosphodiester internucleoside phosphorus linkages are cyano 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) tyleneoxy) substituted sugar intermediate MP-oligomer and 5'-hydroxyl, or between 3'-carbon and 3'-hydroxyl. Modification in which one of the bonds is replaced with an alkyleneoxy group such as an ethyleneoxy group Can be produced using nucleosides.

Figure 5 shows a 3°-(ethyleneoxy) chain or a 5'-(ethyleneoxy) chain. The reaction scheme proposed for the production of modified nucleoside intermediates with either is shown.

In Figure 5, B is a base, Tr and R are protecting groups, and Tr is dimethoxytrityl. a protecting group such as R is t-butyldimethylsilyl, or tetrahydropyranyl. Indicates a protecting group such as

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

(b) Purine base on both mirrors of the 5'-β-hydroxyethyl substituted sugar intermediate When trying to read a double-stranded DNA sequence that has a slightly elongated nucleo Preference is given to using MP-oligomers with interside linkages on the phosphorus backbone.

Such MP-oligomers contain sugar (deoxyribosyl or ribosyl) moieties. modified and 5'-β-hydroxyethyl substituted nucleosides as shown in Figure 6. In the synthesis reaction scheme for the production of 5-hydroxy, β-hydroxyethyl (OH may be prepared using nucleosides substituted with CH2CH2) groups.

Figure 6 shows the reaction equation proposed for the 5'-β-hydroxyethyl substituted sugar analog. show. In Figure 6, DCC is dicyclohexylcarbodiimide and DMSO is dimethylcarbodiimide. Rusulfoxide, B represents a base. A preferred protecting group R is t-butyldimethylsilyl , and tetrahydropyranyl.

(C) Production of MP-ligomers having extended internucleoside linkages in the phosphorus main chain A modified nucleoside unit by replacing the modified nucleoside unit as described above. An MP-oligomer with inserted is produced.

In the production of oligomers containing only purine bases, the same elongated chain is used. Utilization of nucleoside units may be employed. However, pyrimidine (or pyrimidines) In the production of oligomers containing both bases and purine bases, elongation A mixture of unlinked nucleoside units and extended chains is used. sugar part nucleoside units with extended chains on both the 3′-carbon and 5′-carbon of Can be used as appropriate.

(3) Production of derivatized MP-oligomers The derivatized oligomers are produced as desired. It can be easily produced by adding a DNA modifying group to an oligomer. say it first The number of nucleoside units within the oligomer and the DNA within the oligomer as described The position of the modifying group(s) may vary. The DNA modifying group(s) is D It can be placed into an oligomer that can most effectively modify the target sequence of NA. Therefore Such optimal location can be readily determined by conventional techniques known in the art. However, the position of the DNA modifying group is often determined by the included DNA segment and It can vary depending on its primary target site(s).

(a,) Production of psoralen-derivatized MP-oligomers 8-methoxy psorale psoralen, such as The derivatization of MP-oligomers by , J.M., Keene et al. [Biochemistry, Vol. 27, pp. 9113-9121 (1 988)] and B. L. Lee et al. [Biochemistry, vol. 27, 3197 ~3203 (1988)].

(b) Production of EDTA-derivatized MP-oligomers Derivatization of oligomers is described in S, B, Phosphate, which is incorporated herein by reference. [Biochemistry, Vol. 28, pp. 1054-1061 (1989)]. It has been tell.

(F) Usefulness The specific segments of double-stranded DNA according to this invention are Read the purine bases of the double-stranded DNA double helix according to the guidelines for pret base pairing. The third strand of the MP-oligomer can be used to detect or recognize the MP-oligomer. MP-O The third strand of the oligomer is a compound in which the bases of each nucleoside unit are the corresponding salts of the double helix DNA. Generate base pairs and triplets and select sequences to obtain triple helical structures. have. Hybridization assays for detectably labeled oligomers For example, it can be used as a probe to detect the presence of a specific double-stranded DNA sequence. Can be detected.

This invention also provides an MP-origin that reads the DNA sequence and generates the triple helix structure. Use of gomers to prevent expression or function of selected sequences of double-stranded DNA provide a method. Generation of the triple helix prevents the opening of the double helix for transcription, By such aspects as preventing the binding of effector molecules (such as proteins), Expression and/or function may be prevented. Detection using derivatized oligomers target sites within the DNA double helix. larens), or by cleaving one or both chains (EDTA). Can be reversibly modified. By carefully selecting the target site for cleavage, Oligomers can be used as molecular scissors to specifically cut out DNA sequences.

