JP2009215171A - Nucleoside triphosphate derivative - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleoside derivative for accurately recognizing a complementary nucleoside to prevent an uptake error caused in PCR or the like. <P>SOLUTION: Provided is a nucleoside triphosphate derivative having cytosine whose 3-nitrogen atom is oxidized or adenine whose 1-nitrogen atom is oxidized. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel nucleoside triphosphate derivative, and its use and production method. Since the nucleoside triphosphate derivative of the present invention can accurately recognize complementary bases, generation of mismatch base pairs during nucleic acid synthesis can be suppressed.

  It is known that DNA is oxidized by active oxygen generated in a living body to generate an oxidatively damaged base. During DNA replication, if an oxidatively damaged base formed by oxidation of DNA is contained in the template strand, it may cause a strand elongation reaction by DNA polymerase or cause a mutation. Therefore, it is widely recognized as a mutagen that causes errors in cells. On the other hand, when considering the mechanism of oxidative damage of DNA, there is another important issue apart from the case where the template strand contains an oxidative damage base. It is about oxidative damage of the triphosphate that is taken up by the enzyme. In the triphosphate body storage called nucleotide pool, oxidation by active oxygen occurs in the same manner as the oxidation of DNA double strands, so that an oxidative damage body of triphosphate is generated. When an oxidatively damaged body of triphosphate is generated, the enzyme may not recognize the oxidatively damaged base as a substrate when replicating DNA, or even if the base in the template strand is a non-complementary base, it will be incorporated as a substrate and extend the chain Can be considered. As a specific example of the latter, 8-oxoguanine, which is a representative oxidative damage base, will be described (Non-patent Documents 1 to 4).

  The triphosphate form of 8-oxoguanine is generated in the nucleotide pool, and this triphosphate form is incorporated into the adenine base of the template strand and the chain is elongated. This causes the same mutation as when the DNA duplex is oxidized and 8-oxoguanine is present in the template strand.

Shimizu, M., Gruz, P., Kamiya, H., Masutani, C., Xu, Y., Usui, Y., Sugiyama, H., Harashima, H., Hanaoka, F., Nohmi, T. Biochemistry 2007, 46, 5515-5522. Einolf, H. J., Guengerich, F. P. J. Biol. Chem. 2001, 276, 3764-3771. Einolf, H. J., Schnetz-Boutaud, N., Guengerich, F. P. Biochemistry 1998, 37, 13300-13312. Miller, H., Prasad, R., Wilson, S. H., Johnson, F., Grollman, A. P. Biochemistry 2000, 39, 1029-10

  By the way, in a DNA amplification method such as PCR generally used in gene analysis or the like, there is a problem that a nucleoside that is not complementary to a template DNA is incorporated (uptake error). An object of the present invention is to provide a nucleoside derivative that accurately recognizes a complementary nucleoside in order to prevent such uptake errors.

  As a result of intensive studies to solve the above problems, the present inventor has found that N-oxidized cytosine and adenine have complementary bases to natural (non-N-oxidized) cytosine and adenine. The inventors have found that the ability to recognize is high, and have completed the present invention.

  8-Oxoguanine, which is a typical oxidatively damaged base, has a low ability to recognize complementary bases, and will pair not only with cytosine but also with adenine. Therefore, it was completely unpredictable at the time of filing of the present application that N-oxidized cytosine and adenine, which are oxidatively damaged bases as in 8-oxoguanine, have a high ability to recognize complementary bases.

  The present invention has been completed based on the above findings.

  That is, the present invention provides the following (1) to (7).

(1) General formula (I) or general formula (II)

[Wherein, X represents a hydrogen atom or a hydroxyl group. ]
A nucleoside triphosphate derivative represented by:

(2) A method of synthesizing nucleic acid using a substrate, a nucleic acid synthase, a single-stranded nucleic acid as a template, and a primer, wherein the nucleoside triphosphate derivative described in (1) is used as a substrate. Nucleic acid synthesis method.

(3) The method for synthesizing a nucleic acid according to (2), wherein the nucleic acid synthase is a DNA synthase and the single-stranded nucleic acid is a single-stranded DNA.

(4) The method for synthesizing nucleic acid according to (2) or (3), wherein polymerase chain reaction is used.

