Method for producing hairpin nucleic acid

Abstract

To provide a method for efficiently producing a hairpin nucleic acid with a relatively long sequence while avoiding the use of excess third nucleic acid.SOLUTION: The present invention provides a method for producing a hairpin nucleic acid with a protruding region by connecting a first nucleic acid, which comprises a first connection part, and sequentially from the connection end, comprises a stem region 1, a loop region, a stem region 2, and the protruding region, to a second nucleic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing a hairpin nucleic acid and a method for extending a hairpin nucleic acid. [Background technology]

[0002] Nucleic acid medicines are molecular targeted therapeutic drugs that are based on nucleic acid molecules and specifically bind to target nucleic acids or proteins to suppress their functions. Nucleic acid medicines have attracted attention as new medicines for diseases that were previously difficult to treat, and in fact, in the 2010s, groundbreaking new drugs such as Givlaari targeting the liver as a disease site were born both domestically and internationally (Non-Patent Document 1). In addition, the inventors of the present invention have proposed the application of nucleic acid molecules that form hairpin structures (hereinafter often abbreviated as "hairpin nucleic acids" in this specification) to medical uses (Non-Patent Document 2, Patent Document 1). In this way, a variety of nucleic acid medicines have been developed, and their practical use is expected.

[0003] In order to commercialize nucleic acid medicines, the need to manufacture large amounts of nucleic acids is expected to increase rapidly in the future, and improving the efficiency of nucleic acid production is a major challenge. Generally, nucleic acids are synthesized by chemical synthesis such as the phosphoramidite method, but the efficiency decreases as the number of bases increases. This is because the efficiency of nucleic acid synthesis is the value obtained by multiplying the efficiency of a single base extension reaction by the number of bases ((Efficiency of nucleic acid synthesis) = (Efficiency of a single base extension reaction) ^ (number of bases)). Therefore, even if the efficiency of a single base extension reaction is improved, it is not easy to dramatically improve the efficiency of manufacturing relatively long nucleic acids such as hairpin nucleic acids.

[0004] To address this issue, a method for producing long nucleic acids has been widely adopted in which the final product, a long nucleic acid, is divided into relatively short nucleic acid fragments, synthesized, and then linked together. In this method (FIG. 1), when two nucleic acids (the first nucleic acid and the second nucleic acid in FIG. 1) are linked together, a third nucleic acid that is complementary to the base sequence of the linking portion is used (state before step 1 in FIG. 1). First, the first nucleic acid and the second nucleic acid hybridize to the third nucleic acid, and the ends of the two nucleic acids approach each other using the third nucleic acid as a scaffold (step 1 in FIG. 1). Next, the ends of the first nucleic acid and the second nucleic acid that have come close to each other are linked (step 2 in FIG. 1), and the third nucleic acid is removed (step 3 in FIG. 1), to produce a long nucleic acid such as a hairpin nucleic acid.

[0005] However, this production method requires the synthesis of an extra third nucleic acid that is not included in the final product, and it also requires the removal of the extra third nucleic acid after the ligation reaction, which has been one of the causes of reduced efficiency in the production of long nucleic acids. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 2023 / 013329 [Non-patent literature]

[0007] [Non-Patent Document 1] Approved Nucleic Acid Drugs (as of May 2021), National Institute of Health Sciences, Department of Genetic Medicine, Division 2 [Non-Patent Document 2] K. Morihiro et al., J. Am. Chem. Soc. 2023, 145, 1, 135-142 Summary of the Invention [Problem to be solved by the invention]

[0008] An object of the present invention is to provide a method for efficiently producing a hairpin nucleic acid having a relatively long sequence without using an unnecessary third nucleic acid. [Means for solving the problem]

[0009] As a result of intensive research by the present inventors to solve the above problems, they have found that a hairpin nucleic acid can be produced without using a third nucleic acid by devising a nucleic acid fragment design. The present invention is based on the novel findings and provides the following. [1] A method for producing a hairpin nucleic acid having a protruding region by linking a first nucleic acid and a second nucleic acid, the first nucleic acid comprising a first linking part and, in order from a linking end, a stem first region, a loop region, a stem second region and a protruding region, the linking end being the end closer to the first linking part, the stem first region and the stem second region being capable of hybridizing to each other intramolecularly, the second nucleic acid comprising a second linking part and a pairing region having a single-stranded structure, the pairing region comprising the second linking part or being continuous with the second linking part, the pairing region being capable of hybridizing to all or a part of the protruding region, the method comprising: a hybridization step in which the protruding region of the first nucleic acid and the pairing region of the second nucleic acid are hybridized; and a linking step in which the first linking part of the first nucleic acid and the second linking part of the second nucleic acid are linked by an enzymatic reaction. [2] The method according to [1], further comprising a separation step of separating the extended hairpin nucleic acid from unreacted first nucleic acid and second nucleic acid after or simultaneously with the ligation step. [3] The method according to [1] or [2], wherein the hybridization step is carried out at a temperature of 0°C to 40°C. [4] The method according to any one of [1] to [3], wherein the linking step is carried out at a temperature of 0°C to 40°C. [5] The method according to any one of [1] to [4], wherein the first stem region comprises two or more bases. [6] The method according to any one of [1] to [5], wherein the paired region has 5 or more bases. [7] A method according to any one of [1] to [6], wherein the two hybridizing nucleic acid regions consist of any one of the following base sequences (1) to (3): (1) base sequences that are completely complementary to each other, (2) a base sequence containing one or two consecutive non-complementary bases to (1), or (3) a base sequence containing one or several discontinuous non-complementary bases to (1) or (2). [8] The method according to any one of [1] to [7], wherein the free energy change in the hairpin structure-forming reaction in the first nucleic acid is −20 kcal / mol to −10 kcal / mol. [9] The method according to any one of [1] to [8], wherein the second nucleic acid is a nucleic acid having a single-stranded structure.

[10] The method according to any of [1] to [9], wherein the linking portion of the first nucleic acid and the linking portion of the second nucleic acid are linked via a phosphate group and a hydroxyl group.

[11] A method for extending a hairpin nucleic acid by linking a first nucleic acid and a second nucleic acid that form a hairpin structure, the first nucleic acid comprising a first linking part and, in that order from a linking end, a stem first region, a loop region, a stem second region and a protruding region, the linking end being the end closer to the first linking part, the stem first region and the stem second region being capable of hybridizing to each other intramolecularly, the second nucleic acid comprising a second linking part and a pairing region having a single-stranded structure, the pairing region comprising the second linking part or being continuous with the second linking part, the pairing region being capable of hybridizing to all or a part of the protruding region, the method comprising: a hybridization step in which the protruding region of the first nucleic acid and the pairing region of the second nucleic acid are hybridized; and a linking step in which the first linking part of the first nucleic acid and the second linking part of the second nucleic acid are linked by an enzymatic reaction. Effect of the Invention

[0010] According to the method for producing a hairpin nucleic acid of the present invention, a hairpin nucleic acid can be produced efficiently.

