JP7016511B2 - Nucleic acid synthesis method - Google Patents

Description

The present invention relates to a nucleic acid synthesis method, and more specifically, to a nucleic acid synthesis method in which an enzymatic function of RNA polymerase is converted by a simple method to incorporate a modified nucleic acid species that cannot be incorporated by RNA polymerase before conversion.

In recent years, pharmaceutical products using nucleic acids have been developed. When nucleic acids are used as pharmaceuticals, it is known to chemically modify the nucleic acids in order to enhance their chemical stability. However, the original nucleic acid polymerase has no specificity for chemically modified nucleic acids. Therefore, when nucleic acid synthesis is performed by a nucleic acid polymerase using a chemically modified nucleic acid as a substrate, it is necessary to acquire substrate specificity for the chemically modified nucleic acid by modifying the nucleic acid polymerase. As a specific method of modification, there is a method of changing the chemical properties inside the enzyme by changing the amino acid residue of the active site or the like. For example, Non-Patent Document 1 describes a 2'-methoxy or 2'-fluoropartially modified oligonucleotide that cannot be synthesized by the original DNA polymerase by producing a mutant by randomly introducing a mutation into the active site of DNA. It is disclosed that it was synthesized. In addition, Non-Patent Document 2 discloses that a mutation is introduced into T7 RNA polymerase to incorporate a 2'-methoxy-modified or 2'-fluoro-modified ribose moiety.

T. Chen et al., Nat. Chem. 2016, 8, 556 A. J. Meyer et al., Nucleic Acids Res. 2015, 43, 7480

As described above, it is common sense to introduce a mutation into a polymerase as an approach for incorporating a modified nucleic acid that is not the original substrate into the polymerase. However, it is necessary to introduce mutations by such an approach and screen for mutants having the desired activity, and it takes a lot of time and effort to obtain a polymerase having the desired activity from a polymerase having the desired activity. And are required.

Therefore, an object of the present invention is to provide a technique for acquiring an activity of incorporating a nucleic acid species that cannot be incorporated by RNA polymerase before conversion by converting the enzymatic function of RNA polymerase by a simple method.

In the nucleic acid synthesis method using T7 RNA polymerase, the present inventor added high-concentration polyethylene glycol to the reaction solution to suppress RNA synthesis, which is the original substrate of RNA polymerase, and DNA that is not the original substrate. It was discovered that the synthesis of RNA was promoted. That is, it was found that by adding polyethylene glycol in a high concentration to the reaction solution, RNA polymerase changes into an enzyme exhibiting DNA polymerase-like activity behavior.

The present inventor has repeatedly studied RNA polymerase whose activity behavior changes like DNA polymerase in a reaction solution containing such polyethylene glycol. As a result, surprisingly, RNA polymerase whose activity behavior changed like DNA polymerase in a reaction solution containing polyethylene glycol also acquired the activity of incorporating 2'-modified nucleic acid species, which is not found in DNA polymerase. I found that. That is, it has been found that the above-mentioned object of the present invention can be achieved by carrying out a nucleic acid synthesis reaction using RNA polymerase in a reaction solution containing polyethylene glycol at a high concentration. The present invention has been completed by further studies based on this finding.

That is, the present invention provides the inventions of the following aspects.

Item 1. Complementing the (D) template nucleic acid in (i) reaction solution containing (A) RNA polymerase, (B) polyalkylene glycol, (C1) 2'-modified ribonucleotide, and (D) template nucleic acid. Including the step of synthesizing a modified nucleic acid
The (A) RNA polymerase consists of a single subunit protein.
The (B) polyalkylene glycol has an average molecular weight of 10 to 1000, and (i) the concentration in the reaction solution is 10% by weight or more.
A nucleic acid synthesis method in which the (C1) 2'-modified ribonucleotide is selected from the group consisting of 2'-halogenated ribonucleotides and 2'-alkoxyribonucleotides.
Item 2. Item 2. The nucleic acid synthesis method according to Item 1, wherein the (D) template nucleic acid does not contain a promoter sequence, and (i) the reaction solution further contains (E) a primer having a sequence complementary to a part of (D). ..
Item 3. Item 2. The nucleic acid synthesis method according to Item 1 or 2, wherein the RNA polymerase (A) is an RNA polymerase derived from bacteriophage.
Item 4. Item 6. The nucleic acid synthesis method according to any one of Items 1 to 3, wherein the polyalkylene glycol (B) is polyethylene glycol.
Item 5. Item 6. The nucleic acid synthesis method according to any one of Items 1 to 4, wherein the 2'-halogenated ribonucleotide is a 2'-fluororibonucleotide.
Item 6. Item 6. The nucleic acid synthesis method according to any one of Items 1 to 5, wherein the 2'-alkoxy group in the 2'-alkoxyribonucleotide has 1 to 3 carbon atoms.
Item 7. Item 6. The nucleic acid synthesis method according to any one of Items 2 to 6, wherein the primer (E) has a modifying group at the 5'end.
Item 8. From the reaction solution (i), a reaction solution (ii) containing (C2) ribonucleotide and diluted so that the concentration of the polyalkylene glycol (B) was 5% by weight or less was prepared, and the reaction solution (ii) was prepared. Item 6. The nucleic acid synthesis method according to any one of Items 1 to 7, further comprising a step of extending RNA complementary to the (D) template nucleic acid from the modified nucleic acid in a reaction solution.
Item 9. A kit for performing the nucleic acid synthesis method according to any one of Items 1 to 8.
It comprises (A) RNA polymerase, (B) polyalkylene glycol, and (C1) 2'-modified ribonucleotide.
The (A) RNA polymerase consists of a single subunit protein.
The average molecular weight of the (B) polyalkylene glycol is 10 to 1000, and the polyalkylene glycol has an average molecular weight of 10 to 1000.
A nucleic acid synthesis kit in which the (C1) 2'-modified ribonucleotide is selected from the group consisting of 2'-halogenated ribonucleotides and 2'-alkoxyribonucleotides.
Item 10. Item 9. The nucleic acid synthesis kit according to Item 9, further comprising a solid phase carrier and / or ribonuclease H for immobilizing the template nucleic acid.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a technique for acquiring an activity of incorporating a nucleic acid species that cannot be incorporated by RNA polymerase before conversion by converting the enzymatic function of RNA polymerase by a simple method.