(5) A nucleic acid synthesis substrate comprising the nucleoside triphosphate derivative according to (1).

(6) General formula characterized by reacting cytidine-5'-triphosphate, adenosine-5'-triphosphate, deoxycytidine-5'-triphosphate, or deoxyadenosine-5'-triphosphate with an oxidizing agent (I) or general formula (II)

[Wherein, X represents a hydrogen atom or a hydroxyl group. ]
The manufacturing method of the nucleoside triphosphate derivative represented by these.

(7) The method for producing a nucleoside triphosphate derivative according to (6), wherein the oxidizing agent is metachloroperbenzoic acid.

  Since the nucleoside triphosphate derivative of the present invention can accurately recognize complementary bases, it is possible to suppress the occurrence of mismatched base pairs during chain extension reactions and PCR reactions.

  Hereinafter, the present invention will be described in detail.

  The nucleoside triphosphate derivative of the present invention has the general formula (I) or the general formula (II).

[Wherein, X represents a hydrogen atom or a hydroxyl group. ]
It is represented by

  The nucleoside triphosphate derivatives of the present invention include natural nucleoside triphosphates with cytosine or adenine (ie, cytidine-5′-triphosphate, adenosine-5′-triphosphate, deoxycytidine-5′-triphosphate, and deoxyadenosine -5'-triphosphate) with an oxidizing agent.

  As the oxidizing agent, metachloroperbenzoic acid (mCPBA) can be used, but other oxidizing agents such as hydrogen peroxide and monoperphthalic acid may be used.

  The amount of the oxidizing agent to be used is not particularly limited, but it is preferably 1 to 20 molar equivalents, more preferably 5 to 6 molar equivalents with respect to the nucleoside triphosphate.

  The reaction with the oxidizing agent is usually performed in the presence of a solvent. The solvent to be used is not particularly limited, and for example, water, methanol, acetonitrile, N, N-dimethylformamide, dioxane, N-methylpyrrolidone and the like can be used. These solvents may be used as a mixture of two or more.

  Although the temperature at the time of reaction is not specifically limited, It is preferable to set it as 15-60 degreeC, and it is more preferable to set it as 20-25 degreeC.

  The reaction time is not particularly limited, but is preferably 0.5 to 24 hours, and more preferably 3 to 6 hours.

  The pH during the reaction is not particularly limited, but is preferably 4.0 to 8.0, more preferably 5.0 to 6.0.

  The nucleoside triphosphate derivative of the present invention can be used as a substrate for nucleic acid synthesis. More specifically, the nucleoside triphosphate derivative of the present invention can be used as a substrate in a method for synthesizing a nucleic acid using a substrate, a nucleic acid synthase, a single-stranded nucleic acid as a template, and a primer. Examples of such nucleic acid synthesis methods include polymerase chain reaction (PCR). The nucleic acid synthase to be used may be either DNA polymerase or RNA polymerase. For example, DNA polymerase I Klenow Fragment, Taq polymerase, T7 RNA polymerase, DNA polymerase β, DNA polymerase λ and the like can be used.

  By using the nucleoside triphosphate derivative of the present invention as a substrate for nucleic acid synthesis, it is possible to synthesize nucleic acid chains with fewer mismatches than conventional methods.

  Hereinafter, the present invention will be described in more detail with reference to examples.

[Reference Example] Evaluation of base pairing ability of N-oxidized oxidatively damaged bases of DNA oligomers containing deoxycytidine-3-N-oxide (dC 2 ^{O 3} ) and deoxyadenosine 1-N-oxide (dA 2 ^{O 3} ) Their base pairing ability was evaluated by measuring the duplex melting temperature ( _Tm value). The sequence and conditions used are shown in FIG.

The DNA14 mer center (X) arranged dC ^O or dA ^O, was measured T _m value of a base (Y) of its complementary position A, T, G, using DNA duplexes varied with C . The result is shown in FIG.