[0011] According to the method for extending a hairpin nucleic acid of the present invention, a hairpin nucleic acid can be extended. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram showing steps of a conventional method for producing a hairpin nucleic acid by linking relatively short nucleic acid strands. [Diagram 2] FIG. 1 is a schematic diagram showing the steps of the method for producing a hairpin nucleic acid of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] 1. Method for producing hairpin nucleic acid 1-1. Overview A first aspect of the present invention is a method for producing a hairpin nucleic acid. The method of this aspect includes a hybridization step and a ligation step as essential steps. According to the method of this aspect, a hairpin nucleic acid can be produced with high efficiency.

[0014] 1-2.Definition As used herein, the term "hairpin nucleic acid" refers to a single-stranded nucleic acid capable of forming a hairpin structure. As used herein, the term "hairpin structure" refers to a secondary structure of a nucleic acid that includes a set of a stem structure, a loop structure, and a protruding region, formed by a single-stranded nucleic acid.

[0015] As used herein, the term "5'-end overhanging" refers to a type of hairpin nucleic acid that includes a overhanging region at the 5'-end. A 5'-end overhanging hairpin nucleic acid includes, from the 5'-end, a overhanging region, a stem second region, a loop region, and a stem first region. In addition, as used herein, the term "3'-end overhanging" refers to a type of hairpin nucleic acid that includes a overhanging region at the 3'-end. A 3'-end overhanging hairpin nucleic acid includes, from the 5'-end, a stem first region, a loop region, a stem second region, and a overhanging region.

[0016] The "stem structure" is a structure in which two stem regions (Y and Y') containing hybridizable base sequences form a double strand.

[0017] The "loop structure" is a loop-shaped structure formed by a loop region (Z) consisting of a single-stranded nucleic acid.

[0018] As used herein, the term "overhanging region (X)" refers to a nucleic acid region located at the end of a hairpin nucleic acid and remaining in a single-stranded state even when a hairpin structure is formed. The term "overhanging end" refers to a single-stranded nucleic acid region adjacent to either or both of the free ends (ends not adjacent to the loop region) of the stem region.

[0019] As used herein, "stem regions (Y and Y')" refer to nucleic acid regions that hybridize with each other within a molecule to form a stem structure. At least both ends of each stem region consist of complementary bases. "Stem first region (Y)" refers to a stem region that is not adjacent to a protruding region. "Stem second region (Y')" refers to a stem region that is adjacent to a protruding region.

[0020] As used herein, the term "loop region (Z)" refers to a nucleic acid region located between the two stem regions in a single-stranded nucleic acid.

[0021] As used herein, "linking" refers to covalently binding two molecules to form one molecule. As used herein, "linking portion (circled portion in the figure)" refers to a portion having a functional group for linking two nucleic acid chains. As used herein, "linking end" refers to the end of the two ends of a nucleic acid chain that is closer to the linking portion.

[0022] The term "complementary" refers to a relationship in which nucleic acid bases can form base pairs with each other via hydrogen bonds, such as Watson-Crick base pairs (natural base pairs) or Hoogsteen base pairs.

[0023] The terms "hybridize" or "hybridizable" refer to polynucleotides having complementary base sequences base pairing to form a completely or partially complementary double strand. As used herein, "hybridizable" particularly refers to being capable of hybridizing under the reaction conditions of the hybridization step and / or the linking step. As used herein, when two nucleic acid regions hybridize, the two nucleic acid regions consist of, for example, any one of the following base sequences (1) to (3): (1) Completely complementary base sequences; (2) A base sequence containing one or two consecutive non-complementary bases to (1), or (3) A base sequence containing one or more discontinuous non-complementary bases to (1) or (2).

[0024] In the present specification, "plurality" refers to a number of 2 or more. Specifically, for example, it refers to 2 to 60, 2 to 45, 2 to 30, 2 to 14, 2 to 10, 2 to 8, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In the present specification, "several" refers to 2 to 3.

[0025] As used herein, the term "hairpin structure-forming reaction" refers to a reaction in which a single-stranded nucleic acid in a hairpin nucleic acid changes from a linear form to a hairpin form to form a hairpin structure.

[0026] In this specification, the term "free energy change" refers to the net amount of energy supplied from the external environment to a reaction system through a certain reaction when the temperature and pressure conditions are constant. In this specification, the term particularly refers to the net amount of energy supplied from the external environment in a hairpin structure-forming reaction. For example, if the reaction product is more thermodynamically stable than the starting material, the reaction system loses energy through the reaction, and the free energy change is negative. The free energy change of the present invention includes, for example, both the Gibbs free energy change (ΔG) and the Helmholtz free energy change (ΔF).

[0027] 1-3. Method The method of this embodiment is a method for producing a hairpin nucleic acid having a protruding region by linking a first nucleic acid and a second nucleic acid, and includes a hybridization step and a linking step as essential steps, and includes a nucleic acid synthesis step, a phosphorylation step, a separation step, and a purification step as optional steps.

[0028] The method of the present invention differs from conventional methods for producing relatively long nucleic acids (FIG. 1) in that it does not use a third nucleic acid as a scaffold.

[0029] As described above, in the conventional method, first, in addition to the two nucleic acid molecules to be linked (first and second nucleic acids), a third nucleic acid is prepared (state before step 1 in FIG. 1). Next, the first and second nucleic acids hybridize to the third nucleic acid, and the ends of the two nucleic acids approach each other using the third nucleic acid as a scaffold (step 1 in FIG. 1). The ends of the first and second nucleic acids that have come close to each other are linked (step 2 in FIG. 1), and the third nucleic acid is removed (step 3 in FIG. 1), to produce a long nucleic acid such as a hairpin nucleic acid.

[0030] In contrast, in the method of the present invention, first, only two nucleic acid molecules to be ligated (a first nucleic acid and a second nucleic acid) are prepared (state before step 1 in FIG. 2). Next, the second nucleic acid hybridizes to the single-stranded portion of the first nucleic acid, and the ends of the two nucleic acids approach each other using a part of the first nucleic acid as a scaffold (step 1 in FIG. 2). The ends of the first and second nucleic acids that are now in close proximity are ligated (step 2 in FIG. 2) to produce a hairpin nucleic acid.

[0031] In this way, according to the production method of the present invention, a hairpin nucleic acid can be synthesized without synthesizing a third nucleic acid or removing the third nucleic acid. Each step will be specifically described below.

[0032] 1-3-1. Nucleic acid synthesis process The "nucleic acid synthesis step (the state before step 1 in FIG. 2)" is an optional step, which is a step for synthesizing a first nucleic acid and a second nucleic acid.

[0033] The synthesis of the nucleic acid can be carried out enzymatically and / or chemically. The first and second nucleic acids can be synthesized by different methods or by the same method.

