Method for cleaving nucleic acid strands and apparatus for cleaving nucleic acid strands, method for producing double-stranded DNA and apparatus for producing double-stranded DNA

Abstract

A nucleic acid strand cleaving method that is characterized by comprising a nucleic acid preparation step in which a nucleic acid to be cleaved that has a structure indicated in formula (1) is prepared, and a cleaving step in which the nucleic acid to be cleaved is cleaved at X in formula (1) by reacting the nucleic acid to be cleaved with a cleaving agent, and a nucleic acid that has a structure indicated in formula (2) is generated, wherein the cleaving agent is metal nanoparticles that include atoms selected from the group consisting of silver, mercury, and cadmium.　[Formula 1] (B indicates a base, and X indicates sulfur or selenium. NucA comprises at least one nucleotide, is part of the nucleic acid to be cleaved, and indicates a section on the 5'-end side, relative to X. NucB comprises at least one nucleotide, is part of the nucleic acid to be cleaved, and indicates the 3'-end side, relative to X.)

Description

[Technical Field]

[0001] The present invention relates to a method for cleaving nucleic acid strands, a nucleic acid strand cleaving apparatus, and a method for producing double-stranded DNA and an apparatus for producing double-stranded DNA. [Background technology]

[0002] In fields such as molecular biology, vectors containing target DNA incorporated into a host are used for genetic engineering and transformation. Generally, when the amount of target DNA is small, it is amplified using polymerase chain reaction (PCR). In PCR, a template DNA containing the target DNA sequence is used, and the template DNA is amplified by repeating thermal denaturation and annealing multiple times using primers that bind complementaryly to the template DNA.

[0003] The amplification product of template DNA amplified by PCR has blunt ends, and it is necessary to process it to bind (ligate) it to host DNA such as plasmid DNA. In such cases, there is a technique to construct a vector by making the 3' and 5' ends of the amplification product into adherent ends (also called sticky ends or overhanging ends), and similarly forming adherent ends on the host side, and then ligating the two.

[0004] To form adherent ends on DNA, it is necessary to cleave one of the double-stranded DNA strands at a desired position to create an overhang on the DNA strand. In this regard, for example, Non-Patent Literature 1 describes the preparation of oligonucleotides containing a 3'-S-phosphorothiolate bond using an automated DNA synthesizer with 5'-O-monomethoxytritylthymidine 3'-S-(2-cyanoethyl)-N,N-diisopropylphosphorothioamidite, and states that the strand containing the 3'-S-phosphorothiolate bond is Ag + The abstract describes that the tissue was specifically chemically cleaved at this site using [a specific method / tool]. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] “Sequence-and strand-specific cleavage in oligodeoxyribonucleotides and DNA containing 3'-thiothymidine.” Joseph S. Vyle,et al.Biochemistry,31,3012(1992) [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Non-patent document 1 describes silver ions (Ag) as a cleavage agent for cutting single-stranded DNA. + The method used involves using silver ions. Because silver ions have a positive charge, they electrostatically interact with negatively charged functional groups such as the phosphate group of DNA and the nucleic acid base portion. This leads to aggregate formation due to nonspecific interactions, resulting in a low recovery rate of the target DNA.

[0007] The object of the present invention is to provide a nucleic acid strand cleavage method and apparatus that have good cleavage efficiency and high recovery rate of the target nucleic acid. Another object of the present invention is to provide a method for producing double-stranded DNA having adhesive ends and an apparatus for producing double-stranded DNA that have good adhesion end formation efficiency and high recovery rate of double-stranded DNA having the target adhesive ends. [Means for solving the problem]

[0008] The inventors diligently conducted research to solve the above problems. As a result, they discovered that by using metal nanoparticles instead of metal ions as a cleavage agent, the cleavage efficiency of the nucleic acid to be cleaved is improved, and the recovery rate of the target nucleic acid is increased, thus completing the present invention.