It is a schematic diagram explaining an example of the nucleic acid synthesis method of this invention. It is a schematic diagram explaining the nucleic acid synthesis method in Test Example 1. FIG. The results of Test Example 1, a modified PAGE gel electrophoresis photograph (a) when a ribozyme-type polymerase (tC9Y) was used, and a graph (b) of the primer elongation rate with respect to the content of polyethylene glycol (PEG200) having an average molecular weight of 200. Denaturation using T7 RNA polymerase (T7 RNAP) PAGE gel electrophoresis photograph (c) and graph of primer elongation to PEG200 content (d); and denaturation using Escherichia coli DNA polymerase (KF) The PAGE gel electrophoresis photograph (e) and the graph (f) of the primer extension rate with respect to the content of PEG200 are shown. The results of Test Example 2, the graph (a) of the primer extension rate with respect to the content of PEG200 (same as (d) in FIG. 3), the graph (b) of the primer extension rate with respect to the content of ethylene glycol (EG), and the graph. The graph (c) of the primer extension rate with respect to the content of polyethylene glycol (PEG8000) having an average molecular weight of 8000 is shown. It is a schematic diagram explaining the nucleic acid synthesis method in Example 1. FIG. It is the result of Example 1, and shows the modified PAGE gel electrophoresis photograph (a) for each substrate and the graph (b) of the primer extension rate for each substrate. It is a schematic diagram explaining the nucleic acid synthesis method in Example 2. It is the result of Example 2, and the graph of the primer extension rate for each substrate is shown. It is a schematic diagram explaining the nucleic acid synthesis method (1st step: the step of synthesizing a modified nucleic acid, 2nd step: the step of extending RNA from a modified nucleic acid) in Example 3. It is the result of Example 3, and shows the denaturing PAGE gel electrophoresis photograph (a) for each substrate used in 1st step, and the graph (b) of the primer extension rate or RNA elongation rate for each substrate used in 1st step.

[1. Nucleic acid synthesis method]
The nucleic acid synthesis method of the present invention comprises (i) a reaction containing (A) RNA polymerase, (B) polyalkylene glycol at a predetermined high concentration, (C1) 2'-modified ribonucleotide, and (D) template nucleic acid. The step of synthesizing a modified nucleic acid complementary to the (D) template nucleic acid in a liquid is included. Preferably, (i) the reaction solution contains (E) a primer that is complementary to (D) a portion of the template nucleic acid. Further, in the nucleic acid synthesis method of the present invention, (i) a reaction solution containing (C2) ribonucleotides and (B) the concentration of polyalkylene glycol diluted to a predetermined low concentration is prepared from the reaction solution. It further comprises the steps of (ii) extending RNA complementary to (D) the template nucleic acid from the modified nucleic acid in the reaction solution. (ii) Hybrid RNA is obtained by the process in the reaction solution.

Complementary in the present invention means having a sequence capable of hybridizing under stringent conditions. The stringent conditions referred to here can be selected from known conditions and are not particularly limited, but for example, at 42 ° C., 50% (v / v) formamide and 0.1% bovine serum. Conditions such as albumin, 0.1% ficol (trade name), 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride, and 75 mM sodium citrate coexist. Be done.

FIG. 1 shows a schematic diagram illustrating an example of the nucleic acid synthesis method of the present invention. In FIG. 1, the display of (A) RNA polymerase is omitted, (B) polyethylene glycol (PEG) is exemplified as polyalkylene glycol, (C1) 2'-modified ribonucleotide is represented by X, and (C2) ribonucleotide is represented. Nucleotides are represented by N, RNA is exemplified as (D) template nucleic acid, and RNA is exemplified as (E) primer.

[1-1. (A) RNA polymerase]
The (A) RNA polymerase used in the present invention is an RNA polymerase composed of a single subunit protein. RNA polymerase consisting of a single subunit protein can be used without particular limitation because the sequences are similar from an evolutionary point of view and the mechanism of action at the time of nucleic acid synthesis is the same. Among the single subunit proteins, RNA polymerase derived from bacteriophage is preferable from the viewpoint of RNA synthesis efficiency. Examples of the RNA polymerase derived from Bacterophage include T7 RNA polymerase, T3 RNA polymerase, K11 RNA polymerase, and SP6 RNA polymerase, and preferably T7 RNA polymerase.

(A) RNA polymerase has substrate specificity for RNA and (C1) 2'-modified ribonucleotide in the absence of the predetermined (B) polyalkylene glycol used in the present invention. If it is not, it may be a wild type or a gene-engineered mutation introduced, but a wild type is preferable.

The concentration of (A) RNA polymerase (i) in the reaction solution can be determined by (B) the amount of polyalkylene glycol or the like, and examples thereof include 0.01 to 10 μM, preferably 0.1 to 1 μM. ..

[1-2. (B) Polyalkylene Glycol]
The (B) polyalkylene glycol used in the present invention is used to change the substrate specificity of (A) RNA polymerase. Preferably, (B) polyalkylene glycol is used to reversibly change the substrate specificity of (A) RNA polymerase. (B) Polyalkylene glycol is a compound represented by the following formula (I).

In the formula (I), n is an integer and represents the number of added moles of an alkylene oxide unit, and R represents an alkylene group. The alkylene group has preferably 2 or 3 carbon atoms, and more preferably 2 carbon atoms. That is, as the (B) polyalkylene glycol, polyethylene glycol and polypropylene glycol are preferable, and polyethylene glycol is more preferable. (B) The polyalkylene glycol may be used alone or in combination of a plurality of types. Examples of the number of added moles n include 2 to 50, preferably 3 to 20.

(B) The average molecular weight of the polyalkylene glycol is 10 to 1000. The average molecular weight is calculated from the concentration of hydroxyl groups measured by the pyridine phthalic anhydride method (according to JIS K1557). When the average molecular weight is 10 or more, (i) when present at a predetermined high concentration in the reaction solution, the substrate specificity of RNA polymerase for (C1) 2'-modified ribonucleotide can be acquired. , Preferably, while gaining substrate specificity for (C1) 2'-modified ribonucleotides of RNA polymerase, substrate specificity for (C2) ribonucleotides can be lower than intrinsic substrate specificity. , (Ii) When present at a predetermined low concentration in the reaction solution, it is possible to give the intrinsic substrate specificity of RNA polymerase to (C2) ribonucleotide, and preferably RNA polymerase (C2). It can give rise to the intrinsic substrate specificity for ribonucleotides and lower the substrate specificity for (C1). When the average molecular weight is 1000 or less, (i) denaturation and aggregation of RNA polymerase can be prevented even if it is present at a predetermined high concentration in the reaction solution. From the viewpoint of obtaining these effects better, the average molecular weight of the polyalkylene glycol is preferably 50 to 800, more preferably 50 to 400, still more preferably 100 to 300, and even more preferably 150 to 250.