[0034] The synthesis method of oligonucleotides may be a known technique in the art. For example, methods using enzymes include an extension synthesis reaction using a polymerase based on a template strand, a ligation synthesis reaction between shorter nucleic acid strands using a ligase, or a combination thereof. For example, chemical synthesis can be performed using a solid-phase synthesis method based on the phosphoramidite method. Specifically, for example, the synthesis method described in Current Protocols in Nucleic Acid Chemistry, Vol. 1, Section 3, Verma S. and Eckstein F., 1998, Annul. Rev. Biochem., 67, 99-134 can be used. In addition, for example, synthesis can be performed using a nucleic acid synthesizer or a manufacturer's contract manufacturing service.

[0035] It is preferable to purify the nucleic acid after synthesis. Any method known in the art can be used as the purification method. Specific purification methods include, for example, gel purification, affinity column purification, and HPLC.

[0036] 1-3-2. Hybridization process The "hybridization step (step 1 in FIG. 2)" is an essential step in which the protruding region of the first nucleic acid hybridizes with the paired region of the second nucleic acid. This step can be carried out after the nucleic acid synthesis step, if such a step is carried out. If the nucleic acid synthesis step is not carried out, this step can be carried out using an already synthesized nucleic acid. This step hybridizes the first nucleic acid and the second nucleic acid to partially form a double-stranded structure.

[0037] <First nucleic acid> The first nucleic acid is a nucleic acid which includes a first linking portion and, from the linking end, a stem first region, a loop region, a stem second region and a protruding region in this order.

[0038] The type of nucleic acid constituting the first nucleic acid may be any of DNA, RNA, or a combination thereof. Preferably, the first nucleic acid is a nucleic acid containing DNA or a nucleic acid consisting of DNA. The first nucleic acid may also contain natural nucleotides, non-natural nucleotides, unmodified nucleotides, modified nucleotides, nucleotide analogs, or a combination thereof.

[0039] The first nucleic acid may contain any label. The purpose of the label is not particularly limited, but it may be labeled for detection and / or purification. The type of label is not particularly limited. For example, a radioisotope ( 32 The nucleic acid can be labeled with, for example, a fluorescent substance (such as P), a fluorescent substance (such as TAMRA or Cy3), an adsorptive molecule (such as biotin or streptavidin), an antigen, or a magnetic bead.

[0040] The total length of the first nucleic acid is not particularly limited. For example, it can be 9 bases or more, 10 bases or more, 11 bases or more, 12 bases or more, 13 bases or more, 14 bases or more, 15 bases or more, 16 bases or more, 17 bases or more, 18 bases or more, 19 bases or more, or 20 bases or more. In addition, there is no particular upper limit. For example, when a hairpin nucleic acid having a long stem structure is produced by performing the method of this embodiment several times in succession, the total length of the first nucleic acid becomes longer with each time. The first nucleic acid can be, for example, 100 bases or less, 90 bases or less, 80 bases or less, 70 bases or less, 60 bases or less, 50 bases or less, 40 bases or less, 30 bases or less, 29 bases or less, 25 bases or less, 20 bases or less, 19 bases or less, or 18 bases or less.

[0041] The linking end refers to the end closer to the first linking part among the two ends of the first nucleic acid. The first linking part contains a functional group capable of linking with the second linking part. The combination of functional groups constituting the first linking part and the second linking part will be described later in the section on the linking step. When the linking end of the first nucleic acid is the 5' end, the linking end of the second nucleic acid is the 3' end, and conversely, when the linking end of the first nucleic acid is the 3' end, the linking end of the second nucleic acid is the 5' end.

[0042] The stem first region and the stem second region are capable of hybridizing with each other intramolecularly. Each stem region in the first nucleic acid constitutes a part of the stem structure of the target hairpin nucleic acid. The base sequences of the stem first region and the stem second region are not particularly limited as long as they are capable of hybridizing with each other under the reaction conditions of the ligation step.

[0043] Whether the base sequences of the stem first region and the stem second region can hybridize with each other can be determined based on the free energy change in the hairpin structure-forming reaction of the first nucleic acid. The free energy change in the hairpin structure-forming reaction is not particularly limited. In general, the lower the free energy change in the hairpin structure-forming reaction, the more difficult it is for the hairpin structure to cleave, and the higher the free energy change, the more easily the hairpin structure is cleaved. The free energy change is, for example, -20 kcal / mol to -10 kcal / mol. Specifically, for example, it is -20 kcal / mol or more, -19 kcal / mol or more, -18 kcal / mol or more, -17.5 kcal / mol or more, -17 kcal / mol or more, or -16.5 kcal / mol or more. The free energy change is, for example, -10 kcal / mol or less, -11 kcal / mol or less, -12 kcal / mol or less, -12.5 kcal / mol or less, -13 kcal / mol or less, -13.5 kcal / mol or less, -14 kcal / mol or less, -14.5 kcal / mol or less, or -15 kcal / mol or less. The free energy change can be known using known software used for predicting the stability of the higher-order structure of nucleic acids, such as NUPACK.

[0044] For example, as described in the definition section, the two stem regions each have one of the following base sequences (1) to (3): (1) Completely complementary base sequences; (2) A base sequence containing one or two consecutive non-complementary bases to (1), or (3) A base sequence containing one or more discontinuous non-complementary bases to (1) or (2).

[0045] The length of each stem region in the first nucleic acid is not particularly limited as long as it can be located at a position where the first linking part and the second linking part can be linked under the reaction conditions of the linking step. For example, each is independently 2 bases or more, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, or 7 bases or more. In addition, the length of each stem region can be independently, for example, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, 13 bases or less, 12 bases or less, 11 bases or less, 10 bases or less, 9 bases or less, or 8 bases or less. Specifically, the length of each stem region can be independently, 2 to 20 bases, 3 to 15 bases, or 5 to 8 bases. The ends of each stem region are all complementary to each other.

[0046] The loop region of the first nucleic acid constitutes the loop structure of the target hairpin nucleic acid. The length of the loop region in the first nucleic acid is not particularly limited. For example, it is 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, or 8 bases or more. It can also be, for example, 50 bases or less, 40 bases or less, 30 bases or less, 25 bases or less, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, 13 bases or less, 12 bases or less, 11 bases or less, 10 bases or less, 9 bases or less, or 8 bases or less. Specifically, the length of the loop region can be 3 to 50 bases, 3 to 30 bases, 3 to 20 bases, 3 to 15 bases, or 5 to 10 bases.

[0047] The protruding region of the first nucleic acid can hybridize with the whole or part of the pairing region of the second nucleic acid. The protruding region of the first nucleic acid hybridizes with the pairing region to form a part of the stem structure in the target hairpin nucleic acid. The length of the protruding region of the first nucleic acid is not particularly limited. For example, it is 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, or 7 bases or more. The protruding region can be, for example, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, 13 bases or less, 12 bases or less, 11 bases or less, 10 bases or less, 9 bases or less, or 8 bases or less. Specifically, the length of the protruding region can be 3 to 20 bases, 4 to 20 bases, 5 to 15 bases, or 5 to 10 bases.