First, the comparison of the double strand melting temperature between natural dC and oxidatively damaged base dC 2 ^O will be described (FIG. 2a). Since the natural bases dC is to form a dG base pairs, if value of T _m duplex containing dC whereas showed 47.8 ° C. and a high value, including oxidative damage bases dC ^O is 31.0 ° C. The _Tm value decreased significantly. Compared to the natural case, dC ^O and dG base pair formation has become unstable due to the presence of an oxygen atom at the N3 position of the cytosine base, which makes it difficult to form normal Watson-Crick hydrogen bonds. It is possible. On the other hand, the oxidation damage base dA 2 ^O also showed the same result as dC 2 ^O (FIG. 2b). The T _m value of the duplex containing the natural base dA relative to dT was as high as 45.7 ° C, whereas when it contained the oxidatively damaged base dA 2 ^O , it was 11.3 ° C and the T _m value decreased even at 10 ° C or higher. did. In dA ^O as well, the oxygen atom is added to the N1 position, so that the Watson-Crick hydrogen bonding mode is changed, which seems to be the cause of destabilization of base pairing.

Also, paying attention to the ability to form base pairs with other bases, the T _m value of the duplex containing dC 2 ^O or dA 2 ^O is almost equivalent to any of the dA, dT, dG, and dC bases. Indicated. This indicates that N-oxidized bases are less likely to form base pairs as complementary bases to any of the four natural bases. From this result, it was predicted that N-oxidized bases have a low ability to recognize complementary bases, and therefore DNA polymerase may incorporate any bases during DNA replication.

[Example 1] Examination of N-oxidation of triphosphate
N-oxidized bases dC ^O and dA ^O triphosphates (pppdC ^O , pppdA ^O ) are synthesized by N-oxidation of natural dC and dA triphosphates (pppdC, pppdA). Thought to do. First, oxidation conditions were examined using pppdC (Table 1).

pppdC (Compound 1) was reacted with mCPBA in MeOH: H ₂ O, and the product was confirmed by DEAE-HPLC. The production ratio shown in Table 1 was the ratio of the peak areas of Compound 1 and Compound 2 obtained by DEAE-HPLC. When mCPBA was added and reacted for a long time, an increase in the production ratio of N-oxide (compound 2) was observed (entry 1, 2). In addition, we examined the appropriate reaction solvent by changing the composition ratio of the reaction solvent, but no significant change was observed (entry 3). Therefore, an increase in N-oxide (compound 2) was expected by using an excessive amount of mCPBA. As a result, as the equivalent of mCPBA was increased, the production ratio of the N-oxide (compound 2) was improved, and the time was also shortened (entry 4, 5). The reaction was continued for a longer time with the aim of obtaining compound 2 quantitatively, but the production ratio hardly changed. The cause of the reaction not progressing quantitatively is considered to be the pH in the reaction system. The optimum pH for N-oxidation of cytosine base with mCPBA is 6.0, but the above reaction system seems to be more acidic due to the influence of benzoic acid and phosphoric acid part of the reagent residue. Therefore, an aqueous solution of sodium bicarbonate was added to neutralize the acidity and an oxidation reaction was performed. As a result, it was confirmed that the N-oxide (compound 2) was almost completely produced as expected (entry 6).

[Example 2] Synthesis of pppdC ^O and pppdA ^O Using the oxidation conditions studied in Example 1, pppdC ^O and pppdA ^O were synthesized as follows.

Compounds 1 and 3, which are natural triphosphates, were reacted with mCPBA in MeOH: NaHCO ₃ to synthesize N-oxidized base triphosphate compound 2 and compound 4, respectively. These triphosphates were purified using DEAE-HPLC, and the yield was determined from UV (260 nm) absorbance. It was confirmed by ¹ H-NMR and ³¹ P-NMR measurements that the desired triphosphate compound 2 and compound 4 could be synthesized.