[0009] [1] A nucleic acid preparation step for preparing a nucleic acid to be cleaved having the structure shown in the following formula (1), A cleavage step of reacting the nucleic acid to be cleaved with a cleavage agent to cleave the nucleic acid to be cleaved at the portion of X in the formula (1) to generate a nucleic acid having a structure represented by the following formula (2). The nucleic acid strand cleavage method, wherein the cleavage agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium.

Chemical formula

[0010] 〔2〕The nucleic acid strand cleavage method according to 〔1〕, wherein the cleavage agent is silver nanoparticles and X is sulfur.

[0011] 〔3〕The nucleic acid strand cleavage method according to 〔1〕, wherein the average particle diameter of the metal nanoparticles is in the range of 1 to 20 nm.

[0012] 〔4〕The nucleic acid strand cleavage method according to 〔1〕, wherein polyethylene glycol is bound to the surface of the metal nanoparticles.

[0013] 〔5〕The nucleic acid strand cleavage method according to 〔1〕, wherein the nucleic acid preparation step synthesizes a part or all of the nucleic acid to be cleaved by the phosphoramidite method using amidite reagents represented by the following formulas (3) and (4).

Chemical formula

[0014] [6] The nucleic acid chain cleavage method according to [5], characterized in that the nucleic acid preparation step involves synthesizing 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole as an activator by the phosphoramidite method.

[0015] [7] The nucleic acid chain cleavage method according to [5], characterized in that the nucleic acid preparation step involves synthesizing a portion of the nucleic acid to be cleaved by a phosphoramidite method to form a primer, and using a template DNA having a sequence complementary to the nucleic acid to be cleaved as a template, performing multiple polymerase chain reaction cycles to extend the primer along the template DNA and generate the nucleic acid to be cleaved.

[0016] [8] A nucleic acid preparation means for preparing a nucleic acid to be cleaved having the structure shown in the following formula (1), A cleavage means that reacts the nucleic acid to be cleaved with a cleavage agent to cleave the nucleic acid at the X portion of formula (1) and generate a nucleic acid having the structure shown in formula (2) below, Equipped with, A nucleic acid chain severing device characterized in that the severing agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. [ka] (Here, B represents a base, and X represents sulfur or selenium. NucA consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 5' end portion relative to X. NucB consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 3' end portion relative to X.)

[0017] [9] The nucleic acid chain cleavage apparatus according to [8], characterized in that the nucleic acid preparation means synthesizes part or all of the nucleic acid to be cleaved by the phosphoramidite method using amidite reagents represented by the following formulas (3) and (4). [ka] (Here, B represents a base, X represents sulfur or selenium, and DMTr represents a dimethoxytrityl group.)

[0018]

[10] The nucleic acid chain cleavage apparatus according to [9], characterized in that the nucleic acid preparation step involves synthesizing 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole as an activator by the phosphoramidite method.

[0019]

[11] A method for producing double-stranded DNA having adherent ends, A double-stranded DNA preparation step for preparing a double-stranded DNA to be cleaved, comprising a sense strand and an antisense strand having a sequence complementary to the sense strand, wherein at least one of the sense strand and the antisense strand has a structure represented by the following formula (1); The process includes a step of generating an adherent end, in which the double-stranded DNA to be cleaved is reacted with a cleaving agent to cleave the sense strand and / or the antisense strand at the X portion of formula (1), thereby generating a double-stranded DNA having the structure shown in formula (2) below and having adherent ends. A method for producing double-stranded DNA, characterized in that the cleavage agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. [ka] (Here, B represents a base, and X represents sulfur or selenium. NucA consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 5' end portion relative to X. NucB consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 3' end portion relative to X.)

[0020]

[12] The method for producing double-stranded DNA according to

[11] , characterized in that the double-stranded DNA to be cleaved has a plurality of structures represented by formula (1), and the number of nucleotides between each structure is 10 or less.