[0048] The protruding region of the first nucleic acid may contain one or more additional bases in addition to the sequence capable of hybridizing with the pairing region of the second nucleic acid. These additional bases constitute the protruding region in the target hairpin nucleic acid. The specific number of bases is not particularly limited. For example, it may be 1 or more bases, 2 or more bases, 3 or more bases, 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, or 8 or more bases. In addition, the additional bases may be, for example, 20 or less bases, 19 or less bases, 18 or less bases, 17 or less bases, 16 or less bases, 15 or less bases, 14 or less bases, 13 or less bases, 12 or less bases, 11 or less bases, 10 or less bases, or 9 or less bases.

[0049] <Second Nucleic Acid> The second nucleic acid comprises a second linkage and a pairing region consisting of a single-stranded structure. The type of nucleic acid constituting the second nucleic acid may be any of DNA, RNA, or a combination thereof. Preferably, the second nucleic acid is a nucleic acid containing DNA or a nucleic acid consisting of DNA. The second nucleic acid may also contain natural nucleotides, non-natural nucleotides, unmodified nucleotides, modified nucleotides, nucleotide analogs, or a combination thereof.

[0050] The second nucleic acid may contain any label. The purpose of the label is not particularly limited, but it may be labeled, for example, for detection and / or purification. The type of label is not particularly limited. For example, a radioisotope ( 32 The nucleic acid can be labeled with, for example, a fluorescent substance (such as P), a fluorescent substance (such as TAMRA or Cy3), an adsorptive molecule (such as biotin or streptavidin), an antigen, or a magnetic bead.

[0051] The second nucleic acid may have a part that forms a double-stranded structure or a single-stranded structure. Preferably, the second nucleic acid in the present specification is a nucleic acid that has a single-stranded structure.

[0052] The length of the second nucleic acid is not particularly limited. For example, it can be 5 bases or more, 6 bases or more, 7 bases or more, 8 bases or more, 9 bases or more, 10 bases or more, 11 bases or more, 12 bases or more, 13 bases or more, 14 bases or more, 15 bases or more, 16 bases or more, or 17 bases or more. It can also be, for example, 30 bases or less, 29 bases or less, 25 bases or less, 20 bases or less, 19 bases or less, or 18 bases or less.

[0053] The second linking part contains a functional group capable of linking to the first linking part. The combination of functional groups constituting the first linking part and the second linking part will be described later in the section on the linking step. Of the two ends of the second nucleic acid, the end closer to the second linking part is designated as the linked end.

[0054] The paired region includes the second linking portion or is continuous with the second linking portion, and is capable of hybridizing with all or a part of the protruding region of the first nucleic acid. The paired region hybridizes with the first nucleic acid to form a part of the stem structure of the target hairpin nucleic acid. Both ends of the paired region have bases complementary to the protruding region of the first nucleic acid.

[0055] For example, as described in the definition section, the paired region and the overhanging region of the first nucleic acid have the following base sequences: (1) Completely complementary base sequences; (2) A base sequence containing one or two consecutive non-complementary bases to (1), (3) A base sequence containing one or more discontinuous non-complementary bases to (1) or (2).

[0056] When the pairing region is continuous with the second linking part, it contains one or more additional bases between the pairing region and the second linking part. The specific number of bases is not particularly limited. For example, it can be 1 base or more, 2 bases or more, 3 bases or more, or 4 bases or more. In addition, the additional bases can be, for example, 11 bases or less, 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, or 5 bases or less.

[0057] The length of the pairing region is not particularly limited as long as it can be located at a position where the first linking part and the second linking part can be linked under the reaction conditions of the linking step. The specific length of the pairing region is, for example, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, or 7 bases or more. The pairing region can be, for example, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, 13 bases or less, 12 bases or less, 11 bases or less, 10 bases or less, 9 bases or less, or 8 bases or less. Specifically, the length of the pairing region can be 3 to 20 bases, 4 to 20 bases, 5 to 15 bases, or 5 to 10 bases. It is preferably 5 bases or more.

[0058] The second nucleic acid may contain one or more additional bases in addition to the pairing region. These additional bases constitute the protruding region in the target hairpin nucleic acid. The specific number of bases is not particularly limited. For example, it may be 1 or more bases, 2 or more bases, 3 or more bases, 4 or more bases, 5 or more bases, 6 or more bases, 7 or more bases, or 8 or more bases. The additional bases may be, for example, 20 or less bases, 19 or less bases, 18 or less bases, 17 or less bases, 16 or less bases, 15 or less bases, 14 or less bases, 13 or less bases, 12 or less bases, 11 or less bases, 10 or less bases, or 9 or less bases. For example, the additional bases may be 4 to 20 bases.

[0059] <Reaction conditions> This step can be carried out by incubating the first nucleic acid and the second nucleic acid together in an aqueous solvent. The reaction conditions for this step are not particularly limited as long as the protruding region of the first nucleic acid and the paired region of the second nucleic acid can hybridize. In the state before step 1 in FIG. 2, the stem first region and the stem second region of the first nucleic acid are typically shown hybridized to each other. However, in this step, the first nucleic acid does not necessarily have to form a double strand within the molecule as long as the protruding region of the first nucleic acid and the paired region of the second nucleic acid are hybridized.

[0060] As used herein, the term "aqueous solvent" refers to water or an aqueous solution. The type of aqueous solvent is not particularly limited, but is preferably water or a buffer solution. The type of buffer solution is not particularly limited, and any buffer solution known in the art can be used. For example, the buffer solutions exemplified in the linking step can be used.

[0061] The concentrations of the first and second nucleic acids are not particularly limited. The concentrations of each nucleic acid can be 10 μM or more, 40 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, 300 μM or more, 400 μM or more, or 500 μM or more. In addition, the concentrations can be, for example, 20 mM or less, 15 mM or less, 12 mM or less, 11 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, or 1 mM or less. The concentrations of the first and second nucleic acids may each be constant, or may increase or decrease continuously and / or intermittently.

[0062] The first nucleic acid and the second nucleic acid are not particularly limited. For example, the first nucleic acid and the second nucleic acid can be incubated at a molar ratio of 1:10 to 10:1, more specifically, at a molar ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:1.9 to 1.9:1, 1:1.5 to 1.5:1, 1:1.4 to 1.4:1, 1:1.3 to 1.3:1, 1:1.2 to 1.2:1, 1:1.1 to 1.1:1, or 1:1.

[0063] The pH of the aqueous solvent is not particularly limited, and may be within the range of, for example, 5 to 9, and the pH range exemplified in the linking step can be used.

[0064] The reaction temperature can be appropriately determined based on the base sequences and lengths of the overhanging region and the pairing region. For example, m For example, the reaction temperature can be T m Value -1℃ or less, T m Value -2℃ or less, T m Value -5℃ or less, T m Value -10℃ or less, T m The incubation temperature may be set to a value of −15° C. or lower, etc. Alternatively, the incubation may be performed within the temperature range exemplified in the linking step, such as a range of 0° C. to 40° C.

