KR100437193B1 - A molecule to recognize a mismatch base pair - Google Patents

Abstract

The present invention provides a method for detecting mismatches of base pairs in nucleic acids such as DNA and RNA easily and with high sensitivity, and a detection reagent therefor.

The present invention relates to the following general formula (I) in a base pair that cannot form a normal base pair,

A-L-B (I)

(Wherein A is a chemical moiety capable of pairing with one base of a base pair that cannot form a normal base pair, and B is the other side of the pair of base that cannot form a normal base pair) Chemical mismatch moieties represented by chemical structural moieties capable of pairing with bases, L denotes a linker structure combining chemical structural moieties A and B). A method of detecting and identifying a pair of bases that cannot form a normal base pair consisting of pseudobase pair formation on the pair by measuring the formation of the pseudo base pair, kits therefor, and detection of mismatches. The present invention relates to a method for detecting abnormalities in nucleotide sequences of reagents and genes.

Description

A molecule to recognize a mismatch base pair}

In the present invention, a base pair is formed by pseudo-base pair formation on a base pair that cannot form a normal base pair, and the formation of the pseudo base pair is measured. A method for detecting and identifying a pair of bases that cannot form a base pair of the same, a reagent therefor, a kit containing the same, a compound thereof, and a method for detecting abnormalities in the base sequence of DNA or RNA using the method will be.

When nucleic acids such as DNA and RNA are hybridized into two chains, paired bases are determined. For example, guanine (G) has cytosine (C), and adenine (A) has thymine (T). In general, all bases form such a pair and hybridize, but sometimes a pair cannot be formed in a part of the base sequence.

For example, if some DNA and other DNA are placed under conditions that can hybridize, most bases can form these pairs, but one or several portions of bases cannot form such pairs. There is a case. Base pairs which cannot form such ordinary base pairs are hereinafter referred to as mismatches in the present specification.

On the other hand, research on various genetic diseases caused by different one or two or more bases has recently been conducted. For example, there are genetic diseases in which one base has a gene different from the usual one (Single Nucleotide Polymorphism (SNP)), and elucidation of such genetic diseases has attracted attention. If these genes are hybridized with normal genes, most bases can form normal base pairs, but hybridize, but mismatches occur with respect to one base pair.

Currently, a method of detecting such mismatches is a method of comparing hybridization efficiencies of two stranded DNAs. However, in order to use this method, since the base sequence of the DNA containing mismatch must be known in advance, enormous effort is required, and it is not suitable as a method for processing a large number of samples. There is also a method of selectively binding a repair protein of DNA such as MutS to a gene damage site, but it is difficult to maintain the activity of the protein.

As described above, a method of detecting some mismatches in hybridized DNA and the like is extremely difficult and its sensitivity is insufficient, and there is a demand for the establishment of a method capable of detecting this easily and with high sensitivity.

By the way, the present inventors have developed a bulge DNA recognition molecule that binds specifically to and stabilizes DNA (bulge DNA) having an auxiliary base (bulge base) generated in two-stranded DNA (Japanese Patent Application No. 11). -262205). This bulge recognition molecule not only hydrogen bonds with ancillary bases, but also intercars using stacking interactions between aromatic rings and bases near bulges in the space created by the presence of bulged bases. It is stabilized by intercalation.

The inventors of the present invention have further studied the action of the subbase using the stacking effect of the presence of the surrounding base, and thus, even in a place where a mismatch of a base pair occurs, a molecular species capable of pairing with a base It was found that the compound having two of them can be relatively stable by this stacking effect.

1 is a photographic diagram showing the inhibitory effect of the cleavage by DNaseI of the mismatch region by the mismatch recognition molecule of the present invention.

Figure 2 is a graph showing the inhibitory effect of the cleavage by DNaseI when the mismatch recognition molecule of the present invention.

Figure 3 schematically shows the action of the mismatch recognition molecule in the mismatch portion of the present invention.