[0021]

[13] A double-stranded DNA manufacturing apparatus for producing double-stranded DNA having adherent ends, A double-stranded DNA preparation means for preparing a double-stranded DNA to be cleaved, comprising a sense strand and an antisense strand having a sequence complementary to the sense strand, wherein at least one of the sense strand and the antisense strand has a structure represented by the following formula (1); The system includes an adhesive end generating means for reacting the double-stranded DNA to be cleaved with a cleaving agent to cleave the sense strand and / or the antisense strand at the X portion of formula (1), thereby generating double-stranded DNA having the structure shown in formula (2) below and having adhesive ends, A double-stranded DNA production apparatus characterized in that the cleavage agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. [ka] (Here, B represents a base, and X represents sulfur or selenium. NucA consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 5' end portion relative to X. NucB consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 3' end portion relative to X.)

[0022]

[14] The apparatus for producing double-stranded DNA according to

[13] , characterized in that the double-stranded DNA to be cleaved has a plurality of structures represented by formula (1), and the number of nucleotides between each structure is 10 or less. [Effects of the Invention]

[0023] According to the present invention, it is possible to provide a nucleic acid strand cleavage method and apparatus that have good cleavage efficiency and a high recovery rate of the target nucleic acid. Furthermore, according to the present invention, it is possible to provide a method for producing double-stranded DNA and an apparatus for producing double-stranded DNA that have good adhesion end formation efficiency and a high recovery rate of double-stranded DNA having the target adhesion ends. [Brief explanation of the drawing]

[0024] [Figure 1] This is a schematic diagram illustrating the nucleic acid strand cleavage reaction using metal nanoparticles and the ligation reaction for preparing double-stranded DNA with adhesive ends. [Figure 2] This figure shows the synthesis of oligonucleotides containing a 3'-thiophosphate bond using a thiolated amidite reagent, and the results of analysis by reverse-phase HPLC and MALDI-TOF-MS. [Figure 3] This figure shows the results of an investigation into the DNA strand cleavage reaction induced by silver nanoparticle treatment, and the dependence of silver nanoparticle size on reaction time and reaction temperature. [Figure 4] This figure shows the results of improving DNA strand cleavage activity by polymer modification of the surface of silver nanoparticles. [Figure 5] This figure shows the results of preparing 3' overhang adhesive ends of double-stranded short-chain DNA by treating them with silver nanoparticles. [Figure 6] This figure shows the results of PCR studies when thiolated oligonucleotides were introduced into a DNA template. [Figure 7] This is a schematic diagram of DNA amplification by PCR and preparation of adherent fragments by treatment with BsaI or silver nanoparticles. [Figure 8] This figure shows the results of an experiment on linking adherent fragments using T4 DNA ligase. [Figure 9] This figure shows the results of thiolated oligonucleotide chain cleavage using silver nitrate treatment, an existing technology. [Figure 10] This figure shows the molecular weight analysis results of the cleavage products of thiolated oligonucleotide chains cleaved using silver nitrate treatment, an existing technology, as determined by MALDI-TOF-MS. [Figure 11] This figure shows the results of investigating existing technologies: thiolated oligonucleotide chain cleavage by silver nitrate treatment and silver ion removal by thiol addition. [Figure 12] This figure shows the removal of silver nanoparticles by centrifugation (dependence on nanoparticle size). [Figure 13]Comparison of silver nitrate treatment and silver nanoparticle treatment in thiolated oligonucleotide strand cleavage. In the gel image, Lane 1 shows the results of electrophoresis with DNA (15mer) 5'-CACATTAATTGCGTT-FAM-3', which has the same base length as the DNA produced after cleavage. [Figure 14] This figure shows that the dispersibility of silver nanoparticles was improved by surface PEG modification. [Figure 15] This figure shows that surface PEG modification improved the dispersibility of silver nanoparticles. [Figure 16] This figure shows the sequence design for a DNA ligation reaction utilizing the formation of adherent ends by DNA strand cleavage using silver nanoparticles. [Figure 17] This figure shows the results of optimizing coupling conditions in a dimer model synthesis aimed at improving the reaction conditions of 3'-thiolated amidites. [Figure 18] This experimental result shows the effect of reaction time when using 0.25 M BTFTH (13 equivalents) in dimer model synthesis. [Figure 19] This figure shows the estimated mechanism of side reactions in coupling reactions that were revealed in the dimer model synthesis. [Figure 20] This paper presents the results of applying the optimized reaction conditions used in dimer synthesis to oligoDNA synthesis, and improving those conditions. [Figure 21] This figure shows the synthesis results of oligoDNA (sequence: 5'-TAA Ts CATTAATTGCGTT-FAM-3') synthesized using optimized coupling conditions. [Figure 22] This figure shows the synthesis results of a 51-base oligoDNA (5'-ATGAAACGCCGAGTTsAACGCCATCAAAAATsAATTCGCGTCTGGCCTTCCTsG-3') with three 3' thiophosphate bonds introduced. [Figure 23] This figure shows the sequences of PCR primer DNA with four and two 3' thiophosphate bonds introduced, and the results of their synthesis. [Figure 24]This figure shows the synthesis results of 3'-thiophosphorylated oligoDNA (5'-TAACAs CACATTAATTGCGTT-FAM-3') synthesized using a 3'-thiolated amidite adenosine derivative. [Figure 25] This figure shows the results of an investigation into preparing 3'-overhang adhesive ends of 3'-thiophosphate-bound DNA containing adenosine analogs by cleavage with silver nanoparticles. [Figure 26] This figure shows the experimental design and sequence for adhesion end preparation and linkage using primers with multiple modifications. [Figure 27] This figure shows a schematic diagram illustrating the preparation of adherent ends and the ligation reaction using multiple modified primers, as well as the agarose electrophoresis results of the PCR product. [Figure 28] This is a comparison of the ligation efficiency between 34-base-length adherent end DNA sequences obtained by cleaving PCR amplification products at multiple sites and the ligation efficiency of these sequences. [Modes for carrying out the invention]