(B) Polyalkylene glycol is contained in (i) reaction solution at a predetermined high concentration. In the present specification, the predetermined high concentration specifically means (i) a concentration of 10% by weight or more in the solution. This allows the substrate specificity of RNA polymerase for (C1) 2'-modified ribonucleotides to be acquired in (i) reaction solution, preferably the substrate for RNA polymerase for (C1) 2'-modified ribonucleotides. While acquiring specificity, the substrate specificity for (C2) ribonucleotide can be lower than the intrinsic substrate specificity. From the viewpoint of obtaining such an effect better, the concentration of (B) polyalkylene glycol in (i) reaction solution is preferably 12% by weight or more, more preferably 15% by weight or more, still more preferably 18% by weight. It may be the above. The upper limit of the concentration of (B) polyalkylene glycol in (i) reaction solution is not particularly limited, but for example, from the viewpoint of facilitating the prevention of denaturation and aggregation of RNA polymerase, for example, 40% by weight or less, preferably 35% by weight or less. It may be more preferably 30% by weight or less, still more preferably 25% by weight or less.

[1-3. (C1) 2'-modified ribonucleotide]
(C1) The 2'-modified ribonucleotide is used as a substrate in (i) reaction solution. (C1) 2'-modified ribonucleotides are not the intrinsic substrate for (A) RNA polymerase. (C1) 2'-Modified ribonucleotide refers to a 5'-phosphate ester of ribonucleoside having a modifying group at the 2'position of ribose. Specifically, the (C1) 2'-modified ribonucleotide is selected from the group consisting of 2'-halogenated ribonucleotides and 2'-alkoxyribonucleotides.

The 2'-halogenated ribonucleotide is not particularly limited, and for example, those which are effective in nucleic acid pharmaceutical applications for the purpose of improving chemical stability (biodegradation resistance), cell membrane permeability, molecular recognition, and the like. Those skilled in the art can make appropriate choices. For example, 2'-halogenated ribonucleotides include 2'-fluorinated ribonucleotides, 2'-chloride ribonucleotides, 2'-bromated ribonucleotides, and 2'-iodized ribonucleotides, and substrate specificity. From the viewpoint of the above, a fluorinated ribonucleotide is preferable.

The 2'-alkoxyribonucleotide is not particularly limited, and those which are considered to be effective in nucleic acid pharmaceutical applications for the purpose of improving chemical stability (biodegradation resistance), cell membrane permeability, molecular recognition, etc. The vendor can select as appropriate. Examples of the carbon number of the 2'-alkoxy group in the 2'-alkoxyribonucleotide include 1 to 3, preferably 1 and 2, and more preferably 1 from the viewpoint of substrate specificity and the like.

The nucleobase of the (C1) 2'-modified ribonucleotide is not particularly limited, and a nucleobase complementary to the template nucleic acid is appropriately selected. Specific examples thereof include adenine, thymine, guanine, uracil, and cytosine.

As the (C1) 2'-modified ribonucleotide, one type may be used alone, a plurality of types having different modifying groups may be used in combination, or a plurality of types having different bases may be combined. It may be used, or a plurality of species having different modifying groups and bases may be used in combination.

The concentration of (C1) 2'-modified ribonucleotide in (i) reaction solution is, for example, 5 to 100 μM, preferably 10 to 50 μM.

[1-4. (D) Template nucleic acid]
(D) The template nucleic acid is not particularly limited, and any origin, base length, sequence, etc. can be selected. Further, DNA and RNA may be used regardless of whether they are single-stranded or double-stranded. For example, genomic nucleic acids such as genomic DNA and genomic RNA of organisms; nucleic acid fragments obtained by cleaving the genomic nucleic acids by physical means or restriction enzyme digestion; nucleic acid constructs in which nucleic acid fragments are inserted into vectors such as plasmids and phages; Nucleic acid isolated from a possible sample; synthetic nucleic acid synthesized using an automatic nucleic acid synthesizer or the like can be mentioned.

In the nucleic acid synthesis method of the present invention, although RNA polymerase is used, nucleic acid synthesis can be started by using (E) primers even if (D) the template nucleic acid does not contain a promoter sequence. Therefore, from the viewpoint of gaining the degree of freedom in the sequence of the 5'end of the product to be obtained by the nucleic acid synthesis method of the present invention, (i) the reaction solution contains (E) a primer, and (D) the template nucleic acid is a promoter sequence. It is preferable not to contain. The promoter sequence is a promoter sequence of RNA polymerase, and refers to a sequence corresponding to (i) RNA polymerase used together in the reaction solution. For example, when (i) T7 RNA polymerase is used as RNA polymerase in the reaction solution, it means T7 promoter.

(D) The nucleic acid length of the template nucleic acid is not particularly limited, and can be appropriately determined by those skilled in the art according to the nucleic acid length of the product to be obtained by the nucleic acid synthesis method of the present invention. For example, in the case of obtaining a nucleic acid drug as a product, the length of the (D) template nucleic acid may be, for example, 20 to 110 mer, preferably 20 to 70 mer. When the (E) primer is used, it may be longer than the length of the (E) primer, and examples thereof include 21 to 110 mer, preferably 21 to 70 mer.

Examples of the concentration of (D) the template nucleic acid in (i) the reaction solution include 0.01 to 10 μM, preferably 0.1 to 1 μM.

[1-5. (E) Primer]
In the nucleic acid synthesis method of the present invention, although RNA polymerase is used, the (E) primer which is not originally used together with RNA polymerase is used from the viewpoint of acquiring the degree of freedom of the sequence at the 5'end of the product. It is preferable to use it. Since the nucleic acid synthesis method of the present invention extends the nucleic acid from the 3'end of the (E) primer, the (E) primer constitutes a part of the product according to the present invention.

The (E) primer has a sequence complementary to a portion of the (D) template nucleic acid. The 5'end of the complementary sequence is (D) any position on the template nucleic acid (but 3'end closer to the position where the (C1) 2'-modified ribonucleotide should be complementarily bound), preferably (D). ) Corresponds to the position of the 3'end on the template nucleic acid, and the 3'end of the complementary sequence is 3 more than the position where the (C1) 2'-modified ribonucleotide on the (D) template nucleic acid should be complementarily bound. It corresponds to the adjacent position on the'terminal side, preferably the 3'end side of the position where the (C1) 2'-modified ribonucleotide on the (D) template nucleic acid should be complementarily bound. The sequence design of the primer can be determined by examining the sequence of the (D) template nucleic acid in advance. (D) A database such as GeneBank or EBI can be used to investigate the base sequence of the template nucleic acid.