The present invention provides a method that can detect mismatches of such base pairs simply and even more sensitively.

More specifically, the present invention provides a method capable of easily and easily detecting mismatches existing in two-stranded DNA chains with high sensitivity, and a detection reagent therefor.

The present invention relates to the following general formula (I) for a base pair that cannot form a normal base pair,

A-L-B (I)

(Wherein A is a chemical structure moiety capable of pairing with one base of a pair of bases that cannot form a normal base pair, and B is another base of a pair of bases that cannot form a normal base pair) A chemical structural moiety capable of pairing with (L) represents a linker structure that combines chemical structural moieties A and B). By using a compound having B and a linker moiety L which binds the chemical structural moieties A and B, a base pair is pseudo-formed on a base pair that cannot form the normal base pair, thereby forming the pseudo base pair. The present invention relates to a method for detecting and identifying a pair of bases that cannot form a normal base pair consisting of measuring.

In addition, the present invention provides a pair of bases that cannot form a normal base pair consisting of pseudo-base pairs formed on a pair of bases that cannot form a normal base pair described above, and measuring formation of the pseudo base pair. In the detection and identification method, the following general formula (I) for forming a pseudo base pair in a base pair that cannot form a normal base pair,

A-L-B (I)

(Wherein A is a chemical moiety capable of pairing with one base of a base pair that cannot form a normal base pair, and B is the other side of the pair of base that cannot form a normal base pair) A chemical structural moiety capable of pairing with a base, L denotes a linker structure that bonds chemical structural moieties A and B). It relates to a reagent for forming a base pair.

The present invention pseudo-forms base pairs in a pair of bases that cannot form a normal base pair consisting of the reagents of the present invention and detection and identification materials described above, and measures the formation of the pseudo base pairs. The present invention relates to a kit for detecting and identifying a pair of bases that cannot form a normal base pair.

In addition, the present invention is the following general formula (II),

(Wherein R and R ₁ are a hydrogen atom, an alkyl group having 1 to 15 carbon atoms and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, an alkoxy group having 1 to 15 carbon atoms and the alkoxy) An alkoxy group which may be substituted by one or more carbon atoms in the group with an oxygen atom or a nitrogen atom, or a mono or dialkylamino group having 1 to 15 carbon atoms, and one or more carbon atoms in the alkylamino group be an oxygen atom or a nitrogen atom Represents a mono or dialkylamino group which may be substituted,

R ₂ is an alkyl group having 1 to 20 carbon atoms and represents an alkyl group in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group. The present invention relates to a fixed product modified in a chemical structure that can be immobilized in a detection device, or the like.

Furthermore, the present invention hybridizes DNA or RNA of one chain to be a sample and DNA or RNA having a normal base sequence corresponding thereto, and then normal base pairs in the hybridized DNA or RNA. A pair of bases that cannot be formed are represented by the following general formula (I),

A-L-B (I)

(Wherein A is a chemical structural moiety capable of pairing with one base of a pair of bases that cannot form a normal base pair, and B is another base of a pair of bases that cannot form a normal base pair) A chemical structure moiety capable of pairing with, where L denotes a linker structure that combines chemical moieties A and B). By using a compound having a portion B and a linker moiety L which binds the chemical structural moieties A and B, a base pair is pseudo-formed on a pair of bases that cannot form the normal base pair, thereby The present invention relates to a method for detecting an abnormality in base sequence in DNA or RNA, which comprises detecting and identifying a pair of bases that cannot form a normal base pair consisting of measuring formation.

In addition, in the following description, the chemical structure part (A and B in general formula (I) which can form a pair with one base of the pair of bases which cannot form a normal base pair is mentioned above). Part) ”may simply be referred to as a“ base recognition site ”.