[0025] 1. Nucleic acid strand cleavage method The nucleic acid chain cleavage method of the present invention will now be described. The nucleic acid chain cleavage method of the present invention comprises a nucleic acid preparation step of preparing a nucleic acid to be cleaved having the structure shown in formula (1) below, and a cleavage step of reacting the nucleic acid to be cleaved with a cleavage agent to cleave the nucleic acid at the X portion of formula (1) to produce a nucleic acid having the structure shown in formula (2) below. [ka] (Here, B represents a base, and X represents sulfur or selenium. NucA consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 5' end portion relative to X. NucB consists of at least one nucleotide, is part of the nucleic acid to be cleaved, and represents the 3' end portion relative to X.)

[0026] Here, base B can specifically include adenine, guanine, cytosine, thymine, uracil, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoadenine, 7-methylguanine, N-isobutyrylguanine, 5-fluorocytosine, 5-bromocytosine, 5-methylcytosine, 4-N-methylcytosine, 4-N,N-dimethylcytosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, or 5,6-dihydrouracil.

[0027] The cleavage agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. Silver is particularly preferred as the cleavage agent from the viewpoint of low toxicity and low cost. When the cleavage agent is silver nanoparticles, it is preferable that X in formula (1) of the nucleic acid to be cleaved is sulfur.

[0028] The average particle size of the metal nanoparticles is preferably 100 nm or less, more preferably 50 nm or less, and particularly preferably 20 nm or less, from the viewpoint of high cleavage efficiency with respect to the nucleic acid to be cleaved. Furthermore, the lower limit of the average particle size of the metal nanoparticles is not particularly limited, but for example, it is 0.1 nm or more, preferably 0.5 nm or more, and more preferably 1 nm or more.