The (E) primer may be obtained by chemical synthesis by a phosphoramidite method or the like, or (D) restriction enzyme digestion of a complementary nucleic acid of a template nucleic acid. The base length of the (E) primer is appropriately selected by those skilled in the art to be not too short to specifically recognize the (D) template nucleic acid and not too long to not induce a non-specific reaction. .. The length of the (E) primer can be determined depending on many factors such as the sequence information of the (D) template nucleic acid such as GC content, the reaction temperature, and the hybridization reaction conditions such as the salt concentration in the reaction solution. However, for example, 20 to 50 mer can be mentioned.

(E) The primer can be freely designed according to the product to be obtained as long as it has the above-mentioned basic complementary sequence. For example, the (E) primer may be DNA, RNA, or a mixed sequence of DNA and RNA. Further, the (E) primer may have a modifying group at the 5'end. The modifying group is not particularly limited, and examples thereof include a signal group and a reactive functional group.

Examples of the signal group include a group derived from a fluorescent substance, a radioactive element-containing substance, a magnetic substance and the like. From the viewpoint of detectability and the like, the above-mentioned signal substance is preferably a fluorescent substance. Examples of the fluorescent substance include cyanine dyes such as fluorescein dyes and indocyanine dyes, fluorescent dyes such as rhodamine dyes; fluorescent proteins such as GFP; nanoparticles such as gold colloids and quantum dots. Examples of the above-mentioned radioactive element-containing substance include sugars, amino acids, nucleic acids and the like labeled with a radioisotope such as 18F. Examples of the above-mentioned magnetic substance include magnetic substances such as ferrichrome, ferrite nanoparticles, and nanomagnetic particles.

Examples of the reactive functional group include covalent groups capable of chemically reacting with a modifying reagent when the product obtained by the nucleic acid synthesis method of the present invention is subjected to chemical modification or the like. More specifically, examples of the reactive functional group include an amino group and a thiol group.

The concentration of the primer (i) in the reaction solution can be determined by the concentration of the template nucleic acid (D), and examples thereof include 0.01 to 10 μM, preferably 0.1 to 1 μM.

[1-6. Other ingredients]
(i) The reaction solution contains the above-mentioned components in the buffer solution. Examples of the buffer solution generally include a solution containing an appropriate buffer component, a magnesium salt, or the like in water. Examples of the buffer component include phosphates such as trisacetic acid, trishydrochloric acid, sodium phosphate, and calcium phosphate. The final concentration of the buffer component is 5 mM to 100 mM, the pH is pH 6.0 to 9.5, and more preferably pH 7.0 to 8.0. Examples of the magnesium salt include magnesium chloride and magnesium acetate. Further, if necessary, a potassium salt such as KCl, a surfactant such as DMSO, betaine, gelatin, and Triton may be contained.

(i) The reaction solution may further contain deoxyribonucleotides as a substrate. (i) Since (A) RNA polymerase in the reaction solution has substrate specificity for deoxyribonucleotides which are not the original substrate of (A) RNA polymerase, it can be incorporated as a constituent base in the product.

[1-7. (i) Synthesis of modified nucleic acid in reaction solution]
(i) The reaction solution is complementary to (D) the template nucleic acid by being subjected to conditions of, for example, 10 to 45 ° C., preferably 20 to 37 ° C. for 2 to 48 hours, preferably 12 to 24 hours. Modified nucleic acids can be synthesized. When synthesizing using the (E) primer, the modified nucleic acid extends from the 3'end of the (E) primer. (i) The base length of the modified nucleic acid synthesized in the reaction solution (that is, (i) the number of substrates to be incorporated into one (A) RNA polymerase in the reaction solution) is, for example, 1 or more, preferably 1 to 50. It may be adjusted as appropriate depending on the concentration of the substrate (C1) 2'-modified ribonucleotide and the reaction time.

[1-8. (ii) Preparation of reaction solution]
In the present invention, (i) RNA can be further extended from the modified nucleic acid synthesized in the reaction solution. In this case, (i) a reaction solution is prepared from (i) a reaction solution. (ii) The reaction solution contains (i) RNA polymerase and (D) template nucleic acid that were present in the reaction solution, and (C2) ribonucleotide as a substrate. Further, in (ii) the reaction solution, the concentration of (B) polyalkylene glycol is diluted to a low concentration of 5% by weight or less. As a result, (i) RNA polymerase that had acquired substrate specificity for (C1) 2'-modified ribonucleotide in the reaction solution loses the substrate specificity and has the original substrate specificity. That is, it can be restored to a state having substrate specificity for (C2) ribonucleotide. From the viewpoint of more preferably producing such a recovery effect, the concentration of (ii) polyalkylene glycol in the reaction solution may be preferably 3% by weight or less, more preferably 1% by weight or less. Further, the pH of (ii) the reaction solution is pH 7.0 to 8.0 in order to maintain the complementary binding state of (i) the modified nucleic acid synthesized in the reaction solution and (D) the template nucleic acid. Is preferable.

The method for preparing (ii) the reaction solution from the (i) reaction solution is not particularly limited, and examples thereof include the following methods. For example, by preparing a preparation solution containing (B) polyalkylene glycol and (C2) ribonucleotide in a buffer solution for dilution, and mixing with (i) reaction solution after the modified nucleic acid has been synthesized. , (Ii) A reaction solution can be prepared. As the buffer solution, the same buffer solution as described above is used. In (ii) preparation of the reaction solution, it is not necessary to remove the unreacted (E) primer from (i) the reaction solution after the modified nucleic acid is synthesized, and (ii) subsequently mix it in the reaction solution. May be good.

Further, (D) the template nucleic acid is prepared in a state of being immobilized on the solid-phase carrier in advance, and (i) the reaction is carried out in the reaction solution, and after the reaction, the solid-phase carrier is taken out and mixed with the above-mentioned preparation solution. By doing so, (ii) a reaction solution can also be prepared. The material of the solid-phase carrier is not particularly limited, and examples thereof include glass, metal, and a high molecular polymer (for example, polystyrene). The shape of the solid-phase carrier is not particularly limited, and examples thereof include a plate-like (substrate carrier) and a particle-like (bead carrier). A bead carrier made of a high molecular polymer such as polystyrene is preferable. Further, the mode of immobilization between the solid phase carrier and the (D) template nucleic acid is not particularly limited, and covalent or non-covalent bond is not limited. For example, immobilization conditions for binding the (D) template nucleic acid to the solid phase carrier. (D) It is preferable that it is a non-covalent bond from the viewpoint that it has no effect on the template nucleic acid and is easy to operate, and from the viewpoint of obtaining a stronger binding force, biotin-avidins (for example, avidin, streptavidin). It is preferably a bond of avidin or the like). As a method for immobilizing the (D) template nucleic acid on a solid-phase carrier, the (D) template nucleic acid having an immobilization functional group such as a biotinyl group at the 3'end and an immobilization substance such as avidin on the surface. Alternatively, a method of mixing a solid phase carrier to which an immobilization functional group is chemically bonded in a buffer solution can be mentioned.