Example

Best Mode for Carrying Out the Invention

The inventors have developed a bulge DNA recognition molecule that binds specifically to and stabilizes DNA (bulge DNA) having an auxiliary base (bulge base) generated in two-stranded DNA (Japanese Patent Application No. 11-262205). number). The bulge recognition molecule is stabilized by intercalation in the space caused by the presence of the bulge base by using stacking interaction between the aromatic ring and the base near the bulge. Each bulge recognition molecule forms a base pair similar to a bulge base in the mismatched portion of the base pair by binding with a binding chain such as a linker, and moreover, both of these bulge recognition molecules form a two-stranded chain. It was found to be relatively stable in the chain of DNA or RNA. It is surprising that such a relatively large molecular species is relatively stably accepted in the chain of DNA or RNA, and by utilizing such characteristics, it is possible to simplify the location where base pair mismatch occurs in the hybridized nucleic acid. It was found that it could be specified.

For example, using a 1,8-naphthyridine derivative which forms a hydrogen bond with guanine and is stabilized by a stacking effect with a surrounding base, it is bonded by a linker, and a dimer represented by the following formula (III) Synthesized.

This compound pairs with guanine in the 1,8-naphthyridine moiety. When guanine is a bulge base, since there is sufficient space for the guanine and the 1,8-naphthyridine derivative to form a pair, the formation of the pair of this 1,8-naphthyridine derivative and the bulge base is It is sufficient to examine the stability of both, but in the case of mismatches, there is not enough space for other bases already in place to form a pair, and such a relatively large space in the minute space between the base and the adjacent base is insufficient. Whether the molecular species can stably enter becomes a big problem.

Therefore, in the case where there is a guanine-guanine mismatch in two chains of the nucleic acid, a compound having two such 1, 8 naphthyridine moieties forms a pair with each guanine mismatched. We examined whether it was accepted in the chain.

The DNA of the two chains to prepare a two-stranded DNA of GC base pairs, GA mismatched base pair and a label 52 bots in 5'- ³² P has a GG mismatched base pair (mer). The subarray of the corresponding part is shown below.

* 1 * 2 * 3

ACGGT ACTCT TCGGA

TGGCA ... TGGCA ... AGGCT

In the above two chain DNAs, the portion indicated by * 1 is a normal G-C base pair, the portion indicated by * 2 is a mismatch portion of G-A, and the portion of * 3 is a portion of mismatch of G-G.

Inhibition site of DNA cleavage by DNaseI by using DNaseI foot printing titration in the presence of the compound of formula (III) at various concentrations using the DNA of these two chains. Was investigated.

This result is shown in FIG. 1 is a drawing substitute photograph showing the results of electrophoresis.

As it goes from the left side to the right side of FIG. 1, the density | concentration of the compound of Formula (III) is gradually increasing from 0 to 500 micrometers. In the case of cleavage by DNaseI (DNA hydrolase), it appears black, and the place where cleavage by DNaseI is inhibited is shown in white.

For example, in the case of a normal base pair of G-C, it is shown that cleavage is caused by DNaseI even when the concentration of the compound of the formula (III) is increased, that is, black. By the way, it can be seen that at sites of mismatches such as G-G, when the concentration of the naphthyridine derivative of the formula (III) is increased, it gradually becomes white, that is, its cleavage is inhibited. It can be also seen that such a change occurs at a high concentration even at the site of the G-A mismatch.

The cleavage inhibitory effect of DNA on such DNA hydrolase depends on the presence (including concentration) of the compound of formula (III), and is thought to be a specific action by the compound of formula (III).

Fig. 2 is a graph showing the relationship between the strength of the cutting band in Fig. 1 and the concentration of the added naphthyridine. The vertical axis of FIG. 2 is the inhibition ratio of the cutting obtained from the strength of the cutting band, "0.0" is a situation in which the cutting is almost completely, and "1.0" shows the situation in which cutting is almost completely inhibited. The abscissa of FIG. 2 shows the concentration (M) of the compound of formula (III) added. The black circle () in the graph of Fig. 2 is that of mismatch site of G-G, and the black triangle (▲) is of mismatch site of G-A.