Reacts with a DNA section or its target sequence, irreversibly modifies the DNA, and separates it. DNA reactive groups that can be cleaved or destroyed, thus irreversibly inhibiting their function. Alternatively, modifying groups can be inserted to derivatize the oligomer.

Therefore, these oligomers can be investigated in specific genes or in living cells. Irreversibly inactivates or suppresses the target sequence and causes selective inactivation or suppression. It can be used for straining. These oligomers are then deficient or preferred. Genes that produce undesirable products or have undesirable effects when activated Can be used to permanently inactivate, eliminate, or destroy a child.

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 DNA containing a purine MP-oligomer third strand labeled as A kit for detecting the A sequence is provided.

To further understand this invention, we conducted a computer simulation. Examples include reporting of the results of a series of experiments. Needless to say, these implementations The examples are not intended to specifically limit the invention. of the knowledge of a person skilled in the art Modifications of the invention that are now known or that may occur later are within the scope of this invention. It is considered to be included in the

Example Example 1 Computer simulation of triple helix structure These computer simulations The primary purpose of millation is to have a methylphosphonate ffM P J) backbone. Nonionic nucleoside analogues can be separated by Hoogsteen-type base pairing. As the third strand of heavy chain helical DNA, unmodified oligodeoxynucleotides (fOD This was to see if it binds with higher affinity compared to NJ.

Experimental results show that ODN binding to duplex DNA and translation inhibition may undergo duplex formation. (8), but there is a dichotomy between analogs with MP backbones versus intact ODNs. No experimental comparison of heavy chain DNA helix formation was made. Non-iodine with MP backbone In the past, it was unknown whether the analogues fit into the major groove of double-stranded helical DNA. It has not been done. In addition, fully elucidated proteins with native or MP backbones in the third chain The conformation of the double-stranded helical DNA was not determined. double or two Site-specific oligonucleotides that undergo heavy chain [)NA complex formation have recently been developed in vitro. It has been shown that Toro can suppress the development of human cancer. (8,9) Purpose of this example has an MP backbone in a third-strand helical complex using molecular dynamics simulations. We investigated nonionic oligonucleosides and elucidated the molecular mechanism involved in this process. This is to do so.

(G) Simulation method Double stranded poly(dT+o)-poly(dA+o)-poly(dT+o)-[TIAT2 The co-ligand is from the A-DNA X-ray structure of Arnott and Selsing (1o) Obtained. The same coordination is poly(dT,.)-poly(dA,.)-poly(dT+o). It was used as the starting geometry for methylphosphonate [TIATIMP]. dimethyl Geometry optimization and partial atomic charge assignment for phosphonate fragments are From the beginning, Q using 3-21G* and 5TOG* pace sets, respectively. A quantum mechanical method according to UEST (version 1°1) was used. (11) The latter The basis set is the one previously calculated for nucleic acids in the AMBER database. It has been used to support uniform charge assignment with About MP fragment From the final monopolar atomic charge assignment, each base in the third DNA strand, furanose and Neutral total charges for and MPfM were obtained. On the other hand, the main chain phosphor of the T2MP chain Rp and Sp methyl substitutions of lyle oxygens were performed with stereoscopic computer images. If Since the composition cannot be controlled, substitution of MP diastereomers approximately approximates the experimental yield. I went by method. AM using total atomic force field for molecular mechanics and molecular dynamics calculations A fully vectorized version of BER (version 3.1) was used. (1 2,13) All calculations were performed using CRAYX-MP/24 and VAX8600 computers. I went with pewter.

The negative charge on the DNA phosphate backbone is due to the phosphorus atom that divides the phosphate oxygen into three halves. The positive counterion configuration within 4A aids in neutralization. Opposing ion is MP - Not placed on the displacement mirror. Triple helix and opposing ions are periodic boundary conditions was surrounded by an IOA shell of TIP3P water (14) molecules. T, There are 9283 and 10834 atoms in the Ar1 and TIAT2MP systems, respectively. Exists. Box size is T, 101,686.8A3 per Ar1, T , 124,321.1 As per AT2MP.

Initially, the DNA and opposing ion atoms are well constrained, but the surrounding water molecules are 8. to the value (effective value of slope [rms], <0.1 kg force 1 mol/A). O Energy was minimized using an A-free cutoff. D.N. A, opposing ions and water are then minimized by SHAKE. Supplementary 1500 cycles then 220 cycles without geometric constraints energy was minimized (15). Steady temperature and pressure (at 300 and 1 bar) Molecular dynamics using 5HAKE can be applied to each of two molecules without constraints under It followed the orbit for 40 psec.