[0065] Generally, the hybridization reaction proceeds even when the mixture is left to stand, but it may be mixed by stirring, shaking, etc. as appropriate. The above-mentioned reaction conditions do not need to be constant during this process, and may be changed continuously and / or intermittently. In this case, each condition may be changed artificially or naturally.

[0066] The reaction time is not particularly limited. For example, the reaction can be incubated for 1 minute or more, 2 minutes or more, 3 minutes or more, 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, or 2 hours or more. The reaction can also be incubated for, for example, 12 hours or less, 10 hours or less, 8 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, or 3 hours or less.

[0067] 1-3-3.Phosphorylation process The "phosphorylation step" is an optional step in which the 5'-end of the first nucleic acid and / or the second nucleic acid is phosphorylated. It can be carried out before, simultaneously with, or after the hybridization step.

[0068] This step is usually carried out to phosphorylate the 5'-side linking end of the first nucleic acid or the second nucleic acid when no phosphate group is present at the 5'-side linking end, thereby making the linking end ligatable. It may also be carried out for other purposes, such as adding a desired phosphate group (e.g., 32 This step may be carried out in order to introduce a phosphate group (containing P).

[0069] Phosphorylation is usually carried out using a nucleotide kinase. The nucleotide kinase used is not particularly limited. For example, T4 polynucleotide kinase can be used.

[0070] The donor of the phosphate group is not particularly limited, and for example, ATP, its modified form or derivative, etc. can be used.

[0071] The reaction conditions are not particularly limited as long as the kinase used is functional. They can be appropriately determined depending on the kinase used. For example, the reaction can be carried out in a buffer solution having a pH of 7 to 9, at a temperature range of 20 to 50° C., for 1 minute to 24 hours.

[0072] 1-3-4.Connection process The "ligation step (step 2 in FIG. 2)" is an essential step in which the first linkage of the first nucleic acid and the second linkage of the second nucleic acid are linked by an enzymatic reaction. This step can be carried out simultaneously with or separately from the hybridization step.

[0073] In this step, the first and second nucleic acids are incubated with one or more enzymes in an aqueous solvent, resulting in covalent bonding between the first and second linking parts, and the first and second nucleic acids are linked to form the desired hairpin nucleic acid (Figure 2).

[0074] <Hairpin nucleic acid of interest> A hairpin nucleic acid of interest is a nucleic acid molecule formed by linking a first nucleic acid and a second nucleic acid. In this specification, when simply described as "hairpin nucleic acid", it refers to a hairpin nucleic acid of interest.

[0075] The hairpin nucleic acid of interest has a protruding region and forms a hairpin structure. That is, the hairpin nucleic acid of interest contains a nucleic acid region that constitutes a loop structure and a stem structure in addition to the protruding region. The protruding region in the hairpin nucleic acid of interest is composed of a part of the protruding region of the first nucleic acid and / or a part of the second nucleic acid. When the protruding region of the first nucleic acid contains additional bases in addition to the pairing region with the second nucleic acid, and the second nucleic acid contains additional bases in addition to the pairing region, the hairpin nucleic acid of interest contains protruding regions at both ends.

[0076] The stem structure of the target hairpin nucleic acid is composed of the stem first region and stem second region of the first nucleic acid, which are hybridized to each other, and all or a part of the overhanging region of the first nucleic acid and the pairing region of the second nucleic acid, which are hybridized to each other. The loop structure of the hairpin nucleic acid of interest is composed of the loop region of the first nucleic acid.

[0077] As described above, the target hairpin nucleic acid has a protruding region. The length of the protruding region is not particularly limited and can be appropriately determined depending on the target application. The specific length of the protruding region is, for example, 1 base or more, 2 bases or more, 3 bases or more, 4 bases or more, 5 bases or more, 6 bases or more, 7 bases or more, or 8 bases or more. The protruding region can be, for example, 20 bases or less, 19 bases or less, 18 bases or less, 17 bases or less, 16 bases or less, 15 bases or less, 14 bases or less, 13 bases or less, 12 bases or less, 11 bases or less, 10 bases or less, or 9 bases or less. The length of the protruding region can be, for example, 4 to 20 bases.

[0078] Although not limited thereto, it is preferred that the target hairpin nucleic acid forms a hairpin structure in an aqueous buffer solution at pH 6.8 to pH 7.8 and at 35°C to 40°C.

[0079] <Connection part> The functional groups constituting the first linking part and the second linking part are not particularly limited and can be appropriately selected depending on the enzyme to be used, etc.

[0080] Typically, the first linkage is a hydroxyl group and the second linkage is a phosphate group, or conversely, the first linkage is a phosphate group and the second linkage is a hydroxyl group, and a phosphodiester bond is formed by an enzymatic reaction.

[0081] <Enzyme> Any ligase can be used as the enzyme used for ligation. The ligase may be naturally occurring or artificial. One or more types of ligase can be used as the enzyme used in this step. In addition, other enzymes may be additionally used in this step as necessary.

[0082] For example, when the first linking part and the second linking part are DNA nucleotides, a DNA ligase is used. Specific DNA ligases are not particularly limited, but examples thereof include T4 DNA ligase 2 derived from T4 bacteriophage, E. Coli DNA ligase, Ampligase DNA ligase, T3 DNA ligase, T7 DNA ligase, and Taq DNA ligase, mammalian DNA ligase (e.g., DNA ligase I, DNA ligase II, DNA ligase III, and DNA ligase IV, etc.), 9°N (trademark) DNA ligase, Tfi DNA ligase, or modified versions thereof.

[0083] For example, when the first linking part and the second linking part are RNA nucleotides, an RNA ligase is used. Specific RNA ligases are not particularly limited, and examples thereof include T4 RNA ligase 1 derived from T4 bacteriophage, T4 RNA ligase 2, ligase 2 derived from KVP40, ligase of the Rnl2 family, RtcB ligase, AmEPV ligase, AcNPV ligase, XcGV ligase, M. thermoautotrophicum ligase, Trypanosoma brucei RNA ligase, Deinococcus radiodurans RNA ligase, Leishmania tarentolae RNA ligase, and modified forms thereof.

[0084] The method for obtaining these DNA ligases and RNA ligases is not particularly limited. For example, commercially available ligases may be used, or natural ligases may be used as is or after appropriate modification. Ligases are usually modified by genetic engineering, but the method of modification is not particularly limited. The purpose of modification is not particularly limited, and can be, for example, to improve one or more characteristics such as reaction efficiency, thermostability, and reaction specificity.

[0085] <Reaction conditions> As described above, in this step, the first nucleic acid and the second nucleic acid are incubated with one or more types of enzymes in an aqueous solvent. In this case, the method and order of adding the enzymes and each nucleic acid are not particularly limited. For example, the enzyme may be added later in the presence of both nucleic acids, all may be added to the aqueous solvent at the same time, the aqueous solvent may be added to a space where the nucleic acid and the enzyme are separately present, or the enzyme and / or the nucleic acid may be added to the aqueous solvent in multiple batches or continuously.