As is clear from the graph of FIG. 2, the cleavage inhibitory action of the GG on the mismatch site by the compound of formula (III) occurs at a relatively low temperature and almost completely at a concentration of about 10 ⁻⁵ M or more. It can be seen that cleavage to mismatch is inhibited. In addition, the GA mismatch site also showed that inhibition of cleavage began at around 10 ⁻⁶ M concentration and about 90% cleavage inhibition occurred near 5 × 10 ⁻³ M (500 μM). have.

As a result, the binding constant (Ka (GGmis)) to the mismatch of GG of the compound of formula (III) was found to be 1.13 × 10 ⁷ M ¹ , and the binding constant to the mismatch of GA (Ka (GAmis)) was similarly obtained. Was found to be 1.63 × 10 ⁴ M ^−l .

The ratio of the binding constants of both ((Ka (GGmis)) / (Ka (GAmis))) is 696, and it can be seen that Formula (III) acts specifically on the GG mismatch. In addition, the relatively large binding constant for the GG mismatched base pair of the compound of formula (III) in the order of 10 ⁷ shows that the compound of the formula (III) is accepted in the GG mismatched base pair portion more stably than expected. Doing.

In addition, the mismatched base recognition molecules of the present invention accepted by the two chains of DNA form a relatively stable pair, and it is thought that the formation of the pair newly forms a sequence of bases that cannot be recognized by natural enzymes. .

Fig. 3 schematically shows a state in which the compound (mismatch recognition molecule) shown in the general formula (I) of the present invention is relatively stably received in the mismatch portion of the base.

3 shows the mismatched portion of GA in the DNA of the two stranded chains. In other places, normal base pairs are formed, and despite being mismatched in the part of GA, the DNA is hybridized as a whole. If the mismatch recognition molecule of the present invention shown by N _A -N _G is added to this, it is considered to be in the same state as the right side of FIG. That is, the mismatched base guanine (G) is paired with the mismatch recognition molecule guanine recognition site (N _G ), and the mismatched base adenine (A) is mismatched recognition molecule. Paired with the adenine recognition site (N _A ), and the guanine recognition site (N _G ) and adenine recognition site (N _A ) of the mismatch recognition molecule are combined with a linker (-) of suitable length and with appropriate degrees of freedom. It is thought to be taken in the form of a substantially different base pair in the DNA chain of the two-stranded chain (see the right side of FIG. 3).

In addition, another large reason why the mismatch recognition molecule of the present invention is relatively stably accepted in the chains of two-chain DNA is the base recognition region of the mismatch recognition molecule (for example, guanine in the above-described example). The recognition site (N _G ) and the adenine recognition site (N _A ) are stabilized by stacking effects (such as intermolecular forces between bases) by bases before and after. The dotted line on the right of FIG. 3 shows the stacking effect by this base. As one of the factors in which such a stacking effect occurs, since the interaction (pi stacking effect) of (pi) electron system can be considered, the difference of the degree of stacking effect may arise by the kind of base before and after, There is no extreme degradation of the binding of the molecules of the invention and mismatch sites.

Accordingly, the base recognition sites (mis chemical structures of A and B in the general formula (I)) of the mismatch recognition molecules of the present invention are not only capable of hydrogen bonding with desired bases, but also the bases around or around them. It is necessary that it is a chemical structure in which the stacking effect by the is obtained.

As such, the present invention finds that a compound in which two base recognition sites are joined by a linker having an appropriate length and an appropriate degree of freedom forms a specific and stable pairing of mismatched portions of base pairs in a nucleic acid of two strands. The basic concept is not limited to the above-described GG mismatch.

In the above example, as a mismatch of guanine (G) -guanine (G), a mismatch recognition molecule using a l, 8-naphthyridine derivative, which forms a safe hydrogen bond with guanine base, is used as a base recognition site. However, the recognition of mismatches is not limited to GG mismatches. In the present invention, in the mismatch recognition molecule, the base recognition region recognizes one of the mismatched bases to form a Watson-Crick type base pair with the base, and stacking effect by surrounding bases. By selecting the molecular species which can obtain, it is not limited to the guanine illustrated, What is necessary is just to be able to form various bases and base pairs.