(F) 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 bond that was created.

The average hydrogen bond distance and average atomic fluctuation are T r A was consistently smaller in 72MP (Table 1). The mirror-opening phosphorus atomic distance is 9.6A (+/-0,91) for A-72 and 8,3 (+/-0,91) for A-72MP +/-0,58). Reduced mirror-opening phosphorus atomic distance and smaller second The average atomic fluctuation between the It is.

Both triple helical DNA atoms exhibit strand-specific polymorphic conformational features during molecular dynamics. did. Significant conformations in the furanose relative to the starting geometries of both systems (Table 2). T-AT! In the helix, T and T2 strands Furanose ring occupancy is mostly in the A-DNA conformation (C3 end) The largest portion of adenine sugar remains in the B-DNA conformation (C2 end ).

A significant percentage of adenine furanose conformations are T, AT2 and T, 01' endo conformation in AT2MP. MP-substituted helical sugar patch What is an unsubstituted helix in a culling pattern? On the contrary, it has a 01° end and a C2' end conformation. other conjo Analysis of the formation parameters reveals the mixed structural properties of these triple helices. To support. The helical torsion angle (T1 and A chains) averages 39 for the T, AT2 structure. ．． 4 degrees (+\-2,86), and the B-DNA conformation (range 36-4 5) better match. T, AT 2MP helix twist angle gle) is 32°0 degrees (10\-2,19) on average, and the A-DNA conformation is (range 30-32.7). Average repeat unit for all structures ( between T1 and A chain) is 10.2 TIAT! and T, 11°2 per AT2MP degree. Average mirror-opening phosphorus atom distance of 4Qpsec trajectory are shown in Table 3. In both helices, the optical distance of the T1 strand is the same as that of the A-DNA core. conformation (7, OA). The optical distance of the T2MP chain is B -better DNA conformation (matches, and similar to A-DNA for T2 strand) is significantly different from the matching value.

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 4.6 A (10\-0,9) A for T, AT, and MP helices. T, The increase in average coordination distance and atomic motion (0,8A) in ATz is due to the increase in the second chain Rp (possibly due to the proximity of the axially protruding methyl group) to the coordinated counterion. It's large. The average number of water molecules coordinated to the phosphanate group is different from that in the MP-substituted helix. There are no significant changes.

Table 4 shows the DNA main chain and C1°-N (base) dihedral changes of two types of helical systems. . Comparison of the α-diface of the adenine chain shows the dihedral average position with MP substitution. There is a slight change in the position, and the two surfaces become closer to the transformer, but the There is a large overlap during the transient motion of both biplanes during the ec trajectory. Average Sp-M There was a significant change in the β dihedral angle from the baseline in the P diastereomer. S The fluctuation of the β diplane containing the p-MP diastereomer is significantly less than that of Rp-MP. (I) 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 poly(dT)poly(dA)po1badT ), the second strand forms a triple helix complex that is more stable than the original ODN. suggest that something will happen. Enhanced binding of MP chains is achieved by reducing mirror-opening electrostatic repulsion. It is something that The MP-substituted helix reduces the hydrogen bond distance and opens the mirror-opened A-T2 phosphorus. The distance is reduced, and there is less fluctuation due to the atomic position relative to the original triple helix.

Closer fitting and reduced atomic motion during molecular dynamics result in MP-substituted triplex quantitatively consistent with the larger enthalpy of binding and stability of the complex. these The findings show that MP-substitution in the second strand reduces mirror-opening phosphonate repulsion, resulting in more intimate contact. Facilitates the formation of triple helical structures and secondarily Hoogsteen and Watson This indicates close proximity to click hydrogen bond interactions. Fugus Although we can expect a significant effect on teen pairing, these calculations involve MP replacement. There is an unexpected enhancement of the Watson-Crick hydrogen bond.

The latter finding is due to electrostatic repulsion between the T1 and T2MP chains and interference (due to the second chain). This is most likely due to the reduction of heat.

The conformation of these DNA structures is based on the fibril shape (different from experimental data). Ru. The structure of poly(dT) poly(dA)-poly(dT) is Determined by line-diffraction measurements7, this is a loosely resolved structure. on 5structure). (10) Molecular dynamics simulation Has a fully analyzed DNA structure under periodic boundary conditions with opposing ions . In general, B-type DNA is predominant in solution, and under low humidity, A-type DNA is dominant. homeation is believed to be predominant (16). Several double strands of DNA The A-DNA conformation was determined by X-ray diffraction, and the A-DNA conformation was determined under low humidity conditions. It was uniformly observed that ion and salt concentration increased (10, 17, 18).

