Method for producing single-stranded nucleic acid

Abstract

In order to provide a production method capable of obtaining a single-stranded nucleic acid having a target sequence by linking a plurality of single-stranded nucleic acids in a prescribed order, this method for producing a single-stranded nucleic acid comprises (a) a step for adding an adenyl acid group to the 5'end of a single-stranded nucleic acid, the 3'end of which is protected and the 5'end of which is phosphorylated, with an adenylase and (b) a step for adding another single-stranded nucleic acid, the 5'end of which is phosphorylated, to the 5'end of the single-stranded nucleic acid after step (a) with an adenyl acid group-dependent nucleic acid ligase, wherein steps (a) and (b) are alternately performed at least once.

Description

Method for producing single-stranded nucleic acid

[0005]

[0001] The present invention relates to a method for producing single-stranded nucleic acid.

[0002] In recent years, with the development of genome analysis and gene-related technologies, the use of nucleic acid oligomers such as DNA probes, siRNA, antisense DNA, antisense RNA, etc. has been increasing. As a method for synthesizing nucleic acid oligomers, the solid-phase synthesis method by the phosphoramidite method is often used (Patent Document 1).

[0003] International Publication No. 2014 / 077292 pamphlet

[0004] In order to obtain a long nucleic acid of 100 bases or more or a nucleic acid containing a modified base, in the prior art, there is a problem that the coupling efficiency is low. Therefore, it is conceivable to prepare short oligonucleotides and ligate them enzymatically.

[0005] On the other hand, when short oligonucleotides A and B are to be ligated in this order, if nucleotides of A and B are present in the reaction system, in addition to the desired A-B, A-A, B-B, B-A, and by-products formed by their cyclization may occur. The generation of such by-products not only reduces the yield of the target product but also requires additional steps for the separation and purification of the target product.

[0006] An object of the present invention is to provide a production method capable of obtaining a single-stranded nucleic acid having a target sequence by ligating a plurality of single-stranded nucleic acids in a predetermined order.

[0007] [1] A method for producing a single-stranded nucleic acid, comprising the steps of: (a) adding an adenylate group to the 5' end of a single-stranded nucleic acid whose 3' end is protected and whose 5' end is phosphorylated using adenylase; and (b) adding another single-stranded nucleic acid whose 5' end is phosphorylated using an adenylate group-dependent nucleic acid ligase to the 5' end of the single-stranded nucleic acid after step (a), wherein steps (a) and (b) are performed alternately at least once. [2] The method for producing a single-stranded nucleic acid according to [1], further comprising the step of treating with Proteinase K after step (a) and before step (b), or after step (b) and before step (a), at least one of the above. [3] The method for producing a single-stranded nucleic acid according to [1] or [2], wherein the length of the single-stranded nucleic acid in the first step (a) is 200 bases or less. [4] A method for producing a single-stranded nucleic acid according to any one of [1] to [3], wherein at least one of the single-stranded nucleic acid in step (a) and the single-stranded nucleic acid added in step (b) contains a modified base. [5] A method for producing a single-stranded nucleic acid according to any one of [1] to [4], wherein the 3' end of the single-stranded nucleic acid in step (a) is protected by being immobilized on a solid phase.

[0008] According to the present invention, multiple single-stranded nucleic acids can be linked in a predetermined order, thereby obtaining a single-stranded nucleic acid having a desired sequence.

[0009] This is a schematic diagram illustrating a method for producing single-stranded nucleic acids according to one embodiment of the present invention. This is a schematic diagram illustrating a method for producing single-stranded nucleic acids in an example of the present invention.

[0010] The following descriptions of embodiments for carrying out the present invention will be based on the drawings. The following descriptions of preferred embodiments are essentially illustrative and are not intended to limit the present invention, its application methods, or its uses.

[0011] The present invention provides a method for producing single-stranded nucleic acids, comprising: (a) adding an adenylate group to the 5' end of a single-stranded nucleic acid whose 3' end is protected and whose 5' end is phosphorylated using adenylase; and (b) adding another single-stranded nucleic acid whose 5' end is phosphorylated using an adenylate group-dependent nucleic acid ligase to the 5' end of the single-stranded nucleic acid after step (a), wherein steps (a) and (b) are performed alternately at least once.

[0012] <Single-stranded nucleic acids> Both the single-stranded nucleic acid in step (a) and the other single-stranded nucleic acids added in step (b) may be DNA or RNA. Note that "single-stranded nucleic acids" also include those that contain a self-complementary sequence in part and take on a double-stranded-like structure.

[0013] Furthermore, examples of bases included in the single-stranded nucleic acid of step (a) and the other single-stranded nucleic acid added in (b) include cytosine, adenine, guanine, thymine, and uracil, as well as their modifiers (i.e., modified bases). In other words, at least one of the single-stranded nucleic acid of step (a) and the other single-stranded nucleic acid added in (b) may contain modified bases.