[0086] The reaction conditions are not particularly limited as long as the enzyme used can function, the stem first region of the first nucleic acid can hybridize with the stem second region, and the protruding region of the first nucleic acid can hybridize with the pairing region of the second nucleic acid. In this way, the first nucleic acid forms a stem structure, and the first nucleic acid and the second nucleic acid hybridize with each other, bringing the first linking part and the second linking part into close proximity, making linking in this process easier (Step 1 in FIG. 2).

[0087] The reaction conditions may be the same as or different from those in the hybridization step. In either case, all or part of the aqueous solvent used in the hybridization step may be used in this step as is, or the aqueous solvent used in the hybridization step may be replaced with the aqueous solvent used in this step. When the phosphorylation step is performed, the solvent may be used in this step as is, or may be replaced with the aqueous solvent used in this step. Here, the operation of "replacing" a solvent or the like refers to an operation of changing one property to another. Therefore, the operation of "replacing a solvent" includes, for example, an operation of mixing the original solvent with a large excess of a new solvent so that the properties of the original solvent are substantially lost.

[0088] This step can be carried out in an aqueous solution. A buffer solution can be used as the aqueous solution. Specific examples of the buffer solution include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, citrate buffer, and the like. The salt concentration of the buffer solution is not particularly limited, and may be, for example, a low salt concentration of 750 mM or less (e.g., 15 mM to 750 mM, 15 mM to 500 mM, 15 mM to 300 mM, 15 mM to 200 mM, 50 mM to 200 mM, 75 mM to 150 mM, etc.), or a high salt concentration exceeding 750 mM (e.g., 750 mM to 1500 mM, 800 mM to 1400 mM, 1000 mM to 1300 mM, 1100 mM to 1200 mM, etc.).

[0089] The concentrations of the first and second nucleic acids are not particularly limited. The concentrations of each nucleic acid can be 10 μM or more, 40 μM or more, 100 μM or more, 150 μM or more, 200 μM or more, 300 μM or more, 400 μM or more, or 500 μM or more. In addition, the concentrations can be, for example, 20 mM or less, 15 mM or less, 12 mM or less, 11 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 5 mM or less, 4 mM or less, 3 mM or less, 2 mM or less, or 1 mM or less. The concentrations of the first and second nucleic acids may each be constant, or may increase or decrease continuously and / or intermittently.

[0090] The first nucleic acid and the second nucleic acid are not particularly limited. For example, the first nucleic acid and the second nucleic acid can be incubated at a molar ratio of 1:10 to 10:1, more specifically, at a molar ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, 1:1.9 to 1.9:1, 1:1.5 to 1.5:1, 1:1.4 to 1.4:1, 1:1.3 to 1.3:1, 1:1.2 to 1.2:1, 1:1.1 to 1.1:1, or 1:1.

[0091] The concentration of the ligase (units (U)) is not particularly limited. For example, the concentration can be 0.01 U / μL or more, 0.02 U / μL or more, 0.08 U / μL or more, 0.2 U / μL or more, or 0.35 U / μL or more. In addition, the concentration can be, for example, 10 U / μL or less, 1 U / μL or less, or 0.5 U / μL or less.

[0092] The concentration of the ligase relative to the nucleic acid is not particularly limited. For example, the concentration (nmol) of the smaller nucleic acid of the first nucleic acid and the second nucleic acid can be 0.001 U / nmol or more, 0.01 U / nmol or more, 0.1 U / nmol or more, 0.2 U / nmol or more, or 1 U / nmol or more. In addition, the concentration (nmol) of the smaller nucleic acid of the first nucleic acid and the second nucleic acid can be 10 U / nmol or less, 5 U / nmol or less, 4 U / nmol or less, 2 U / nmol or less, or 1 U / nmol or less.

[0093] In the incubation, an auxiliary agent for assisting the function of the enzyme can be added. Specific examples of the auxiliary agent include Tris-HCl, adenosine triphosphate (ATP), polyamines, nicotinic aminoadenine (NAD), animal serum proteins, dithiothreitol (DTT), divalent metal ions, and combinations thereof. The concentration of the auxiliary agent is not particularly limited. For example, the molar concentration can be 1 μM or more, 2 μM or more, 5 μM or more, 10 μM or more, 50 μM or more, 100 μM or more, 200 μM or more, 500 μM or more, 1 mM or more, 2 mM or more, 3 mM or more, 4 mM or more, 5 mM or more, or 6 mM or more, and can be added at a concentration of, for example, 1 mM to 20 mM, 2 mM to 10 mM, 3 mM to 8 mM, or 4 mM to 7 mM. For example, the mass percent concentration can be 0.0001 mass % or more, more specifically, 0.01 to 0.05 mass %.

[0094] Divalent metal ions include, for example, magnesium ions (Mg 2+ ), manganese ion (Mn 2+ ), calcium ion (Ca 2+ These divalent metal ions can be added in the form of any salt, such as hydroxides, fluorides, chlorides, sulfates, nitrates, borates, carbonates, or organic acid salts.

[0095] Examples of animal serum proteins include serum proteins such as albumin and globulin derived from mammals such as rabbits, cows, and mice.

[0096] The pH is not particularly limited and can be appropriately set according to the nucleic acid concentration and the type of ligase. For example, it can be 5 to 9, 6 to 8, 6.5 to 8, 7 to 8, 7 to 7.9, 7.3 to 8, 7.3 to 7.9, 7.4 to 8, 7.4 to 7.9, 7.5 to 8, or 7.5 to 7.9. For example, when the nucleic acid concentration is high, the pH can be set higher (for example, 7.5 to 9.0, 8.0 to 8.5).

[0097] The buffer solution may contain additives as necessary. Specific examples of additives include dimethyl sulfoxide (DMSO), polyethylene glycol (PEG), 1,2-propanediol, glycerol, Tween (registered trademark)-20, and combinations thereof. The specific type of polyethylene glycol is not particularly limited. For example, PEG6000, PEG8000, PEG20000, etc. may be used. The concentration of the additive is not particularly limited. For example, the molar concentration may be 1 μM to 40 mM, 10 μM to 30 mM, 100 μM to 20 mM, 1 mM to 15 mM, or 5 mM to 10 mM. In addition, the mass volume percent concentration may be, for example, 3 to 30 w / v%, 5 to 20 w / v%, 5 to 15 w / v%, or 10 to 30 w / v%.

[0098] The temperature of this step is not particularly limited as long as the ligase used can function at the temperature, and can be appropriately set depending on the type of ligase. For example, the temperature can be 0°C or higher, 4°C or higher, 10°C or higher, 15°C or higher, or 16°C or higher. In addition, the temperature can be, for example, 60°C or lower, 50°C or lower, 40°C or lower, 37°C or lower, 35°C or lower, 33°C or lower, 30°C or lower, 27°C or lower, 26°C or lower, 20°C or lower, 22°C or lower, 18°C ​​or lower, or 16°C or lower. For example, the step can be performed at a temperature of 0°C to 40°C, 4°C to 37°C, or 4°C to 16°C, but it can also be performed at 0°C to 4°C or 0°C depending on the ligase used.