[Example 3] Single-base chain extension reaction using DNA polymerase
To examine the substrate specificity of bases that are N- oxides of the chain elongation reaction with DNA polymerase, it was a single nucleotide chain elongation reaction using a synthesized PppdC ^O and pppdA ^O. As a result, it was expected that the effects of pppdC ^O and pppdA ^O obtained by oxidizing cytosine base or adenine base in the nucleotide pool upon DNA replication would be clarified. The enzyme used in the single base chain extension reaction was DNA polymerase I Klenow Fragment lacking 5 ′ → 3 ′ exonuclease activity. Detailed reaction conditions and sequences used are shown in FIG. 3 (Shimizu, M., Gruz, P., Kamiya, H., Masutani, C., Xu, Y., Usui, Y., Sugiyama, H., Harashima, H., Hanaoka, F., Nohmi, T. Biochemistry 2007, 46, 5515-5522., Taniguchi, Y., Kool, ETJ Am. Chem. Soc. 2007, 129, 8836-8844., Wu, Y ., Ogawa, AK, Berger, M., McMinn, DL, Schultz, PG, Romesberg, FEJ Am. Chem. Soc. 2000, 122, 7621-7632., Hirao, I., Harada, Y., Kimoto, M Mitsui, T., Fujiware, T., Yokoyama, SJ Am. Chem. Soc. 2004, 126, 13298-13305.).

As shown in FIG. 3, 25-mer DNA was used as the template, and four types of bases (A, G, C, T) serving as templates were arranged at the 19th position from the 3 ′ end. On the other hand, 18-mer DNA was used as the primer, and a FAM group, which is a fluorescent modifying group, was introduced at the 5 ′ end so that it could be observed by fluorescence detection. The evaluated triphosphates were obtained using pppdC ^O and pppdA ^O , which are oxidative damage bases, and natural pppdC and pppdA as controls. First, triphosphoric acid as a substrate was added to the template-primer duplex and reacted at 37 ° C., the optimal temperature for DNA polymerase, for 10 minutes. Thereafter, the reaction was stopped by adding 98% formamide. Electrophoresis was performed using polyacrylamide gel denatured with 7 M urea, and the state of single base chain elongation was observed by fluorescence detection. The result is shown in FIG.

First, a description will be given of an nucleotide chain elongation reaction of pppdC ^O (Fig. 4a). Enzymatic incorporation of PppdC ^O are template that was not expected chain extension was observed only for dG. From this, it was shown that the tendency was similar to that of natural pppdC triphosphate. Although also slightly chain elongation in addition to the natural in PppdC of dG is going was observed, a chain extension reaction in the oxidative damage body PppdC ^O In other bases was observed. Next, the single base chain elongation reaction of pppdA 2 ^O will be described (FIG. 4b). It was the same result as if also uptake by enzymes PppdA ^O using pppdC ^O. Chain elongation was observed only when the template was dT, and no chain elongation occurred for other bases. The above results are considered from the reference example (results of double-strand melting temperature), “Because there is no base discrimination ability, it is incorporated into any base and causes chain elongation”, or “base formation ability is This is contrary to the fact that the enzyme does not recognize any of the bases and chain elongation does not occur.

(Synthesis example)
A synthesis method for a new compound or a compound synthesized by a method different from the conventional method will be described.

(1) 2′-Deoxycytidine-3-N-oxide-5′-triphosphate (Compound 2)
Compound 1 was dissolved in water in advance to prepare 0.4 M stock solution A. Further, mCPBA was dissolved in MeOH to prepare 1.2 M stock solution B.

Saturated sodium bicarbonate water (50 μL) and stocl solution B (100 μL, 120 μmol) were added to 0.4 M stock solution A (50 μL, 20 μmol), and reacted at room temperature for 6 hours. Water (500 μL) was added to the reaction solution, and the mixture was allowed to stand at 0 ° C. for 10 minutes, and the precipitate was removed by filtration. Purification by DEAE-HPLC (eluted by TEAB-buffer) gave Compound 2 (42%). The yield was calculated by measuring the absorbance of UV (260 nm) and using the ε value of dC 2 ^O (3910).

UV (H ₂ O) λ _max 272 nm, λ _max 224 nm. ¹ H NMR (D ₂ O) δ 1.14 (t, 27H, J = 7.5 Hz), 2.20-2.30 (m, 1H), 2.33-2.43 ( m, 1H), 3.03-3.11 (m, 18H), 4.10-4.11 (m, 3H), 4.48-4.53 (m, 1H), 6.20 (t, 1H, J = 6.4 Hz), 6.27 (d, 1H, J = 7.9 Hz), 7.84 (d, 1H, J = 7.9 Hz); ³¹ P NMR (D ₂ O) δ -22.6 (t, J = 21.0 Hz, J = 20.0 Hz), -10.9 (d, J = 20.0 Hz), -10.1 (d, J = 21.0 Hz).
(2) 2'-deoxyadenosine-1-N-oxide-5'-triphosphate (compound 4)
Compound 3 was dissolved in water in advance to prepare 0.4 M stock solution A. Further, mCPBA was dissolved in MeOH to prepare 1.2 M stock solution B.