These computer simulations show that different DNA conformations coexist within a triple helix, with individual strands of the helix having dominant conformational occupancy. However, the Hoogsteen pairing/MP-substituted chain predicts that the B-type is predominant. . The majority of the 01' endosugars in both triplets are in this furanose conformation. 02' endo and C3, 0.6 from endo DNA Sugar Packers (16) I'm interested in it because it's 1 mole of kilocalories higher. DNA-dodecamer crystal structure (1 9) has a remarkable OL' end occupancy, and the molecular dynamics of dsDNA by Siebel et al. In the mechanical simulation, significant endosugar puckers were observed. (2 0) Both helical structures are commonly associated with purine nuclei that adopt a C2' end geometry. This follows the classic observation of pyrimidines adopting an octide and C3' end geometry.

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. S The p-MP group is very close to the thymine methyl group (in the natural groove) on the same DNA mirror. and interact with each other according to van der Waalska, which means that Partially destabilizes the helix by interlocking thymine to the Sp-MP main chain can be converted into There is a large deflection in the β-biface of the Rp-MP group. Because these The group protrudes into the solvent water and is located far away from the thymine/methyl group. This is because that. Between the orientation of methyl substituents on phosphorus atoms and DNA duplex stability It is known that there is a correlation, and the stability of Sp-MP diastereomer It has been proposed that the mechanism of decrease is due to local steric interactions (2 1).

The inventor's calculations show that steric interactions are highly sensitive to local helical destabilization. There is a slight contribution, and the likely mechanism is that the methyl group and chain of the Sp-MP backbone on the same mirror This is transmitted by non-bonding interactions between mins, which locally destabilizes the DNA. suggests that it is.

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) a E”=9.2X 10’M-’cm-’n: d(AGAGAGAGAGAGAG GAG) abbreviation d(AG)s E”-1,45X IQ’M-’am-’m: d(CI)TI)CpTI )CpTpCpTpCpTpCpTpCpTpCpTp) Abbreviation d(CpT p)++ E”=8.0X10’M-’cm-’ (a) 2:1d (CT) s・d (AG) a and (b) 1:1:ld (CT) Double-stranded helical structure formed by g・d(AG)a・d(CpTp)s Chromaticity (CD) spectra are calculated using the CD fraction in 0.1M phosphate buffer at the specified pH. Measured using an optical polarimeter.

Figure 7 shows (a) [2:1d(CT)a”d(AG)s()ko and (b) [1:　 1: 1 Example 3 Cross-linking of triple helical structures using psoralen-derivatized MP-oligomers Psoralen derivatized dTp(T)a oligomer beam, B, L, et al. Biochemis It was produced in the same manner as described in Tory 27+3197-3203 (1988).

T7 oligomer is 15-mer poly A sequence: 5' 10 20 30 40 3 ” Cor 51 4'-(aminomethyl)aminomethyl-4,5',8-1-lianethyl psorale Ambi(ae)AMT"], 4'-(aminobutyl)aminomethyl-4°5°, 8 -trimethylpsoralen [“(ab)AMT” and 4°-(aminohexyl) Aminomethyl-4,5',8-trimethylpsoralen [″(ab)　A　MT”] MP-oligomers induced using (a) single-stranded DNA of the above DNA sequence or and (b) piperidized with double-stranded DNA having the above sequence at 4°C, and as described in Lee et al. The results of crosslinking caused by irradiation in the same manner as above are shown in FIGS. 8A and 8B. Figure 8 is , crosslinking of psoralen-derivatized T7 oligomers with single-stranded (polyA-containing) DNA sequences. Showing a bridge.

Figure 8B shows cross-linking of double-stranded DNA and double-stranded DNA sequences.

Table 1 Average hydrogen bond distance (RMS) Watson-Crick Without MP With MP ADEHN6B-THYO42,33 (+/-0,31) 1.98 (+/-0,15)＾DENI-THYH32 ,10(+/-0,17) 1.95(+/-0,13) Hoogsteen ADEHN6A-THYO42,12(+/-0,22) 2.09(+/-0 ,19) ADEN7 -THYH31,94(+/-0,16) 1.92(+ /-0,12) T, AT, and T, AT2MP helix average Watson-Cli Chock and Hoogsteen hydrogen bond distances (Angstroms). these distances was calculated for a triple helix DNA complex. Oscillation due to atomic position (effective value [rm s]) is (person).