For example, 2-aminonaphthyridin-4-one or a derivative thereof as a base recognition site in the case of a mismatched base is cytosine, or a 2-quinolone derivative such as 3 when a base of a mismatch is adenine. In the case of-(2-aminoethyl) -2-quinolone or a derivative thereof, and the mismatch base is thymine, 2-aminonaphthyridin-7-one or a derivative thereof is used.

In the mismatch recognition molecule of the present invention that is specifically recognized for a specific mismatch base, the base recognition site is a complex having a hydrogen bond site for forming a hydrogen bond and a planar structure for stacking in a nearby base. Although it is preferable to have a cyclic aromatic group, Furthermore, in order to enhance selectivity with respect to a base, the heterocyclic aromatic group which has a substituent which has some degree of steric hindrance is preferable.

As such a substituent, it is C1-C15, Preferably it is 1-10, More preferably, it is a linear or branched alkyl group of 1-7, C1-C15, Preferably it is 1-10 More preferably, the alkoxy group which consists of a linear or branched alkyl group of 1-7, C1-C15, Preferably it is 1-10, More preferably, the linear or branched alkyl group of 1-7 is mono Or a di-substituted mono or dialkyl amino group.

In these alkyl groups, alkoxy groups or mono or dialkylamino groups, one or more carbon atoms may be substituted with an oxygen atom or a nitrogen atom.

In addition, as linker part L in the compound represented by General formula (I) of this invention, although it does not restrict | limit especially to providing suitable degree of freedom to two base recognition sites in a suitable length, For example, C1-C20 And preferably 1 to 15, more preferably 1 to 12, straight or branched, saturated or unsaturated alkyl groups, and even if one or more carbon atoms in the alkyl group is substituted with an oxygen atom, a nitrogen atom or a carbonyl group Good alkyl group is mentioned. Preferred linkers include those having an amide bond portion at both ends and a nitrogen atom at the central portion as in the compound of formula (III).

This linker moiety not only binds two base recognition sites, but can also bind a branch for immobilization to the carrier from the linker moiety. For example, a branch such as an alkylene group having a functional group for bonding with the carrier and the like can be further extended from the nitrogen atom near the linker center and immobilized on the carrier as necessary.

The bond between the base recognition site A or B in the general formula (I) of the present invention and the linker portion L may be a carbon-carbon bond, but is preferably bonded by a functional group for simplicity of synthesis. As a bond by a functional group, although various types, such as an ether bond, an ester bond, an amide bond, and a phosphoric acid bond, can be selected, an amide bond is preferable.

In the mismatched base recognition molecule of the present invention, as a preferable compound of general formula (I) for G-G mismatch, the following general formula (II),

(Wherein R and R ₁ are a hydrogen atom, an alkyl group having 1 to 15 carbon atoms and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, an alkoxy group having 1 to 15 carbon atoms and in the alkoxy group) One or more carbon atoms of may be substituted with an oxygen atom or a nitrogen atom, or an alkoxy group or mono or dialkylamino group having 1 to 15 carbon atoms, and one or more carbon atoms in the alkylamino group may be replaced with an oxygen atom or a nitrogen atom. Represents a good mono or dialkylamino group,

R <_2> is a C1-C20 alkyl group and represents the alkyl group in which one or more carbon atoms in this alkyl group may be substituted by the oxygen atom, the nitrogen atom, or the carbonyl group. As used herein, the term "fixed product" refers to a compound in a state in which the above-mentioned compound is immobilized on a carrier or in a state in which the above-mentioned "branch" is extended so as to be immobilized.

In addition, the alkyl group in R <_2> is a bivalent alkyl group as shown to general formula (II).