[1-9. (ii) RNA synthesis in the reaction solution]
(Ii) Substrate specificity for (C1) 2'-modified ribonucleotide obtained in (i) reaction solution by diluting the concentration of (B) polyalkylene glycol to 5% by weight or less in the reaction solution. (A) Restores the original substrate specificity of RNA polymerase. Therefore, (A) RNA polymerase that has restored the original substrate specificity can take up (C2) ribonucleotide, which is the original substrate in (ii) reaction. Therefore, (ii) the reaction solution is subjected to conditions of, for example, 10 to 45 ° C., preferably 20 to 37 ° C. for 2 to 48 hours, preferably 12 to 24 hours, from the 3'end of the modified nucleic acid (D). RNA complementary to the template nucleic acid can be extended. This makes it possible to synthesize a hybrid RNA having a modifying group in a part of the RNA chain as a synthetic product.

Although not shown, after (ii) RNA elongation to some extent in the reaction solution (specifically, (D) before elongation to the 5'end of the template nucleic acid), (B) poly Alkylene glycols and optionally (C1) 2'-modified ribonucleotides and / or deoxyribonucleotides as non-intrinsic substrates may be added to the reaction solution (ii). In this case, by increasing the concentration of (B) polyalkylene glycol to a concentration of 10% by weight or more again, (C1) 2'-modified ribonucleotide and / or with respect to (A) RNA polymerase as in (i) reaction solution. Substrate specificity for deoxyribonucleotides can be acquired. Therefore, a nucleobase derived from a non-intrinsic substrate can be further extended from the 3'end of the RNA extension chain. In this way, by varying the concentration of (B) polyalkylene glycol, (A) the activity of RNA polymerase is changed, and the synthesis of non-intrinsic substrates (2'-modified ribonucleotide, deoxyribonucleotide) and intrinsic Substrate (ribonucleotide) synthesis can also be alternated. The synthetic product thus obtained is a hybrid RNA having a nucleobase derived from a non-intrinsic substrate (at least one 2'-modified ribonucleotide) at a plurality of sites in the RNA chain.

As described above, according to the method of the present invention, (A) without changing the sequence of RNA polymerase, (B) the concentration of polypropylene glycol is changed to change the chemical environment in the reaction solution, which is an extremely simple method. Hybrid RNA can be easily synthesized by changing the reaction efficiency of the substrate and using a simple combination of reagents.

After completion of the reaction, (ii) recovery of the hybrid RNA from the reaction solution is performed by (ii) breaking the double strand between the template nucleic acid and the hybrid RNA by (ii) raising the pH in the reaction solution, etc., and performing chromatography. This can be done by separating and purifying the hybrid RNA with or the like. Enzymes such as ribonuclease H can be used to break the double chain. Further, in order to perform separation and purification, any nucleic acid purification method such as chromatography and gel electrophoresis can be mentioned. (D) When the template nucleic acid is used in a state of being immobilized on a solid phase carrier in advance, the double chain is broken, the solid phase is removed by solid-liquid separation, and then the above-mentioned arbitrary nucleic acid purification method is performed. be able to. The nucleic acid length of the hybrid RNA obtained by the present invention is not particularly limited because it may vary depending on the intended use, but is not particularly limited, for example, from several mer to several hundred mer, specifically from 10 to 100 mer, preferably from 10 to 60 mer, and more preferably from 10 to. 40mer can be mentioned.

[1-10. Hybrid RNA]
The hybrid RNA synthesized by the nucleic acid synthesis method of the present invention has improved chemical stability (biodegradation resistance), cell membrane permeability, and / or molecular recognition, etc., as compared with the case without modification. Has a function. For example, it is stable to endogenous nucleases in cells and exhibits high affinity for complementary target sequences. In particular, when the hybrid RNA contains a 2'-fluorolated base, the affinity and stability are significantly improved. Hybrid RNA is useful as a nucleic acid drug such as ribozyme, aptamer, and oligonucleotide. Hybrid RNA is also useful as a research tool. Therefore, the usefulness of unnatural nucleic acid can be screened by preparing a hybrid RNA having various sequences using the nucleic acid synthesis method of the present invention to prepare an unnatural nucleic acid library.

[2. Nucleic acid synthesis kit]
The nucleic acid synthesis kit of the present invention is a kit for carrying out the above-mentioned nucleic acid synthesis method, and contains (A) RNA polymerase, (B) polyalkylene glycol, and (C1) 2'-modified ribonucleotide. .. These components are used to construct (i) reaction solution with (D) template nucleic acid, or (D) template nucleic acid and (E) primer.

The nucleic acid synthesis kit of the present invention may further contain any other component for carrying out the nucleic acid synthesis method described above. Other components that may be further included include (D) a solid phase carrier for immobilizing the template nucleic acid, ribonuclease H, (D) the template nucleic acid, and (E) primers, and (ii) constructing a reaction solution. Examples thereof include (B) a buffer solution for dilution containing no polyalkylene glycol, and (C2) ribonucleotides, etc., which may be used one or more. Among these, it is preferable to contain at least a solid phase carrier and / or a ribonuclease H for immobilizing the (D) template nucleic acid. Considering the versatility of the nucleic acid synthesis kit, it is preferable to configure the template nucleic acid (D) and the primer (E) so that they can be prepared by the user without being included in the nucleic acid synthesis kit. Thereby, the user can freely select any (D) template nucleic acid and / or (E) primer, and by using the nucleic acid synthesis kit of the present invention, the selected (D) template nucleic acid and / or (E) The desired modified nucleic acid according to the primer can be obtained.

In the nucleic acid synthesis kit of the present invention, the above-mentioned components may be contained in the form of individually packaged items. Alternatively, a plurality of components selected from the above-mentioned components may be mixed in one item. Other than the solid phase carrier, they may be in a liquid state (for example, a solution in which a component is dissolved in a buffer solution, a glycerol stock, etc.) or a solid state (for example, a crystal, a lyophilized product, etc.). For example, one item can be composed of (A) RNA polymerase and (B) polyalkylene glycol as a mixed solution. (A) Considering the stability of RNA polymerase, it is preferable to configure it as a separate item.