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) T2 6.3 (±10.29) 6.8 (±10 ．． 26) T, AT! and T, intrastrand phosphate distance of the AT2MP helix. 4Q Calculated intrachain phosphate distance averaged over psec orbitals (Angstro ) is shown for a complete triple helix system. The standard mirror-opening phosphorus atomic distance is Ki6. 7. for OA and B-DNA. It is OA (15).

Table 4 Without MP With MP α03’-P-05°-C5° 71 280, 5 (18, 5) 288.4 (11, 3) A 252.9 (28 ,7) 233.2 (28,5) 72 285,1 (11,6) 28g, 5 (11,3)βP-05'-C5°-04' 71 161, 2 (11, 5) 168.7 (8, 7) A 151.5 (10 ,7) 159.3 (2t5) T2 140.8 (8,8) FOR158, 8 (21,9) POS 167, 8 (8,7) YO5'-C5'-C4°-C3 7170, 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°-〇3' 71 90, 3 (12, 8) 82.5 (11, 1) A 112.5 (16, 7 ) 111.6 (17,2) 72 88,6 (10,4) 104.5 (16, 5) εC4°-C3'-03°-P 71 196, 4 (10, 1) 199.1 (10, 6) A 200.1 (10 ,9) 198.0 (13,3) T 2 197.5 (10,5) 187.9 (7,8)C3'-03'-P-05' T 1 290.8 (10,6) 290.6 (9,6) A 82.9 (12 , 0) 282.0 (18,5) T 2 285.0 (10,5) 280.5 (11,4) A D H215,3 (14,7) 212.1 (14,2) YR 71212, 3 (11,0) 205.9 (10,0) T 2 206.3 (1 0,7) Triple helix DNA structure between 213.9 (13,2) 40 psec orbitals Average main chain dihedral angle (rms) Table 5 Cytidine analog structure References (manufacturing) Godard, A. J., et al. Tetrahedron Letters, Doposusausuke, B., et al. Page 4234 (1980) Parchenal, J. H., et al. Cancer Research Volume 36 Page 1520 (1976) literature 1. Moscou, H.E., et al., Science Vol. 238, pp. 645-650 (19 1987) 2. Bobitz, T. J., et al., Journal on American Chemical Sosa. Yeti Vol. 111, pp. 3059-3061 (1989) 3. Wells, R.D., et al., FASEB Journal Vol. 2, No. 2939-29 49 pages (1988) 4, Durban, P1 Science Vol. 232, pp. 464-471 (1988) 5. Miller, p, s, et al. Anti Cancer Drug Design 2nd Shungiku pp. 117-128 (1987) 6, Yachong et al., “AIDS Treatment”, Scien. Tiffic American pp. 110-119 (October 1988) 7, Sari Proceedings on Natural Academy on Science IS (USA> Vol. 85, pp. 7448-7451 (1988) 8, Cooney, M. et al., Science Vol. 241, pp. 456-459 (1988) ) 9. Wickstrom, E. et al., Prosweeding on Natural Aca. Demy on Sciences (USA) Vol. 85, pp. 1028-1032 (1) 988) 10, Arnoff, S., et al., Journal of Molecule Bio. 88, pp. 509-521 (1974) 11, Schimpf, U.C., et al. , Journal on Combatational Chemistry Volume 5, No. 129-1 p. 45 (1984) 12, Schimpf, U.C., et al., AMBER (UC8F) - John 3゜1 (1988) 13. Uniinar, S. J., et al., Journal on Combatational Studies. Mistry Vol. 7, pp. 230-252 (1986) 14. Jorgenson, W. J., et al., Journal on Chemical Physics. 79, pp. 926-935 (1983) 15, Blendsen, H.J. C, et al., Journal on Chemical Physics, Vol. 81, No. 3684-36. 90 pages (1984) 16, Sanger, W9, Principal on Nucreek. ・Acid Strafture (Sbringer Bell Rag, New York, 198 4 years) 17, Arnott, S. et al., Nature, Vol. 244, pp. 99-101 (197 3 years) 18, Arnott, S. et al., Science Vol. 181, pp. 68-69 (1973) ) 19. Toru, H.R. et al. Prosweeding on Natural Aca Demy on Sciences (U.S.A.) Vol. 78, pp. 2179-2183 (1981) 20, Sabel, G. L., et al., Prosweeding on Na. Natural Academy on Sciences (USA) Volume 82 6537 -6540 pages (1985) 21, Bower 1M, et al. Nucleitsk Acid Research Vol. 1 No. 4915 -4930 pages (1987) 22, Donohini, J. et al., Journal on Molecular Biology Vol. 2, p. 363 (1960) 22a, Ts’o ,　P,O,P,,Basic Principal in Nucleitsk Acid ・Chemistry pp. 453-584 (P, O, P, Ts'o, Academic Press (Res, New York, 1974) 23, Sandaralingam 9M1, Piopolymers Vol. 7, p. 821 (1969 Year) 24. Arnott, S., et al. Progress in Nucleitsk Acid Lisa Molecule Biology Vol. 10, p. 183 (1970) 25, Sashisekharan, ■, et al., Conformation on biopolymer, PE. Per International Symposium 1967, Volume 2, Page 641 (19 67) 26, Lakshminarayanan, A. V., et al., Biochemical Biof. Physical Acta Volume 204, Page 49 (1970) 27° Lakshminarayana N, A, V, et al., Pionopolymers Kan, Vol. 8, pp. 475 and 489 (1970 ) 28, Lee, B. L., et al., Nucleitsk Acid Research Vol. 16, No. pp. 10681-10697 (1988) 29, Barone, A. D., et al., Nucle. Itsuku Acid Research Vol. 12, pp. 4051-4060 (1984) Special Omotehira 4-506152 (24) C'G Oe Gri 9G◎ /] Kuf / Lid l- Crosslinking kinetics Time (minutes) international search report