In addition, the mismatched base recognition molecule of the present invention may be used alone, but a radioactive element is introduced into a suitable position in the molecule, for example, a linker moiety or an extended branch for immobilization from the linker, or the like, or chemiluminescence or It can also be used by labeling, for example, introducing a molecular species that emits fluorescence. In addition, labeling as a measuring means can also be carried out by labeling nucleic acid parts, such as DNA and RNA of a detection object.

Furthermore, the mismatched base recognition molecule of the present invention may be bonded to a polymer material such as polystyrene at a suitable position by using a direct or alkylene group or the like and immobilized.

The mismatched base recognition molecule of the present invention is a low molecular organic compound, and can be appropriately prepared by ordinary organic synthesis. For example, the above-mentioned 1,8-naphthyridine derivatives may be 2-amino-1, 8-naphthyridine or 2 amino7methyl-1, 8naphthyridine as N-protecting 4-amino-butyric reactive derivatives, eg For example, an acid chloride can be made to react and acylated the 2nd amino group, and an amino group can be manufactured by deprotecting a protecting group. As a protecting group at this time, the amino protecting group used in peptide synthesis, such as a hydrochloride, an acyl group, or an alkoxycarbonyl group, can be used.

The desired mismatched base recognition molecule can be obtained by reacting the obtained base recognition site with a compound for linker having a carboxyl group or a reactive derivative group thereof at the sock end. At this time, when reactive groups, such as a nitrogen atom, exist in the molecule | numerator of a linker compound, it can use suitably protecting by the above-mentioned protecting group.

The mismatched base recognition molecule of the present invention may be used as a reagent or a detection agent for detecting a pair of mismatched bases, and may be used as a composition for detecting a pair of mismatched bases by combining with a suitable carrier. Can be. Moreover, it can also be used as a base pair generating agent for forming base pairs pseudologically in the base pair which cannot form a normal base pair. Here, "pseudo base pair" means a pair of bases different from the pair of bases existing in nature, and does not mean the strength of the base pair. In addition, the "normal base pair" used in this specification is a pair of base which exists in nature, and means a base pair of G-C, A-T, or A-U.

The present invention further comprises a base which cannot form a normal base pair consisting of the mismatched base recognition molecule of the present invention and a material for detection and identification, for example, a material such as a reagent or buffer for chemiluminescence or fluorescence. The present invention provides a kit for detecting and identifying a pair of bases that cannot form a normal base pair consisting of forming a base pair pseudoly in a pair and measuring the formation of the pseudo base pair.

Furthermore, the present invention provides a method for detecting, identifying or quantifying a pair of mismatched bases in DNA using a mismatched base molecule of the present invention or a mismatched base molecule of the present invention labeled or immobilized. To provide.

By using the base recognition sites of mismatch recognition molecules of the present invention, DNAs having one or more pairs of mismatched bases can be used to identify pairs of bases of a specific mismatch, for example, GG mismatch, GA Hydrogen bonds are formed with bases that have a particular mismatch, such as mismatches, to stabilize them, as well as being stacked in nearby, preferably adjacent base pairs, to provide relatively stable DNA despite the presence of mismatched base pairs. You can get it.

Therefore, the present invention is that the base recognition site of the mismatch recognition molecule of the present invention forms a hydrogen bond with each of the bases in the pair of bases of the specific mismatch, and is stacked on the base pairs near the base. The present invention provides a DNA containing mismatched base pairs in which pairs of mismatched bases are stabilized.

In this DNA of the present invention, a pair of mismatched bases forms a "pair" (a pseudo base pair) similar to a base pair by hydrogen bonding with the base recognition site of the mismatch recognition molecule of the present invention, and also with a mismatched base. The base recognition site of the mismatch recognition molecule of the present invention forming a "pair" is sandwiched in a sandwich form with a base forming an adjacent base pair, preferably adjacent.

By using the mismatch recognition molecule of the present invention, it is possible to easily detect, identify, or quantify mismatched base pairs that could not be achieved in the prior art with high sensitivity, and is specific to mismatched base pairs. In addition, since stable DNA is formed, it is also possible to apply to the treatment, prevention or diagnosis of various diseases caused by DNA damage.