[0029] From the viewpoint of improving the cleavage activity of metal nanoparticles against target nucleic acids, it is preferable to surface-treat the metal nanoparticles with polyethylene glycol to bind polyethylene glycol to their surface. By binding polyethylene glycol to the surface of the metal nanoparticles, oxidation of the metal nanoparticle surface is prevented and the dispersibility of the metal nanoparticles in aqueous solution is improved. Therefore, compared to metal nanoparticles that have not been surface-treated with polyethylene glycol, metal nanoparticles surface-treated with polyethylene glycol exhibit improved cleavage activity against target nucleic acids. Polyethylene glycol can be used with thiol modification at the ends to bind to the metal nanoparticles. The average molecular weight of polyethylene glycol is preferably in the range of 1,000 to 10,000.

[0030] (1) Nucleic acid preparation process Next, each step of the present invention will be described with reference to Figure 1. The upper part of Figure 1 ("Nucleic Acid Chain Severing Reaction by Metal Nanoparticles [Step 1]") schematically shows the nucleic acid chain severing method of the present invention. In Figure 1, X in formula (1) is sulfur, the severing agent is silver nanoparticles, and the nucleic acid to be severed (thiolated oligonucleotide) is used as a PCR primer, but the present invention is not limited to this. For example, the nucleic acid to be severed can be severed by a similar mechanism when X is selenium, or when the severing agent is mercury or cadmium.

[0031] First, the nucleic acid preparation process will be explained. The nucleic acid preparation process is a process for preparing the nucleic acid to be cleaved, which has the structure shown in formula (1). In this process, part or all of the nucleic acid to be cleaved can be synthesized by the phosphoramidite method using the amidite reagents shown in formulas (3) and (4) below. [ka] (Here, B represents a base, X represents sulfur or selenium, and DMTr represents a dimethoxytrityl group.)

[0032] The amidite reagent represented by formula (3) (thiolated amidite reagent or selenated amidite reagent) can be synthesized by the following method. First, a nucleoside derivative is prepared by protecting the amino group of a base with an amide bond and protecting the 5' carbon with a dimethoxytrityl group (DMTr group) or a tert-butyldimethylsilyl group (TBDMS group). Next, this nucleoside derivative is reacted with triphenylphosphine (PPh3), diisopropyl azodicarboxylic acid (DIAD), etc. to stereochemically invert the 3' hydroxyl group, and this 3' hydroxyl group is reacted with thiobenzoic acid or selenobenzoic acid. Subsequently, by alkaline hydrolysis of the benzoic acid, nucleosides are obtained in which the oxygen of the 3' hydroxyl group of the nucleoside derivative is replaced with sulfur or selenium. Finally, the desired amidite reagent can be obtained by reacting with amidite such as 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite.

[0033] The nucleic acid to be cleaved can be synthesized using a known automated nucleic acid synthesizer with a thiolated or selenized amidite reagent shown in formula (3) and a conventional (i.e., unthiolated or unselenolated) amidite reagent shown in formula (4). By synthesizing a nucleic acid with a desired sequence using the amidite reagent shown in formula (4) and then using the amidite reagent shown in formula (3) at the desired position, a nucleic acid to be cleaved can be synthesized in which the thiolated or selenized nucleotide is positioned at the desired location.

[0034] When synthesizing the nucleic acid to be cleaved, it is preferable to add an activator. Examples of activators include 1H-tetrazole (1H-tet), 5-benzylthio-1H-tetrazole (BTT), 4,5-dicyanoimidazole (DCI), and 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (BTFTH). Of these, BTFTH is the most preferred because it reduces the amount of by-products (dithiolates) and improves the yield of the target nucleic acid to be cleaved. Furthermore, when using BTFTH as an activator, the coupling time is preferably in the range of 60 to 120 seconds, and it is preferable to perform the coupling reaction multiple times (e.g., 2 to 7 times) within this coupling time from the viewpoint of improving the yield.