[0014] "Modification" includes methylation (methylcytosine is an example of a methylated base); base rearrangement (e.g., pseudouridylation); double bond saturation (e.g., dihydrouridine for uridine); deamination; atom substitution such as replacing an oxygen atom with a sulfur atom (e.g., 4-thiouridine, in which the oxygen atom of nucleosidouridine is replaced with a sulfur atom); and the addition of other functional groups.

[0015] Furthermore, the bases also include artificial nucleic acid bases. Examples of artificial nucleic acid bases include labels such as fluorescent probes. In addition, by introducing artificial nucleic acid bases, it is possible to construct nucleic acids that can be used as drugs such as anticancer drugs and antiviral drugs.

[0016] The length of either the single-stranded nucleic acid in step (a) or the other single-stranded nucleic acid added in step (b) is not limited to a specific numerical value.

[0017] The length of the single-stranded nucleic acid in the first step (a), i.e., before step (b) is performed, can be determined according to conditions such as the type of protection at its 3' end, and is not particularly limited, but may be, for example, a few kilobases or less. Since shorter single-stranded nucleic acids often result in better protection reaction efficiency, the length of the single-stranded nucleic acid is preferably 200 bases or less or 100 bases or less, and more specifically, examples include those of 50 bases or less. The length of the single-stranded nucleic acid may be, for example, 5 bases or more, 10 bases or more, 40 bases or less, or 30 bases or less. More specifically, examples of lengths include 5 bases or more and 40 bases or less, 10 bases or more and 40 bases or less, 20 bases or more and 40 bases or less, 10 bases or more and 30 bases or less.

[0018] Furthermore, the length of the single-stranded nucleic acid in step (b) can be selected from the range exemplified as the length of the single-stranded nucleic acid in the first step (a) described above. The single-stranded nucleic acid in step (b) may also be longer, for example, 100b or more, or 1kb or more.

[0019] The lengths of the single-stranded nucleic acids in the first step (a), the first step (b), and the second and subsequent steps (b) may be the same or different, and can be set arbitrarily. For example, if the length of the single-stranded nucleic acid in the first step (a) is 20b and the length of the single-stranded nucleic acid in step (b) is 20b, then 100b of single-stranded nucleic acid can be obtained by performing steps (a) and (b) alternately four times. Also, if the length of the single-stranded nucleic acid in the first step (a) is 20b, the length of the single-stranded nucleic acid in the first step (b) is 20b, and the lengths of the second and third steps (b) are 30b, then 100b of single-stranded nucleic acid can be obtained. The sequences of the single-stranded nucleic acids in the first step (a), the first step (b), and the second and subsequent steps (b) are not particularly limited and can be set arbitrarily according to the desired sequence.

[0020] The length of the single-stranded nucleic acid ultimately obtained by this manufacturing method is not particularly limited, but may be, for example, 2 kb or less, 1 kb or less, 100 b or less, etc.

[0021] The 5' end of the single-stranded nucleic acid in step (a) is phosphorylated. By phosphorylating the 5' end of the single-stranded nucleic acid, an adenylate group can be added via this phosphate group. If a phosphate group is not present at the 5' end of the single-stranded nucleic acid, a step of phosphorylating the 5' end may be performed before step (a).

[0022] Furthermore, the 5'-terminal phosphate group of other single-stranded nucleic acids used in step (b) may be protected. Commonly used protecting groups can be applied to the phosphate group. Examples of protecting groups include the trityl group, p-methoxyphenyldiphenylmethyl group, di(p-methoxyphenyl)phenylmethyl (DMTr) group, cyanoethyl group, and ethylthio group.

[0023] <Protection of the 3' End> The protection should be such that it can prevent the single-stranded nucleic acid from linking with other single-stranded nucleic acids at its 3' end in step (a). For protection, techniques known in the field of nucleic acid production can be used. For example, the 3' end of the single-stranded nucleic acid may be fixed (bound) to a solid phase. The solid phase can be any carrier that can hold the 3' end, and examples of solid phases include beads (glass, resin), columns, membranes, etc. More specifically, streptavidin beads (for example, magnetic beads coated with streptavidin) are given as examples. Alternatively, the hydroxyl group at the 3' end of the single-stranded nucleic acid may be chemically protected by converting it to another functional group.

[0024] The method for producing single-stranded nucleic acid may further include a step of protecting the 3' end of the single-stranded nucleic acid before step (a).