In addition, since the DNA of the present invention can exist relatively stably in the state of having mismatched base pairs, stabilization of DNA containing mismatched base pairs, causes of mismatches, and mismatch repair mechanisms. It can also be used as a research material for elucidation.

The present invention also hybridizes DNA or RNA of one chain to be a sample and DNA or RNA having a normal base sequence corresponding thereto, and then normal base pairs in the hybridized DNA or RNA. By using the mismatch recognition molecule of the present invention as described above, a base pair can not be formed to form a base pair pseudo-base pair on a base pair that cannot form a normal base pair, thereby forming the pseudo base pair. It also provides a method for detecting an abnormality in the base sequence in DNA or RNA, which is to detect and identify a pair of bases that cannot form a normal base pair. This method can be used for the purpose of investigating the presence or absence of gene abnormalities.

For example, DNA having a possible probability of DNA or its transcription product, RNA is taken, and hybridized with complementary DNA or RNA having a normal nucleotide sequence to it to generate a double stranded nucleic acid. . If there is an abnormality in the nucleotide sequence of the collected gene, a mismatch occurs in the pair of bases in the base of the abnormal position. By adding the mismatch recognition molecule of the present invention to the nucleic acid of the two-stranded chain where this mismatch occurs, the above-described pseudo base pair is formed, and the new pair is formed by measuring the presence or absence of the molecule. The abnormality can be detected and identified easily and with high sensitivity.

As a means for measuring said new pair (pair of mismatch recognition molecule of this invention and base of mismatch), it can also carry out by labeling, such as chemiluminescence, fluorescence, a radioisotope. The mismatched recognition molecule of the present invention is a low molecular organic compound, and when a new pair is formed, the molecule is accepted in the nucleic acid, so that the unreacted mismatched recognition molecule of the present invention and nucleic acids can be separated relatively easily. Can be.

As described above, the mismatch recognition molecules of the present invention may be immobilized on a carrier and used. For example, a mismatch recognition molecule specific to various mismatches of the present invention is immobilized on a plate such as a titer plate, and the nucleic acid of the above two chains, preferably a labeled nucleic acid, is added thereto. After incubation for a minute, the nucleic acid is removed, and the nucleic acid that specifically reacts with the mismatch recognition molecule of the present invention is trapped by the immiscible mismatch recognition molecule of the present invention, and can be detected and identified by a label.

It is also possible to fix the mismatch recognition molecules of the present invention on the metal thin film of the chip for detecting surface plasmon resonance (SPR). In the case of this SPR, it is possible to specifically detect the presence or absence of mismatch by simply flowing the sample liquid containing the nucleic acids of the two chains above on the surface of the detection chip.

Moreover, the mismatch recognition molecule of the present invention can be applied to many other detection means, and the present invention is not limited to these specific detection means.

Hereinafter, the present invention will be described in detail by way of specific examples, but the present invention is not limited to these specific examples.

Example 1 Synthesis of Compound of Formula (III)

The compound of the title was synthesized according to the chemical reaction shown in the following formula.

(Boc in a formula represents a t-butoxycarbonyl group.)

N-Bocylated dicarboxylic acid rosinimidyl ester (313 mg, 0.74mmo1) was dissolved in chloroform (15 mL), 2-amino-7-methyl-1, 8-naphthyridine (294mg, 1, 85mmo1) Added. After 48 hours of reaction at room temperature, Bocylated dinaphthyridinamide was obtained by workup. When this was dissolved in ethyl acetate containing 4N hydrochloric acid and reacted at room temperature for 2 hours, the indicated dinaphthyridinamide was obtained in 13% yield.