[0035] The nucleic acid to be cleaved can be synthesized in its entirety using the automated nucleic acid synthesis apparatus described above. However, the entire nucleic acid can also be synthesized by synthesizing only a portion of the nucleic acid to be cleaved using the automated nucleic acid synthesis apparatus, and then using this as a primer and a template DNA having a complementary sequence to the nucleic acid to be cleaved as a template, performing multiple cycles of polymerase chain reaction (PCR). In other words, the nucleic acid to be cleaved is generated by extending the primer along the template DNA using PCR. The number of nucleotides constituting the primer can be set appropriately depending on the purpose, but for example, it can be 5 or more and 50 or less.

[0036] As described later, vectors and the like can be constructed by forming adherent ends in the nucleic acid to be cleaved according to the present invention and ligating it with other DNA strands having adherent ends. When the length of the single-stranded DNA on the protruding side of the adherent end is long, for example, 20 bases or more, it is preferable to introduce the structure shown in formula (1) above at multiple locations on the complementary strand side. In this case, as shown in the examples described later, from the viewpoint of ligation efficiency, the number of nucleotides nt (number of bases: b) between the structures of formula (1) is preferably 15 or less, and more preferably 10 or less. When this number of nucleotides is 15 or less, the dissociation of DNA fragments after strand cleavage by metal nanoparticles is accelerated, and the ligation efficiency tends to be high.

[0037] (2) Cutting process Next, the cleavage process will be explained. In the cleavage process, the nucleic acid to be cleaved is reacted with a cleavage agent to cleave the nucleic acid at the X portion of equation (1), generating a nucleic acid having the structure shown in equation (2) below. In Figure 1, silver nanoparticles are used to cleave the nucleic acid to be cleaved (thiolated oligonucleotide), and after cleavage, the oligonucleotide on the 5' end side of the X portion is removed, and the cleaved portion becomes a phosphate group.

[0038] The cleavage agent is preferably used in the form of a dispersion of metal nanoparticles. The cleavage reaction is preferably carried out at 70°C or higher, more preferably at 90°C or higher, and particularly preferably at 95°C or higher, from the viewpoint of high cleavage activity. There is no particular upper limit to the temperature of the cleavage reaction, but it is preferably 100°C or lower. The reaction time of the cleavage reaction can be appropriately set according to the reaction temperature, and the higher the reaction temperature, the shorter the reaction time can be set. The reaction time is preferably in the range of 5 minutes or more and 120 minutes or less.

[0039] 2.Nucleic acid strand cutting device Next, a nucleic acid chain cleavage apparatus will be described. The nucleic acid chain cleavage apparatus of the present invention is an apparatus for carrying out the nucleic acid chain cleavage method described above. The nucleic acid chain cleavage apparatus comprises a nucleic acid preparation means for preparing a nucleic acid to be cleaved having the structure shown in formula (1), and a cleavage means for reacting the nucleic acid to be cleaved with a cleavage agent to cleave the nucleic acid at the X portion of formula (1) and generate a nucleic acid having the structure shown in formula (2).

[0040] Nucleic acid tone Made The means include the amidite reagent shown in formulas (3) and (4) above, an automated nucleic acid synthesizer, and various reagents used for nucleic acid synthesis. Furthermore, when using nucleic acids synthesized by an automated nucleic acid synthesizer as primers, template DNA, a PCR machine, and reagents used for PCR are also included. Made It is included in the means.

[0041] As a means of cutting, nucleic acid preparation Made This includes nucleic acids to be cleaved synthesized by the means, the cleaving agent mentioned above, various devices (such as incubators) for carrying out the cleavage reaction of the nucleic acids to be cleaved by the cleaving agent, and various reagents used in the reaction.