Saturated sodium bicarbonate water (50 μL) and stocl solution B (100 μL, 120 μmol) were added to 0.4 M stock solution A (50 μL, 20 μmol), and reacted at room temperature for 6 hours. Water (500 μL) was added to the reaction solution, and the mixture was allowed to stand at 0 ° C. for 10 minutes, and the precipitate was removed by filtration. Purification by DEAE-HPLC (eluted by TEAB-buffer) gave Compound 4 (48%). The yield was calculated by measuring the absorbance of UV (260 nm) and using the ε value (7930) of dA 2 ^O.

UV (H ₂ O) λ _max 261 nm, λ _max 232 nm. ¹ H NMR (D ₂ O) δ 1.09 (t, 27H, J = 7.4 Hz), 2.38-2.46 (m, 1H), 2.63-2.73 ( m, 1H), 2.97-3.05 (m, 18H), 4.01-4.11 (m, 3H), 6.36 (t, 1H, J = 6.6 Hz), 8.41-8.42 (2s, 2H); ³¹ P NMR (D ₂ O) δ -22.3 (t, J = 19.5 Hz), -10.7 (d, J = 19.5 Hz), -7.9 (d, J = 19.5 Hz).
(3) Single base chain extension reaction The pppdC ^O and pppdA ^O used were obtained by dissolving compounds 2 and 4 in MeOH (100 μL), adding 0.6 M NaClO ₄ / acetone (600 μL), and centrifuging the white powder. After precipitation, the solution was removed and washed 4 times with acetone to form Na ⁺ -form. A primer whose 5 ′ end was fluorescently labeled with FAM was used. The arrangement of primer and templet used is as shown in FIG.

100 nM templete-primer duplex, DNA polymerase Klenow fragment exo - (0.01 unit), 10 μM triphosphate containing acid material buffer (50 mM Tris-HCl (pH 7.2), 10 mM MgSO 4, 0.1 mM DTT) 10 μL Incubated at 37 ° C for 10 minutes. 30 μL of a reaction terminator (98% formamide, 20 mM EDTA) was added, and electrophoresis was performed using a modified 20% polyacrylamide gel containing 7 M urea. Detection was carried out by fluorescence detection with a fluoro image analyzer (FUJIFILM FLA-2000G).

shows the sequences and conditions used in the base-pairing ability evaluation experiment of a DNA oligomer containing dC ^O and dA ^O. It shows the results of evaluation experiments dC ^O and dA ^O base pairing capability with duplex melting temperatures. The figure which shows the arrangement | sequence and conditions used by the single base chain extension reaction. The figure which shows the experimental result of a single base chain extension reaction.

Claims (7)

General formula (I) or general formula (II)
[Wherein, X represents a hydrogen atom or a hydroxyl group. ]
A nucleoside triphosphate derivative represented by:   A method of synthesizing a nucleic acid using a substrate, a nucleic acid synthase, a single-stranded nucleic acid as a template, and a primer, wherein the nucleoside triphosphate derivative according to claim 1 is used as a substrate. Method.   The nucleic acid synthesizing method according to claim 2, wherein the nucleic acid synthase is a DNA synthase and the single-stranded nucleic acid is a single-stranded DNA.   The method for synthesizing a nucleic acid according to claim 2 or 3, wherein a polymerase chain reaction is used.   A nucleic acid synthesis substrate comprising the nucleoside triphosphate derivative according to claim 1. General formula (I) characterized by reacting cytidine-5'-triphosphate, adenosine-5'-triphosphate, deoxycytidine-5'-triphosphate or deoxyadenosine-5'-triphosphate with an oxidizing agent Or general formula (II)
[Wherein, X represents a hydrogen atom or a hydroxyl group. ]
The manufacturing method of the nucleoside triphosphate derivative represented by these.   The method for producing a nucleoside triphosphate derivative according to claim 6, wherein the oxidizing agent is metachloroperbenzoic acid.