¹ H NMR (CD ₃ OD, 400 MHz) δ:

8.26 (d, 2H, J = 8.8 Hz), 8.14 (d, 2H, J = 8.8 Hz), 8.11 (d, 2H, J = 8.0 Hz),

7.34 (d, 2H, J = 8.0 Hz), 3.20 (t, 4H, J = 6.0 Hz), 2.84 (t, 4H, J = 6.0 Hz),

2.68 (s, 6 H);

FABMS (NBA), m / e (%): 444 [(M + H) ⁺ , 10),

246 (40), 154 (100);

HRMS calcd: C ₂₄ H ₂₆ O ₂ N ₇ [(M + H) &lt; + &gt;

Found: 444.2148.

Example 2

52 base DNAs labeled with ³² P at the 5 'end were hybridized to generate mismatches of GG and GA, resulting in double stranded DNA (see right of FIG. 1).

The compounds obtained in Example 1 at various concentrations were added to the DNAs of these two chains and examined by DNaseI footprinting titration.

That is, in Example 1, the DNA of the two chains (<4 nM strand concentration) was adjusted to various concentrations with Tris hydrochloric acid buffer solution (10 mM, pH7.6) containing NaC1 (100 mM) and MgC1 ₂ (5 mM). With the obtained compound, it was incubated at 4 degreeC for 12 hours. 0.2 U DNaseI (DNA hydrolase) was added thereto and incubated at 25 ° C for 8 minutes. Thereafter, DNA was recovered by ethanol precipitation, which was electrophoresed using a gel containing 12% polyacrylamide and 7M urea.

This result is shown in FIG.

By using the mismatch recognition molecule of the present invention, pairs of mismatched bases such as guanine-guanine mismatch, which have not been achieved in the prior art, can be detected easily and with high sensitivity.

Claims (14)

For base pairs that cannot form a normal base pair, the following general formula (II), (Wherein R and R ₁ represent a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, an alkyl group in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, and R ₂ represents an alkyl group having 1 to 20 carbon atoms) And base pairs pseudo-base pairs to base pairs which cannot form the normal base pair by using a compound represented by one or more carbon atoms in the alkyl group, which represents an alkyl group which may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group. Forming and measuring the formation of the pseudo base pair, thereby detecting and identifying a pair of bases that cannot form a normal base pair. delete delete delete delete delete The method according to claim 1, wherein the compound represented by formula (II) is immobilized on a carrier. The method according to claim 1, wherein the compound represented by the general formula (II) is labeled. A normal pair consisting of forming base pairs pseudo-base pairs in base pairs which cannot form the normal base pairs according to any one of claims 1, 7, and 8, and measuring the formation of the pseudo base pairs. In the method for detecting and identifying a pair of bases that cannot form a base pair, the following general formula (II) for pseudo-forming a base pair pseudoly on a pair of bases that cannot form a normal base pair, (Wherein R and R ₁ represent a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, an alkyl group in which one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, and R ₂ represents an alkyl group having 1 to 20 carbon atoms) A reagent consisting of a compound represented by one or more carbon atoms in the alkyl group represents an alkyl group which may be substituted with an oxygen atom, a nitrogen atom or a carbonyl group. The reagent according to claim 9, wherein the reagent is a base pair generating agent for pseudoforming base pairs pseudoly on a pair of bases which cannot form a normal base pair. A normal base pair which consists of forming a base pair pseudo-wise in the base pair which cannot form a normal base pair which consists of the reagent of Claim 9, and a material for detection and identification, and measuring the formation of the said pseudo base pair. Kit for detecting and identifying a pair of bases that can not form. The following general formula (II), (Wherein R and R ₁ represent a hydrogen atom or an alkyl group having 1 to 15 carbon atoms and one or more carbon atoms in the alkyl group may be substituted with an oxygen atom or a nitrogen atom, and R ₂ represents an alkyl group having 1 to 20 carbon atoms) And an alkyl group in which one or more carbon atoms in the alkyl group may be substituted with a nitrogen atom or a carbonyl group.). The compound according to claim 12, wherein the compound represented by the general formula (II) is represented by the following general formula (III), Phosphorus compounds or their fixed substances. delete