[0025] <Step (a): Adenylation> In step (a), an adenylate group is added to the 5' end of a single-stranded nucleic acid whose 3' end is protected and whose 5' end is phosphorylated, using adenylase. In the embodiment shown in Figure 1, in step (a), pyrophosphate is cleaved from ATP (adenosine triphosphate) by adenylase, and the remaining AMP (adenylate) is added to the phosphate group at the 5' end of the single-stranded nucleic acid. In this way, a single-stranded nucleic acid with an adenylate 5' end is obtained.

[0026] Specifically, Mth RNA ligase can be used as the adenilase.

[0027] After step (a) and before adding the single-stranded nucleic acid in step (b), unwanted adenilase, ATP, and pyrophosphate can be removed by washing.

[0028] <Step (b): Single-stranded nucleic acid addition (ligation)> In step (b), another single-stranded nucleic acid with a phosphorylated 5' end is added (ligated) to the 5' end of the single-stranded nucleic acid after step (a) using an adenylate-dependent nucleic acid ligase. As shown in Figure 1, another single-stranded nucleic acid with a phosphorylated 5' end (shown as a dotted line in the figure) is added to the reaction system after step (a) and washing. The hydroxyl group at the 3' end of the added other single-stranded nucleic acid is not protected and is in a reactive state. The adenylate-dependent nucleic acid ligase recognizes the adenylate group and binds to the 5' end of the single-stranded nucleic acid from step (a), cleaving the AMP and adding (ligating) the 3' end of the other single-stranded nucleic acid to this 5' end. In this way, the single-stranded nucleic acid is extended.

[0029] Specifically, mutant Mth RNA ligase is used as an adenylate-dependent nucleic acid ligase.

[0030] Washing removes the adenylate-dependent nucleic acid ligase and unreacted single-stranded nucleic acids from the reaction system, and the extended single-stranded nucleic acids can be further extended by subjecting them to steps (a) and (b).

[0031] During step (b), the protection of the 3' end of the single-stranded nucleic acid from step (a) is maintained. For example, if the 3' end of the single-stranded nucleic acid is fixed to a solid phase, steps (a) and (b) can be repeated on the solid phase to extend the nucleic acid molecule.

[0032] Furthermore, the adenylation in step (a) and the ligation in step (b) are preferably carried out under temperature conditions in which the single-stranded nucleic acid does not take on a three-dimensional structure. This is because, under such temperature conditions, the reaction sites at the ends of the single-stranded nucleic acid are exposed. Such a temperature is, for example, around 65°C.

[0033] For adenylation and ligation, conditions such as buffer composition, buffer pH, reaction time, enzymes used, and temperature can be appropriately selected and modified by those skilled in the art, within the scope of producing single-stranded nucleic acids.

[0034] Furthermore, the method for producing single-stranded nucleic acids may include a step after step (a) and before step (b), or after step (b) and before step (a), in which the activity of the enzyme used in step (a) or step (b) is reduced. "Reducing the activity of the enzyme" means, for example, reducing the activity of the enzyme or removing the enzyme from the reaction system.

[0035] Steps to reduce enzyme activity include heating to a degree that does not interfere with the next step, adding a drug, decomposition by a protease, or any combination thereof. Examples of proteases include proteinases. An example of a step to remove the enzyme from the reaction system is washing with a buffer.

[0036] More specifically, a method for producing single-stranded nucleic acids may further include a step of treatment with Proteinase K. Proteinase K can remove the enzymes used in each step from the single-stranded nucleic acids. That is, Proteinase K may be applied to adenylase after step (a) and before step (b), or Proteinase K may be applied to an adenylate-dependent nucleic acid ligase after step (b) and before step (a). These steps are shown in the figure as "Adenylase Desorption" and "Ligase Desorption."

[0037] If, in step (b), a single-stranded nucleic acid with a 5' end protected is used as the other single-stranded nucleic acid, and further steps (a) and (b) are performed to extend the nucleic acid, a step of removing the 5' end protecting group may be performed before step (a).

[0038] Furthermore, the number of cycles performed is not particularly limited, and the series of operations including step (a) and the subsequent step (b) constitutes one cycle. The number of cycles can be determined according to the desired nucleic acid sequence. Depending on the desired sequence and length, it may be completed in one cycle, or two, three, four, or more cycles may be performed.

[0039] The single-stranded nucleic acid production method described above can be carried out in an aqueous system without the use of organic solvents.

[0040] (1) Binding of oligonucleotides to magnetic beads The specific procedure for preparing oligonucleotide-bound magnetic beads was as follows. In the following, four samples were prepared from one type of oligonucleotide (sequence A) to be initially bound to the magnetic beads by performing the adenylation and subsequent steps in four tubes.

[0041] 84 μL of 10 mg / mL magnetic beads were washed twice with 400 μL of 2× B&W buffer, and then suspended in 50 μL of 2× B&W buffer. 200 μL of 1× TE buffer solution (concentration 2 pmol / μL) of oligonucleotides consisting of sequence A (i.e., 400 pmol of oligonucleotide) was added to the suspension and mixed. This was allowed to stand at room temperature for 10 minutes, and then washed twice with 400 μL of 1× B&W buffer.