[0099] Usually, the ligation reaction proceeds even when the mixture is left to stand, but it may be mixed by stirring, shaking, etc. as appropriate. The above-mentioned reaction conditions do not need to be constant during this process, and may be changed during the process. In this case, each condition may be changed artificially or naturally.

[0100] The incubation time is not particularly limited as long as it is sufficient for the ligation reaction to occur, and can be appropriately set depending on the type of ligase used and the temperature conditions. For example, the incubation can be performed for 5 minutes or more, 10 minutes or more, 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, or 12 hours or more. In addition, there is no particular upper limit. For example, the incubation can be performed for 48 hours or less, 36 hours or less, 30 hours or less, 24 hours or less, 20 hours or less, 19 hours or less, 18 hours or less, 17 hours or less, or 16 hours or less.

[0101] The reaction conditions can be determined based on the ligation efficiency, which is calculated by the following formula: Ligation efficiency (%) = (concentration of the desired hairpin nucleic acid) / (total concentration of the desired hairpin nucleic acid and unreacted nucleic acid) x 100

[0102] The ligation efficiency can be calculated by a method for quantifying nucleic acids known in the art. For example, it can be calculated by chromatography, electrophoresis, absorbance measurement, or a combination thereof. For example, the reaction conditions can be determined so that the ligation efficiency is 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, or 90% or more. For example, the ligation efficiency may be determined so that it is more than 90%.

[0103] After incubation, the solution can be used as the target hairpin nucleic acid sample without stopping the ligation reaction, or the next step can be started. The ligation reaction may be stopped as necessary. The ligation reaction can be stopped by any method, for example, by inactivating the ligase by heating or adding a denaturant, or by reducing the concentration of the ligase by washing, dilution, or the like.

[0104] This process can be carried out multiple times, in which case the reaction conditions used can be the same or different each time.

[0105] 1-3-5. Separation process The "separation step" is an optional step in which the extended hairpin nucleic acid is separated from the unreacted first and second nucleic acids. This step can be carried out after or simultaneously with the ligation step. This step allows the preparation of a hairpin nucleic acid sample that does not contain the unreacted first and second nucleic acids as impurities.

[0106] "Unreacted" refers to nucleic acids that have not undergone a ligation reaction, specifically, a second nucleic acid that has not ligated to a first nucleic acid and a first nucleic acid that has not ligated to a second nucleic acid.

[0107] The separation method is not particularly limited as long as it can separate the unreacted nucleic acid from the target hairpin nucleic acid. For example, separation can be performed by a method based on molecular size and / or polarity, a method based on labeling, or a combination thereof.

[0108] The method based on molecular size and / or polarity is not particularly limited, and examples of the method that can be used include chromatography such as high performance liquid chromatography, ion exchange chromatography, size exclusion chromatography, reverse phase chromatography, hydrophilic interaction chromatography, electrophoresis, or a combination thereof.

[0109] The label-based method is not particularly limited, but may involve, for example, chromatography such as affinity chromatography, capture with magnetic beads, density gradient centrifugation, or a combination thereof.

[0110] 1-3-6. Purification process The "purification step" is an optional step in which the extended hairpin nucleic acid is purified. This step can be performed after or simultaneously with the ligation step. If a separation step is performed, this step can be performed before, simultaneously with, and / or after the separation step.

[0111] The purification method used in this step is not particularly limited. Many purification methods are known in the art. Specific examples include phenol-chloroform extraction, ethanol precipitation, capture with magnetic beads, adsorption to silica membrane, chromatography (reverse phase chromatography, reverse phase high performance liquid chromatography (RP-HPLC), ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography, etc.), gel filtration, column purification, electrophoresis (polyacrylamide gel electrophoresis (PAGE), etc.), and combinations thereof.

[0112] If the purification method used can also be used for separation based on the molecular size or label of nucleic acids, the separation step and purification step can be carried out simultaneously by using the method.

[0113] 1-4.Effects According to the method of the present invention, hairpin nucleic acids can be produced with extremely high efficiency compared to synthesizing the entire length of a hairpin nucleic acid once. The method of the present invention does not require a third nucleic acid, which is required for normal linking of nucleic acid chains (FIG. 1). Therefore, it is not necessary to remove the third nucleic acid after synthesis and linking of the third nucleic acid, and hairpin nucleic acids can be produced more efficiently.

[0114] For example, a hairpin nucleic acid having a longer stem structure can be produced by carrying out the method of the present invention using a hairpin nucleic acid produced by the method of the present invention as the first nucleic acid and another second nucleic acid. In this case, after carrying out the method of the present invention using the first nucleic acid and the first second nucleic acid, the method of the present invention may be repeated using the product and the second or subsequent second nucleic acids, or the method of the present invention may be carried out using the first nucleic acid, the first second nucleic acid, and the second or subsequent second nucleic acids all at once.

[0115] 2. Method for extending hairpin nucleic acid 2-1. Overview The second aspect of the present invention is a method for extending a hairpin nucleic acid by linking a first nucleic acid and a second nucleic acid that form a hairpin structure. The method includes a hybridization step and a linking step as essential steps, and a phosphorylation step as an optional step. According to the method of this aspect, the hairpin nucleic acid can be extended easily.

[0116] 2-2. Method 2-2-1. Hybridization process The "hybridization step" is an essential step in which the overhanging region of a first nucleic acid hybridizes with the matching region of a second nucleic acid. This step may be carried out in accordance with the description of the first embodiment.

[0117] 2-2-2.Phosphorylation process The "phosphorylation step" is a step of phosphorylating the 5'-end of the first nucleic acid and / or the second nucleic acid. It can be carried out before, simultaneously with, or after the hybridization step.

[0118] 2-2-3.Connection process The "ligation step" is a step of linking the first linkage of the first nucleic acid to the second linkage of the second nucleic acid by an enzyme reaction. This step can be carried out simultaneously with or separately from the hybridization step. When a phosphorylation step is carried out, this step can be carried out after the phosphorylation step.

[0119] This step may be carried out in accordance with the description of the first embodiment. EXAMPLES

[0120] Example 1: Preparation of hairpin nucleic acid (the purpose) The efficiency of the method for producing hairpin nucleic acids of the present invention is verified using a hairpin nucleic acid (HP) having the base sequence represented by sequence number 1 and nucleic acids (hp-1 and hp-2, respectively) having its partial sequences (sequence numbers 2 and 3).

[0121] (method) 1. Chemical synthesis of nucleic acids Three types of nucleic acid molecules having the sequences shown in Table 1 were chemically synthesized by the phosphoramidite method.

[0122] [Table 1]

[0123] The nucleic acids were synthesized using a DNA synthesizer (Nihon Techno Service Co., Ltd.) according to the manufacturer's protocol. The 5' end of hp-2 (corresponding to the second nucleic acid) was phosphorylated during this synthesis process.