Claims (44)

[Claims] 1. Test specific sections of double-stranded DNA without interfering with double-stranded DNA base pairing. A method for detecting or recognizing the DNA fragment, the method comprising: olefins having at least approximately 7 nucleosidyl units that are fully complementary in part A method characterized by contacting with a oligomer to form a triple helical structure. 2. 2. The oligomer according to claim 1, wherein the oligomer is a methylphosphonate oligomer. Method. 3. 3. The method according to claim 2, wherein the oligomer contains only purine bases. 4. oligomeric guanine or substituted guanine to one of the strands of double-stranded DNA The adenine or substituted adenine is used to read the guanine in the double-stranded DNA. The method according to claim 3, which is used for reading adenine in one of the chains. Law. 5. Oligomeric guanine nucleosidyl units in syn-conformation or Substituted guanine nucleosidyl units are substituted for guanine in parallel strands in double-stranded DNA. Anti-conformational oligomeric guanine nucleotides used for reading Dyl unit or substituted guanine nucleosidyl unit to the antiparallel of double-stranded DNA. used for reading guanine in the chain; and Sesame adenine nucleosidyl unit or substituted adenine nucleosidyl unit Used to read adenine in parallel strands of double-stranded DNA, anti-conformity adenine nucleosidyl units or substituted adenine nucleosidyl units in the oligomers of the Osidyl units are used to read adenine in the antiparallel strand of double-stranded DNA. 5. The method according to claim 4. 6. The above oligomer has the formula: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ [wherein B contains a purine or pyrimidine base and alk has 2-6 carbon atoms] alkylene or alkyleneoxy of 2-6 carbon atoms] The claimant contains a modified nucleosidyl unit having a silibosyl analog moiety. The method described in claim 2. 7. 7. The method according to claim 6, wherein the oligomer contains only purine bases. 8. A claim that the oligomer contains a purine base and a pyrimidine base. The method described in Section 2. 9. In the above oligomer, a cytosine analog replaces cytosine, and the above cytosine analogue is substituted for cytosine. ・The analog has water available for hydrogen bonding at the position on the ring corresponding to N-3 of cytosine. and is capable of forming two hydrogen bonds with guanine bases at neutral pH. Claim 8, which has a nucleosidyl unit containing a certain 6-membered heterocyclic ring. the method of. 10. The above cytosine analog is 5,6-dihydro-5-azacytosine, pseudo- Doisocytosine; 6-amino-3-pyrimidine-24-dione; or 1-amino 10. The method of claim 9, wherein the compound is not-1,2,4-triazin-3[4H]-one. 11. The above oligomers are as follows: ▲There are mathematical formulas, chemical formulas, tables, etc.▼,▲There are mathematical formulas, chemical formulas, tables, etc.▼,▲Numbers There are formulas, chemical formulas, tables, etc.▼and▲There are mathematical formulas, chemical formulas, tables, etc.▼ , alk is alkylene of 2-6 carbon atoms, or alkylene of 2-6 carbon atoms and R' is hydrogen, hydroxy or methoxy] 10. The method according to claim 2, 3, 8 or 9, which comprises a modified sugar moiety. 12. Characteristics of duplex DNA with a given sequence of each duplex in a complementary DNA duplex A method for inhibiting the expression or function of a DNA fragment, the method comprising: inhibiting the expression or function of a DNA fragment; at least approximately 7 nucleosides that are sufficiently complementary to the NA section or portion thereof. It is characterized by forming a triple helical structure when brought into contact with an oligomer having dill units. How to make it a sign. 13. The method according to claim 12, wherein the oligomer contains only purine bases. Law. 14. Oligomeric guanine or substituted guanine in one of the strands of a DNA duplex Used to read guanine in the book, adenine or substituted adenine is 14. The method according to claim 13, which is used for reading adenine in one of the chains of the method of. 15. The guanine nucleosidyl unit of the oligomer in the syn-conformation or replaces the substituted guanine nucleosidyl unit with the guanine in the parallel strand in the DNA duplex. used to read oligomeric guanine nucleotides with anti-conformation. cidyl units or substituted guanine nucleosidyl units to the antiparallel of the DNA duplex. used to read guanine in the parallel chain; and for syn-conformational orientation. Adenine nucleosidyl units or substituted adenine nucleosidyl units of oligomers is used to read adenine in parallel strands in DNA duplexes, and Adenine nucleosidyl units or substituted adenine nucleosidyl units in oligomers of mation The leosidyl unit is used to read adenine in the antiparallel strand of a DNA duplex. 