(B) Items containing polyalkylene glycol are usually in a liquid state (polyalkylene glycol solution). The concentration of (B) polyalkylene glycol in the polyalkylene glycol solution is (i) in the reaction solution when (i) the reaction solution is constructed by mixing with other components when using the nucleic acid synthesis kit of the present invention. (B) The concentration of the polyalkylene glycol is adjusted so that it can be easily set to a predetermined concentration (10% by weight or more). For example, in the polyalkylene glycol solution, the concentration of (B) polyalkylene glycol may be (i) twice the predetermined concentration set in the reaction solution. In this case, by mixing with the same volume of liquid containing other components, (i) reaction liquid containing (B) polyalkylene glycol having a predetermined concentration can be easily prepared.

Further, (ii) a buffer solution for dilution containing no polyalkylene glycol for constructing a reaction solution and (C2) a ribonucleotide may be included in the kit as separate items. It may be included in the kit as one item (preparation liquid) in which both components are mixed. In this case, the user can add (i) a diluting buffer solution containing no polyalkylene glycol and (C2) a ribonucleotide to the reaction solution to obtain a predetermined concentration (preferably) of the polyalkylene glycol. (Ii) The reaction solution diluted to 5% by weight or less) can be easily prepared.

The above-mentioned (A) RNA polymerase, (B) polyalkylene glycol, (C1) 2'-modified ribonucleotide, (D) template nucleic acid, (E) primer included in or may be included in the nucleic acid synthesis kit of the present invention. , (C2) Details of the ribonucleotide, solid phase carrier, ribonuclease H, buffer solution, and adjusting solution and the method of use are as described in "1. Nucleic acid synthesis method" described above.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited thereto.

[reagent]
Regarding RNA polymerase, T7 RNAP was purchased from Takara Bio Inc., KF was purchased from New England Biolab Japan Inc., and tC9Y was prepared by in vitro transcription from DNA by PCR amplification.
EG, PEG200, and PEG8000 were purchased from Wako Pure Chemical Industries, Ltd. and used without purification. 2'-F NTPs and 2'-OMe NTPs were purchased from Trilink.
The NTP solution was purchased from Thermo Fisher Scientific Co., Ltd., and the dNTP solution was purchased from Toyobo Co., Ltd.
The template RNA strand was obtained in vitro by an in vitro transcription method using the DNA strand as a template. All DNA was HPLC grade and purchased from Eurofins Genomics, Inc.
The FITC-labeled primer was purchased from Nippon Bioservice Co., Ltd.
Other reagents were purchased from Wako Pure Chemical Industries, Ltd.

[Test Example 1: Nucleic acid synthesis using different RNA polymerases and different concentrations of PEG200]
For ribozyme-type polymerase (tC9Y), T7 RNA polymerase (T7 RNAP), and Escherichia coli DNA polymerase (KF), as shown in FIG. 2, 39mer RNA strand (5'-GUCAAUGACACGCUUCGCXCGGUUGGCAG (SEQ ID NO: 1)) was used as a template. A primer extension assay was performed using FITC-labeled 10 mer RNA (5'-CUGUUAACCG (SEQ ID NO: 2)) as a primer. The name of the template (template X) corresponds to the base X in the template sequence. That is, if the base X in the template sequence is A (adenine), the name of the template is template A.

Polymerase (0.5 μM), 0-20 wt% polyethylene glycol 200 (PEG200, average molecular weight 200), 0.5 μM FITC-labeled RNA primer (complementary to template), 0.5 μM RNA template A, They were also incubated with 50 mM Tris-HCl (pH 8.0) and 100 μM NTPs or dNTPs in 10 mM MgCl ₂ at 25 ° C. for 12 hours. The product was added with 5 μL of loading solution (3x loading die), 125 mM EDTA, 4.8 M of urea and 2.6 ng / μL of competing template and separated using modified polyacrylamide gel electrophoresis (modified PAGE). .. Bands corresponding to the elongated and unreacted primers were quantified by measuring the fluorescence intensity.

The results are shown in FIG. 3 (a) and 3 (b) show a modified PAGE gel electrophoresis photograph using a ribozyme-type polymerase (tC9Y) and a graph of the primer elongation rate with respect to the content of PEG200, respectively.

3 (c) and 3 (d) show a denatured PAGE gel electrophoresis photograph using T7 RNA polymerase (T7 RNAP), and a graph of primer elongation rate with respect to the content of PEG200, respectively. 3 (e) and 3 (f) show a modified PAGE gel electrophoresis photograph using Escherichia coli DNA polymerase (KF) and a graph of primer extension rate with respect to the content of PEG200, respectively. In the PAGE analysis, the conditions in each lane were that lane 1 was an unreacted primer, lane 2 was NTPs and 0% by weight PEG200, lane 3 was NTPs and 5% by weight PEG200, and lane 4 was NTPs and 10% by weight. PEG200, lane 5 is NTPs and 15% by weight PEG200, lane 6 is NTPs and 20% by weight PEG200, lane 7 is dNTPs and 0% by weight PEG200, lane 8 is dNTPs and 5% by weight PEG200, lane 9 is dNTPs and 10% by weight PEG200, lane 10 is dNTPs and 15% by weight PEG200, lane 11 is dNTPs and 20% by weight PEG200, and lane 12 is an unreacted primer. In addition, the band positions of the product (n + 1, 2, 3, 4, 5, 6: n represent the primer base length) are also shown.

The primer elongation rate was derived by dividing the fluorescence intensity of the extension product band (LAU / mm ² ) by the total fluorescence intensity of all detection bands (LAU / mm ² ). The line graph represented by the broken line shows the results when NTP, which is the original substrate, is used, and the line graph represented by the solid line shows the results when dNTP, which is not the original substrate, is used. As for the data points, the data of T7 RNAP is the average of 6 samples, and the other data is the average of 3 samples (hereinafter, the average of 3 samples in all examples). The error represents the standard deviation.

As shown in FIGS. 3 (a) and 3 (b), RNA-dependent RNA synthesis by ribozyme-type polymerase (tC9Y) is inadequate when PEG200 is not contained in the buffer, only 3.3%. Primer was only elongated. The reaction was clearly accelerated in the presence of PEG200, and the primer extension fraction increased with increasing concentration of PEG200. When PEG200 was 20% by weight, 87% of the primers were extended, leading to the polymerization of 6 NTPs.
When dNTPs were used as the substrate, only slight elongation was observed in the absence of PEG200. Elongation of dNTPs was promoted when 20% by weight PEG200 was included, and 82% of the primers were extended.
As described above, in the ribozyme-type polymerase (tC9Y), the polymerization efficiency of both dNTPs and NTPs increased as the concentration of PEG200 increased. (In addition, when the same test using ethylene glycol instead of PEG200, the degree of elongation of the primer is lower than that when PEG200 is used, and when the same test is performed using PEG8000 instead of PEG200, either NTPs or dNTPs are used. It was separately confirmed that even when PEG200 was used as a substrate, primer elongation was promoted to a degree comparable to that when PEG200 was used.) In addition, PEG reduces the stability of RNA helix and reduces the efficiency of primer elongation. Thus, the ribozyme-type polymerase (tC9Y) synthesizes both RNA and DNA with molecular environment-dependent efficiency.