[0124] The concentration of the synthesized nucleic acid molecule was measured by measuring the absorbance at a wavelength of 260 nm using a spectrophotometer.

[0125] 2. Nucleic acid ligation The synthesized hp-1 (corresponding to the first nucleic acid) and hp-2 (corresponding to the second nucleic acid) were added to the ligation buffer at a concentration of 0.5 μM, and incubated at room temperature (16° C.) for 16 hours. The ligation solution used was a buffer (pH 7.5-7.9) containing T4 DNA ligase at a final concentration of 500 U / μL, Tris-HCl at a final concentration of 50 mM, DTT at 20 mM, MgCl2 at 10 mM, ATP at 1 mM, etc.

[0126] 3. Nucleic Acid Isolation and Purification The produced hairpin nucleic acid was separated from unreacted nucleic acid and purified by the following procedure. Separation and purification were performed using gel electrophoresis or high performance liquid chromatography.

[0127] Gel electrophoresis was performed using 8% polyacrylamide gel in TBE buffer (89 mM Tris / Tris-borate, 2 mM EDTA) under denaturing conditions at a voltage of 150 V for 30 minutes.

[0128] High performance liquid chromatography was carried out under conditions typically used for the separation of oligonucleotides, using 20 mM TEAA buffer (acetic acid-triethylamine buffer) and acetonitrile as solvents.

[0129] The concentration of the hairpin nucleic acid was determined by measuring the absorbance at a wavelength of 260 nm using a spectrophotometer for the unpurified sample and the purified sample.

[0130] The efficiency of the ligation reaction was calculated by the following formula. Ligation efficiency (%) = (absorbance of purified hairpin nucleic acid) / (absorbance of ligated hairpin nucleic acid + absorbance of unligated nucleic acid raw material (hp-1 and hp-2 that remained unreacted)) × 100

[0131] (result) The synthesis efficiency results are shown in Table 1. As expected, the synthesis efficiency of HP, which had the longest length, was the lowest (0.0108%), while that of hp-2, which had the shortest length, was the highest (0.0729%).

[0132] When hp-1 (corresponding to the first nucleic acid) and hp-2 (corresponding to the second nucleic acid) were ligated, the reaction efficiency was 90.2%.

[0133] Based on these results, the efficiency improvement rate of the production method of the hairpin nucleic acid of the present invention was calculated compared to full-length chemical synthesis.

[0134] First, the overall efficiency of full-length chemical synthesis was 0.0108%, which was the synthesis efficiency of HP. Next, the efficiency of the method for producing a hairpin nucleic acid of the present invention was calculated by the following procedure.

[0135] Of the synthesis efficiencies of hp-1 and hp-2, the lower one was determined as the efficiency of the nucleic acid synthesis reaction. Here, the lower synthesis efficiency of hp-1, 0.0551%, was determined as the efficiency of the nucleic acid synthesis reaction.

[0136] The overall efficiency when using the production method of the present invention was calculated as the product of the efficiency of the nucleic acid synthesis reaction and the efficiency of the ligation reaction. The efficiency of the nucleic acid synthesis reaction was 0.0551%, and the efficiency of the ligation reaction was 90.2%, so the efficiency of the production method of the present invention was 0.0497% (=0.0551%×0.902).

[0137] By using the production method of the present invention, the efficiency of producing the same hairpin nucleic acid (HP) improved dramatically from 0.0108% to 0.0497%, a 4.6-fold increase in efficiency.

[0138] From the above, it was demonstrated that the production method of the present invention makes it possible to produce nucleic acid molecules with high efficiency without synthesizing nucleic acid molecules that are unnecessary for the final product.

Claims

1. A method for producing a hairpin nucleic acid having a protruding region by linking a first nucleic acid and a second nucleic acid, The first nucleic acid comprises a first linkage portion and, in order from the linkage end, a first stem region, a loop region, a second stem region, and a protruding region. The aforementioned connecting end is the end closer to the first connecting portion. The first stem region and the second stem region are capable of hybridizing with each other within the molecule. The second nucleic acid includes a second linkage region and a pairing region consisting of a single-stranded structure. The aforementioned matching region includes the second connecting portion or is continuous with the second connecting portion. The opposing region can hybridize with all or part of the protruding region. A hybridization step in which the protruding region of the first nucleic acid and the pairing region of the second nucleic acid hybridize, and A linking step in which the first linking portion of the first nucleic acid and the second linking portion of the second nucleic acid are linked by an enzymatic reaction. The method, including the method described above.

2. The hairpin nucleic acid produced has protruding regions at both ends, The above method does not use a third nucleic acid, The protruding region of the first nucleic acid includes one or more bases in addition to a sequence that can hybridize with the pairing region of the second nucleic acid, and these one or more bases constitute one of the protruding regions of the hairpin nucleic acid obtained after ligation. The second nucleic acid contains one or more bases in addition to the pairing region, and these one or more bases constitute the other protruding region of the hairpin nucleic acid obtained after ligation. The method according to claim 1.

3. The method according to claim 1 or 2, wherein the change in free energy of the hairpin structure formation reaction in the first nucleic acid is -20 kcal / mol to -10 kcal / mol.

4. The method according to claim 1 or 2, further comprising a separation step of separating the extended hairpin nucleic acid from the unreacted first nucleic acid and the second nucleic acid after or simultaneously with the coupling step.

5. The method according to claim 1 or 2, wherein the hybridization step is carried out at a temperature of 0°C to 40°C.

6. The method according to claim 1 or 2, wherein the coupling step is carried out at a temperature of 0°C to 40°C.

7. The method according to claim 1 or 2, wherein the first stem region has two or more bases.

8. The method according to claim 1 or 2, wherein the pairing region is 5 bases or more.

9. The method according to claim 1 or 2, wherein the two nucleic acid regions to be hybridized consist of any one of the following base sequences (1) to (3): (1) Base sequences that are completely complementary to each other, (2) A base sequence containing one or two non-complementary bases in succession to (1), or (3) A base sequence containing one or more non-complementary bases discontinuously with respect to (1) or (2).

10. The method according to claim 1 or 2, wherein the second nucleic acid is a nucleic acid having a single-stranded structure.

11. The method according to claim 1 or 2, wherein the linking portion of the first nucleic acid and the linking portion of the second nucleic acid are linked by a phosphate group and a hydroxyl group.

12. The method according to claim 1 or 2, wherein in the hybridization step, the first nucleic acid and the second nucleic acid are incubated in a molar ratio of 1:10 to 10:

1.

13. The method according to claim 1 or 2, wherein the concentration of polyethylene glycol in the coupling step is 15 w / v% or less.

14. The method according to claim 13, wherein the coupling step is carried out under conditions that do not contain polyethylene glycol.

15. A method for extending hairpin nucleic acid, comprising performing the method according to claim 1 or 2 at least once more, using a hairpin nucleic acid obtained by the method according to claim 1 or 2 as a first nucleic acid and another second nucleic acid.