15. The method of claim 14. 16. In the above oligomer, a cytosine analog replaces cytosine, and the above cytosine analog is substituted for cytosine. analogues are available for hydrogen bonding at the ring position corresponding to N-3 of cytosine. Contains hydrogen and can form two hydrogen bonds with guanine bases at neutral pH Claim 12 has a nucleosidyl unit containing a 6-membered heterocyclic ring. Method described. 17. The above cytosine analog is 5,6-dihydro-5-azacytosine, pseudo- Doisocytosine; 6-amino-3-pyrimidine-2,4-dione; or 1-a The method according to claim 16, which is mino-1,2,4-triazin-3[4H]-one. Law. 18. The above nucleosidyl unit is a phosphodiester group or an alkyl or alkyl group. The method according to claim 12, which is linked by a lylphosphonate group. Law. 19. The above nucleosidyl units are shown below: ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, alk is alkylene having 2 to 6 carbon atoms, or alkyl having 2 to 6 carbon atoms. and R' is hydrogen, hydroxy or methoxy]. 19. The method of claim 18, wherein the method comprises a modified sugar moiety that is 20. The oligomer is modified to cause a reaction with the DNA, and the oligomer is modified to cause a reaction with the DNA. Claim 12, which contains a DNA modifying group capable of irreversibly modifying the structure of How to put it on. 21. the oligomer reacts with the DNA segment or a target sequence therein; may irreversibly modify said DNA, thereby inhibiting the expression of said DNA segment. or contains a DNA modification group that irreversibly inhibits the function. How to put it on. 22. The above modification may also result in covalent binding of the oligomer to a single strand of DNA. 22. The method of claim 21, wherein said step comprises crosslinking said DNA. 23. 22. The method of claim 21, wherein said modification is to cleave said DNA. 24. The above-mentioned DNA section contains the gene in living cells and contains the triple-helix structure. homeation permanently inhibits or inactivates the above gene, 22. The method according to claim 21. 25. Read the selected double-stranded DNA sequence and the purine sequence of the double-stranded DNA above. an alkyl or aryl phosphatide that is sufficiently complementary to form a triple strand. Triple helical structure containing phonate oligomers. 26. Oligomeric guanine or substituted guanine in one of the strands of double-stranded DNA Used to read guanine in this book, adenine or substituted adenine is converted into double-stranded DNA. according to claim 25, which is used for reading adenine in one of the chains of structure. 27. The guanine nucleosidyl unit of the oligomer in the syn-conformation or replaces the substituted guanine nucleosidyl unit with the guanine in the parallel strand in double-stranded DNA. used to read oligomeric guanine nucleotides with anti-conformation. cidyl units or substituted guanine nucleosidyl units to the antiparallel of double-stranded DNA. used to read guanine in the parallel chain; and for syn-conformational orientation. Adenine nucleosidyl units or substituted adenine nucleosidyl units of oligomers is used to read adenine in parallel strands of double-stranded DNA, and Adenine nucleosidyl units or substituted adenine nucleosidyl units in oligomers of mation The leosidyl unit is used to read adenine in the antiparallel strand of double-stranded DNA. 27. The structure of claim 26, wherein the structure comprises: 28. the below described: ▲There are mathematical formulas, chemical formulas, tables, etc.▼, ▲There are mathematical formulas, chemical formulas, tables, etc.▼, an d▲There are mathematical formulas, chemical formulas, tables, etc.▼[In the formula, Bp is a purine base; R is Alkyl or are independently selected from the aryl groups; R' is hydrogen, hydroxy or methoxy; Yes; alk is alkylene with 2-6 carbon atoms or alkyl with 2-6 carbon atoms at least approximately 7 interconnected elements selected from Oligomers containing nucleosidyl units. 