As shown in FIGS. 3 (c) and 3 (d), for primer extension using T7 RNA polymerase (T7 RNAP), 45% of the primers are extended using NTPs as a substrate when PEG200 is not contained. Four or more NTPs were polymerized. In the presence of 10 wt% PEG200, the primer elongation was comparable to that without PEG200. In the presence of 20% by weight PEG200, only 23% of the primers were extended. On the other hand, with respect to the substrate dNTP, the extended fraction of the primer increased with increasing concentration of PEG200. In the absence of PEG200, only 13% of the primers were extended, whereas in the presence of 20% by weight PEG200, 69% of the primers were extended. (The suppression of RNA synthesis and the promotion of DNA synthesis by PEG200 is the effect of PEG200 on the conformation of T7 RNA polymerase, the dissociation of the complex consisting of T7 RNA polymerase, primers, template nucleic acids, and nucleic acids, and / Alternatively, it may be due to a change in substrate specificity. It was separately confirmed by circularly polarized dichroism analysis that PEG200 did not denature T7 RNA polymerase. Furthermore, T7 RNA polymerase was added in the presence of PEG200 before the reaction started. It was also separately confirmed that the product did not change even after preincubation. From these results, it is considered that PEG200 has an important influence on the substrate specificity.)

As shown in FIGS. 3 (e) and 3 (f), RNA-dependent DNA elongation from RNA primers by E. coli DNA polymerase (KF) was promoted by PEG200. In the absence of PEG200, 67% of the primers were extended with dNTPs as the substrate. In the presence of 20% by weight PEG200, 93% of the primers were extended. On the other hand, no elongation of NTPs was observed under any conditions of PEG200.

[Test Example 2: Nucleic acid synthesis using PEG (or EG) with different numbers of added moles]
For T7 RNA polymerase (T7 RNAP), instead of 0-20% by weight polyethylene glycol 200, 0-20% by weight ethylene glycol (EG) or 0-20% by weight polyethylene glycol 8000 (PEG8000, average molecular weight 8000). The primer was extended in the same manner as in Test Example 1 except that the above was used. That is, T7 RNA polymerase (T7 RNAP) (0.5 μM), 0-20 wt% EG or 0-20 wt% PEH8000, 0.5 μM FITC-labeled RNA primer (complementary to the template), 0. Incubated at 25 ° C. for 12 hours with .5 μM RNA template A and 100 μM NTPs or dNTPs in 50 mM Tris-HCl (pH 8.0) and 10 mM MgCl ₂ . The obtained product was separated by using modified polyacrylamide gel electrophoresis (modified PAGE) in the same manner as in Test Example 1, and the bands corresponding to the extended and unreacted primers were measured for fluorescence intensity. Quantified.

The results are shown in FIG. FIG. 4 (a) is a graph of the primer extension rate with respect to the content of PEG200 shown in FIG. 3 obtained in Test Example 1, and FIG. 4 (b) is a primer with respect to the content of EG obtained in this test example. The graph of the elongation rate, FIG. 4 (c), shows the graph of the primer extension rate with respect to the content of PEG8000 obtained in this test example. In each case, the line graph represented by the broken line shows the results when NTP, which is the original substrate, is used, and the line graph represented by the solid line shows the results when dNTP, which is not the original substrate, is used.

As shown in FIG. 4 (b), when EG was used, primer elongation was promoted for both NTPs and dNTPs as the content increased. On the other hand, as shown in FIG. 4 (c), when PEG8000 was used, primer extension was suppressed for both NTPs and dNTPs. From this result, it is considered that PEG8000 denatured or aggregated T7 RNA polymerase (T7 RNAP).
(It was separately confirmed that the effect of the difference in the molecular weight of PEG shown in this way is stronger with T7 RNA polymerase (T7 RNAP) than with ribozyme-type polymerase (tC9Y).)

[Example 1: Nucleic acid synthesis using 2'-fluorinated ribonucleotide as a substrate]
As shown in FIG. 5, T7 RNA polymerase (T7 RNAP), template A and FITC-labeled primers are used, and 2'-fluorinated ribonucleotide (2'-FUTP or 2'-F TTP) is used as a substrate. Using the same primer extension assay as in Test Example 1.

T7 RNA polymerase (T7 RNAP) (0.5 μM), 20 wt% polyethylene glycol 200, 0.5 μM FITC-labeled RNA primer (complementary to the template), 0.5 μM RNA template A, and 50 mM Tris. Incubated at 25 ° C. for 12 hours with -HCl (pH 8.0) and 100 μM 2'-F UTP or 2'-F TTP in 10 mM MgCl ₂ . Similar to Test Example 1, the product was separated by using modified polyacrylamide gel electrophoresis (modified PAGE), and the bands corresponding to the extended and unreacted primers were quantified by measuring the fluorescence intensity. did.

The results are shown in FIG. In FIG. 6, the results obtained in Test Example 1 using 2'-F UTP or 2'-F TTP as the substrate as well as the results obtained in Test Example 1 using dTTP as the substrate (for comparison) are also shown. Also described. 6 (a) and 6 (b) show a modified PAGE gel electrophoresis photograph for each substrate and a graph of the primer elongation rate for each substrate, respectively. In the PAGE analysis, the conditions in each lane were that the left lane was before the reaction (for comparison), the middle lane was free of PEG200 (for comparison), and the right lane contained 20% by weight of PEG200.

As shown in FIG. 6, in both 2'-F UTP and 2'-F TTP, primer extension was promoted by 20% by weight of PEG200. Of these, 2'-FTTP having thymidine, which is a canonical base of DNA, had better elongation efficiency.

[Example 2: Nucleic acid synthesis using different 2'-modified ribonucleotides as substrates and different template nucleic acids]
As shown in FIG. 7, using T7 RNA polymerase (T7 RNAP), template nucleic acid (template U, template G, template C or template A) and FITC-labeled primers, 2'-fluorinated ribonucleotide (2) Primer extension assay was performed in the same manner as in Example 1 using'-F NTPs) or 2'-methoxyribonucleotides (2'-OMe NTPs). Also, for reference or comparison, a primer extension assay was similarly performed using unmodified NTPs or dNTPs as a substrate.