[0042] Magnetic beads: Dynabeads MyOne Streptavidin C1 (Invitrogen) Binder and Washing (B&W) buffer (2x): 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl Sequence A: 5'-Phos-rArArArArGrUrGrGrCrArCrCrCrArGrUrCrGrGrUrGrC-PC_spacer-AAAA-BioTEG-3'

[0043] Note that in Array A, "Phos" represents a phosphate group, "PC" represents "Photocleavage", and "BioTEG" represents "Biotin-TEG". Thus, the oligonucleotide of Array A has a phosphate group at the 5' end of the 22-nucleotide RNA and has Biotin-TEG bound via "PC" and a spacer at the 3'. With this configuration, as will be described later, by cleaving the "PC" portion with light, the extended oligonucleotide was separated and recovered from the magnetic beads.

[0044] (2) Adenylated oligonucleotide-bound magnetic beads were suspended by adding 420 μL of 1× adenylation buffer. This suspension was dispensed into four 0.2 mL tubes at 100 μL each. That is, the amount of magnetic beads per tube was 200 μg.

[0045] The magnetic beads were separated from the adenylation buffer using a magnet, and 50 μL of the adenylation solution was added to the separated magnetic beads and mixed. The mixture was treated at 65°C for 60 minutes and at 85°C for 5 minutes, and the temperature was maintained at 12°C.

[0046] 1 μL of 0.8 units / μL Proteinase K was added to the mixture after adenylation and mixed, and the mixture was treated at 25°C for 15 minutes and at 95°C for 10 minutes, and the temperature was maintained at 12°C.

[0047]

[0048]

[0049] (3) Ligation and repetition of the process The magnetic beads after adenylation were washed twice with 100 μL of 1× ON-ligation buffer. 50 μL of the ligation solution was added to the washed magnetic beads and mixed. The mixture was treated at 65°C for 60 minutes and at 85°C for 5 minutes, and the temperature was maintained at 12°C. Then, 1 μL of 0.8 units / μL Proteinase K was added and mixed, and the mixture was treated at 25°C for 15 minutes and at 95°C for 10 minutes, and the temperature was maintained at 12°C. <00 , 00102>

[0051]

[0052] The buffer was discarded, and the sample was washed twice with 100 μL of 1x adenylation buffer before being subjected to further adenylation.

[0053] In the first ligation, sequence B was used as the phosphorylated oligonucleotide; in the second ligation, sequence C was used; and in the third ligation, sequence D was used, thus changing the sequence of the phosphorylated oligonucleotide. For sequences B through D, the phosphate group at the 5' end has been omitted from the notation.

[0054] Sequence B: 5'-rGrArGrCrUrArGrArArArUrArGrCrArArGrUrUrArArArArUrA-3' Sequence C: 5'-rArGrGrCrUrArGrUrCrCrGrUrUrArUrCrArArCrUrUrGrA-3' Sequence D: 5'-rUrCrArUrUrArCrUrGrGrArUrCrUrCrUrCrArUrCrArArCrGrUrUrUrU-3'

[0055] After performing adenylation and subsequent ligation three times each, 50 μL of 1×TE buffer was added to complete the synthesis.

[0056] Finally, the Dynabeads suspended in this 1×TE buffer were irradiated with 300-350 nm UV light to cleave at the PC portion, and the 1×TE buffer containing the extended RNA was recovered.

[0057] The recovered RNA was examined by electrophoresis to confirm that products of the desired length had been produced.

Claims

1. A method for producing single-stranded nucleic acids, comprising: (a) adding an adenylate group to the 5' end of a single-stranded nucleic acid whose 3' end is protected and whose 5' end is phosphorylated using adenylase; and (b) adding another single-stranded nucleic acid whose 5' end is phosphorylated using an adenylate-dependent nucleic acid ligase to the 5' end of the single-stranded nucleic acid after step (a), wherein steps (a) and (b) are performed alternately at least once.

2. A method for producing a single-stranded nucleic acid according to claim 1, further comprising a step of performing treatment with Proteinase K after step (a) and before step (b), or after step (b) and before step (a).

3. The method for producing a single-stranded nucleic acid according to claim 1, wherein the length of the single-stranded nucleic acid in the first step (a) is 200 bases or less.

4. The method for producing a single-stranded nucleic acid according to claim 1, wherein at least one of the single-stranded nucleic acid in step (a) and the single-stranded nucleic acid added in step (b) contains a modified base.

5. The method for producing a single-stranded nucleic acid according to claim 1, wherein the 3' end of the single-stranded nucleic acid in step (a) is protected by being immobilized on a solid phase.

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