[0042] 3. Method for producing double-stranded DNA with adherent ends Next, a method for producing double-stranded DNA having adherent ends will be described. In the production method of the present invention, adherent ends are formed on one or both ends of the double-stranded DNA. First, a double-stranded DNA preparation step is performed. In this step, a double-stranded DNA to be cleaved, consisting of a sense strand and an antisense strand, is prepared. The antisense strand has a sequence complementary to the sense strand. At least one of the sense strand and the antisense strand has the structure shown in formula (1). The lower part of Figure 1 (the area enclosed by the rectangle) shows the case where both the sense strand and the antisense strand each have the structure of formula (1). In this way, by introducing the structure of formula (1) to both strands, DNA having adherent ends at both ends can be produced in the adherent end generation step described later.

[0043] Next, the adherent end formation process is performed ("Step 1: Strand Breaking" in Figure 1). In this process, the double-stranded DNA to be cleaved is reacted with a cleaving agent to cleave the sense strand and / or antisense strand at the X portion of equation (1). As a result, double-stranded DNA having the structure shown in equation (2) and adherent ends is produced. The cleavage conditions are as described in "(2) Cleavage Process" above.

[0044] 3. Apparatus for manufacturing double-stranded DNA with adherent ends Next, an apparatus for producing double-stranded DNA having adherent ends will be described. The apparatus of the present invention is for carrying out the above-described method for producing double-stranded DNA having adherent ends. Specifically, it comprises a double-stranded DNA preparation means for preparing double-stranded DNA to be cleaved, and an adherent end generation means for reacting the double-stranded DNA to be cleaved with a cleaving agent to produce double-stranded DNA having the structure shown in formula (2) and having adherent ends.

[0045] As a method for preparing double-stranded DNA, the above-mentioned "nucleic acid preparation" Made This includes the amidite reagents shown in formulas (3) and (4), an automated nucleic acid synthesizer, template DNA, a PCR machine, and various reagents, as described in the "Means" section. As a means of generating adhesive ends, the nucleic acid preparation described in the "cutting means" above is used. Made This includes nucleic acids to be cleaved synthesized by the method, cleavage agents, various devices such as incubators, and various reagents.

[0046] In this invention, metal nanoparticles are used as the cleavage agent instead of conventionally used metal ions. This allows for a higher recovery rate of the DNA cleavage product while maintaining high cleavage activity equivalent to that of metal ions, and enables efficient formation of adherent ends. Furthermore, DNA sequences with such adherent ends can be freely ligated together. For example, adherent ends common to both target DNA and a vector can be formed using the cleavage agent, and these can be ligated to prepare recombinant DNA, which can then be used for cloning, library creation, and construction of high-level expression systems. Additionally, by ligating multiple genome sequences with adherent ends, genome build-up reactions can be performed in vitro (see "Step 2" in the lower part of Figure 1). Alternatively, double-stranded DNA can be introduced into cells in a blunt-ended state, and deprotection treatment can be performed intracellularly to carry out genome build-up reactions. [Examples]

[0047] The present invention will be described in detail below based on examples, but these examples are not intended to limit the object of the present invention. In addition, in the following examples, the "%" notation refers to mass-based percentages unless otherwise specified.

[0048] 1. Improved synthesis of thiolated amidite reagents and their introduction into oligonucleotides. (1) Synthesis of thiolated amidite reagents (thymidine derivative: T, cytidine derivative: C) The synthesis scheme for thiolated amidite reagents (thymidine derivative: T, cytidine derivative: C) is shown below. [ka]