The results are shown in FIG. FIG. 8 shows a graph of primer elongation rate for each substrate. As shown in FIG. 8, when unmodified NTPs other than unmodified UTP were used as the substrate (for comparison), the elongation improving effect by 20% by weight of PEG was hardly observed, but dNTPs were used as the substrate. When the case (for reference) and 2'-modified NTPs (2'-F NTPs and 2'-OMe NTPs) were used, the elongation improving effect by 20% by weight of PEG was obtained. In particular, 2'-F CTP, 2'-OMe CTP, dGTP, 2'-F GTP, 2'-OMe GTP, dUTP, 2'-OMe UTP, dTTP or 2'-F TTP was used as the substrate. In some cases, the degree of polymer elongation was more than double that of the case without PEG (for comparison). From these data, it was shown that T7 RNA polymerase can incorporate 2'-modified NTPs for any template nucleic acid by simply changing the molecular environment in the reaction solution.

[Example 3: Synthesis of hybrid RNA (chimeric nucleic acid)]
As shown in FIG. 9, 2'-fluorinated ribonucleotide (2'-FUTP, or 2'-" using T7 RNA polymerase, template A and FITC-labeled primers in the presence of 20 wt% PEG Modified nucleic acid synthesis was performed by performing the same primer extension as in Example 1 using FTTP (1st step), and then NTPs solution was added and diluted 5-fold to reduce the concentration of PEG to 4% by weight. Then, a reaction solution newly containing NTPs as a substrate was prepared, and natural nucleic acid synthesis was performed by continuing the primer extension reaction in the prepared reaction solution (2nd step).

The results are shown in FIG. FIGS. 10 (a) and 10 (b) show a modified PAGE gel electrophoresis photograph for each substrate used in the 1st step, and a graph of the primer elongation rate or RNA elongation rate for each substrate used in the 1st step, respectively. show. In the PAGE analysis, the conditions for each lane are that the left lane is after the 1st step and the right lane is after the 2nd step.

As shown in FIG. 10, in the 1st step, modified nucleic acids were synthesized by extending dTTP (for reference), which is not the original substrate of T7 RNA polymerase, 2'-FUTP and 2'-FTTP, while the 2nd step. Then, a natural nucleic acid chain was synthesized by extending NTPs, which are the original substrates. From this, in the 1st step, 20% by weight of PEG formed a substrate enzyme complex inactive with respect to the substrate UTP, while in the 2nd step, the PEG concentration was diluted to 4% by weight. The inactive substrate enzyme complex was altered and activated with respect to the substrate UTP, and the uptake of the substrate UTP proceeded. Furthermore, during RNA elongation in the 2nd step, the inactive substrate-enzyme complex as formed in the 1st step was not formed, and the natural nucleic acid chain was elongated. In this way, a chimeric RNA containing 2'-F NTPs that cannot be synthesized by T7 RNA polymerase (5'-FITC-CUGCCAACCGNGCGAAGCGUGUCAUUGAC-3'(SEQ ID NO: 3); in the sequence, N is a 2'-fluorinated base. Represented) could be obtained using the same T7 RNA polymerase.

The unreacted primer in the 1st step was not removed by the time the 2nd step was performed. However, when either dTTP (for reference) or 2'-FTTP was used as the substrate, the band of nucleic acid with one base extended was produced in the 2nd step more than in the product obtained in the 1st step. It was confirmed that there was a clear decrease in the thing. From this result, it was shown that the natural nucleic acid chain subsequently extended in the 2nd step is extended from the deoxyribonucleotide or 2'-modified ribonucleotide which is not the original substrate.

Preferred embodiments of the present invention are as described above, but the present invention is not limited thereto, and various embodiments that do not deviate from the gist of the present invention are made elsewhere.

SEQ ID NO: 1 is a synthetic nucleic acid, and the 19th base is A, C, G, or U.
SEQ ID NO: 2 is a synthetic nucleic acid.
SEQ ID NO: 3 is a synthetic nucleic acid, the 11th base is A, C, G, or U, and the 11th nucleotide is a 2'-fluororibonucleotide.

Claims (9)

Complementing the (D) template nucleic acid in (i) reaction solution containing (A) RNA polymerase, (B) polyalkylene glycol, (C1) 2'-modified ribonucleotide, and (D) template nucleic acid. Including the step of synthesizing a modified nucleic acid
The (A) RNA polymerase is a bacteriophage-derived RNA polymerase .
The (B) polyalkylene glycol has an average molecular weight of 100 to 1000, and (i) the concentration in the reaction solution is 10% by weight or more.
A nucleic acid synthesis method in which the (C1) 2'-modified ribonucleotide is selected from the group consisting of 2'-halogenated ribonucleotides and 2'-alkoxyribonucleotides. The nucleic acid synthesis according to claim 1, wherein the (D) template nucleic acid does not contain a promoter sequence, and (i) the reaction solution further contains (E) a primer having a sequence complementary to a part of (D). Law. The nucleic acid synthesis method according to claim 1 or 2 , wherein the polyalkylene glycol (B) is polyethylene glycol. The nucleic acid synthesis method according to any one of claims 1 to 3 , wherein the 2'-halogenated ribonucleotide is a 2'-fluororibonucleotide. The nucleic acid synthesis method according to any one of claims 1 to 4 , wherein the 2'-alkoxy group in the 2'-alkoxyribonucleotide has 1 to 3 carbon atoms. The nucleic acid synthesis method according to any one of claims 2 to 5 , wherein the primer (E) has a modifying group at the 5'end. From the reaction solution (i), a reaction solution (ii) containing (C2) ribonucleotide and diluted so that the concentration of the polyalkylene glycol (B) was 5% by weight or less was prepared, and the reaction solution (ii) was prepared. The nucleic acid synthesis method according to any one of claims 1 to 6 , further comprising a step of extending RNA complementary to the (D) template nucleic acid from the modified nucleic acid in a reaction solution. A kit for performing the nucleic acid synthesis method according to any one of claims 1 to 7 .
It comprises (A) RNA polymerase, (B) polyalkylene glycol, and (C1) 2'-modified ribonucleotide.
The (A) RNA polymerase is a bacteriophage-derived RNA polymerase .
The average molecular weight of the (B) polyalkylene glycol is 100 to 1000, and the polyalkylene glycol has an average molecular weight of 100 to 1000.
A nucleic acid synthesis kit in which the (C1) 2'-modified ribonucleotide is selected from the group consisting of 2'-halogenated ribonucleotides and 2'-alkoxyribonucleotides. The nucleic acid synthesis kit of claim 8 , further comprising a solid phase carrier and / or ribonuclease H for immobilizing the template nucleic acid.

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