29. 29. The oligomer of claim 28, comprising a methylphosphonate oligomer. 30. Cytosine is used for hydrogen bonding at the position on the ring corresponding to N-3 of the pyrimidine ring. formation of two hydrogen bonds with the guanine base at neutral pH. The nucleosidyl unit is replaced by a heterocyclic cytosine analog that is capable of An oligomer containing purine bases in a double-stranded DNA sequence that can be read. 31. The above nucleosidyl unit is 2'-deoxy-5,6-dihydro-5-aza -deoxycytidine; pseudocytidine; 6-amino-3-(β-D-riboflura nosyl)-pyrimidine-2,4-dione and 1-amino-1,2,4-(β- D-deoxyfuranosyl)triazin-3[4H]-one 31. The oligomer of claim 30. 32. 32. The oil according to claim 31, which comprises a methylphosphonate oligomer. Ligomer. 33. At least one nucleosidyl unit is ▼、▲There are mathematical formulas, chemical formulas, tables, etc.▼and▲There are mathematical formulas, chemical formulas, tables, etc. ▼ [In the formula, B is a base; R' is hydrogen, hydroxy, or methoxy. Yes: alk is independently alkylene of 2-6 carbon atoms or 2-6 carbon atoms alkyleneoxy of 2 to 6 carbon atoms, and alk' is an alkyleneoxy of 2 to 6 carbon atoms. at least one selected from of purine bases in a piece of double-stranded DNA, containing approximately 7 nucleosidyl units. Oligomers that can be read. 34. 34. The oligomer according to claim 33, which contains only purine bases. 35. Oligomeric guanine or substituted guanine in one of the strands of double-stranded DNA Used to read guanine in this book, adenine or substituted adenine is converted into double-stranded DNA. 35. The method according to claim 34, which is used for reading adenine in one of the chains of oligomers. 36. The guanine nucleosidyl unit of the oligomer in the syn-conformation or replaces the substituted guanine nucleosidyl unit with the guanine in the parallel strand in double-stranded DNA. used to read oligomeric guanine nucleotides with anti-conformation. cidyl units or substituted guanine nucleosidyl units to the antiparallel of double-stranded DNA. used to read guanine in the parallel chain; and for syn-conformational orientation. Adenine nucleosidyl units or substituted adenine nucleosidyl units of oligomers is used to read adenine in parallel strands of double-stranded DNA, and Adenine nucleosidyl units or substituted adenine nucleosidyl units in oligomers of mation The leosidyl unit is used to read adenine in the antiparallel strand of double-stranded DNA. 36. The oligomer of claim 35. 37. The above internucleoside phosphorus bond is an alkyl or aryl phosphonate. Claims that are internucleoside bonds or phosphodiester internucleoside bonds The oligomer according to item 33. 38. 38. The oligomer according to claim 37, which contains only purine bases. 39. Oligomeric guanine or substituted guanine in one of the strands of double-stranded DNA Used to read guanine in this book, adenine or substituted adenine is converted into double-stranded DNA. according to claim 38, which is used for reading adenine in one of the chains of oligomers. 40. The guanine nucleosidyl unit of the oligomer in the syn-conformation or replaces the substituted guanine nucleosidyl unit with the guanine in the parallel strand in double-stranded DNA. used to read oligomeric guanine nucleotides with anti-conformation. cidyl units or substituted guanine nucleosidyl units to the antiparallel of double-stranded DNA. used to read guanine in the parallel chain; and for syn-conformational orientation. Adenine nucleosidyl units or substituted adenine nucleosidyl units of oligomers is used to read adenine in parallel strands of double-stranded DNA, and Adenine nucleosidyl units or substituted adenine nucleosidyl units in oligomers of mation The leosidyl unit is used to read adenine in the antiparallel strand of double-stranded DNA. 40. The oligomer according to claim 39, which is 41. the below described: ▲Contains mathematical formulas, chemical formulas, tables, etc.▼ 41. The oligomer of claim 40, comprising nucleosidyl units of. 42. 42. The oligomer of claim 41, wherein R' is hydrogen. 43. 42. The oligo according to claim 41, wherein alk is ethylene or ethyleneoxy. Ma. 44. 44. The oligomer of claim 43, wherein alk is ethylene.