[0049] (a) Compound 2T 5'-O-(4,4'-dimethoxytrityl)thymidine (compound 1T) (1.0 g, 1.8 mmol) was mixed with triphenylphosphine (0.72 g, 2.8 mmol), and then dissolved in dichloromethane (6.0 mL). Under an ice bath, diisopropyl azodicarboxylic acid (0.54 mL, 2.8 mmol) was slowly added dropwise. After stirring at room temperature for 3 hours, the reaction solution was concentrated using a rotary evaporator. The solution was purified by column chromatography (neutral flash silica, developing solvent: methanol / ethyl acetate mixed solvent containing 1% triethylamine = 0 / 1 → 1 / 4) to obtain 0.89 g (1.7 mmol) of compound 2T as a white solid (yield 94%). 1 ¹H NMR (400MHz, DMSO-d6) δ 7.59 (d, J=1.3Hz, 1H), 7.37-7.30 (m, 2H), 7.30-7.11 (m, 7H), 6.85-6.76 (m, 4H), 5.85 (d, J=3.7Hz, 1H), 5.27 (q, J=2.2Hz, 1H), 4.37 (ddd, J=7.8, 4.8, 2.5Hz, 1H), 3.68 (d, J=2.5Hz, 6H), 3.13-2.98 (m, 2H), 2.54 (dd, J=12.8, 1.5Hz, 1H), 2.44-2.37 (m, 1H), 1.73 (d, J=1.1Hz, 3H) ppm. The spectral data showed good agreement with literature values.

[0050] (b) Compound 3T Compound 2T (1.0 g, 1.9 mmol) and cesium thiobenzoate (1.8 g, 7.0 mmol) were suspended in 1,4-dioxane (40 mL) and stirred overnight at 120°C. Further addition of cesium thiobenzoate (1.8 g, 7.0 mmol) was added, and the mixture was stirred overnight at 130°C. After cooling the reaction solution to room temperature, it was diluted with ethyl acetate and washed twice with saturated sodium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate and then concentrated using a rotary evaporator. Purification by column chromatography (neutral flash silica, developing solvent: methanol / dichloromethane mixed solvent containing 1% triethylamine = 0 / 1 → 1:60) yielded 1.1 g (1.7 mmol) of compound 3T as a brown solid (yield 90%). 1 H NMR(600MHz,CDCl3)δ7.93-7.86(m,2H),7.71(d,J=1.4Hz,1H),7.61-7.56(m,1H),7.47-7.43(m,4H),7.34-7.31( m,4H),7.26(dd,J=8.4,7.0Hz,2H),7.22-7.17(m,1H),6.82-6.77(m,4H),6.30(dd,J=6.6,5.2Hz,1H),4.49(q,J=7 The spectral data showed good agreement with literature values. (0.4Hz, 1H), 4.14(dt, J=7.2, 2.7Hz, 1H), 3.73(d, J=4.4Hz, 6H), 3.52(dd, J=10.8, 2.4Hz, 1H), 3.45(dd, J=10.8, 3.1Hz, 1H), 2.75(ddd, J=13.8, 8.3, 5.2Hz, 1H), 2.53-2.48(m, 1H), 1.48(d, J=1.3Hz, 3H) ppm.

[0051] (c) Compound 4T Compound 3T (840 mg, 1.26 mmol) was dissolved in a mixed solution of ethanol (30 mL) and 10 M sodium hydroxide aqueous solution (4 mL) that had been deoxygenated by argon bubbling for 30 minutes. The mixture was then stirred at room temperature for 2 hours under argon bubbling. The reaction solution was cooled on an ice bath, and the reaction was stopped by slowly adding 1 M hydrochloric acid aqueous solution (40 mL) dropwise. The resulting precipitate was collected by suction filtration and washed three times with water on filter paper. The obtained solid was dissolved in dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The compound was purified by column chromatography (neutral flash silica, developing solvent: hexane / ethyl acetate mixed solvent containing 1% triethylamine = 1 / 1 → 1 / 3) to obtain 560 mg (0.995 mmol) of compound 5T as a brown solid (yield 79%). 1H NMR(600MHz,CDCl3)δ7.68(d,J=1.4Hz,1H),7.41(dd,J=7.5,1.7Hz,2H),7.34-7.20(m,7H),6.83(dd,J =8.8,2.0Hz,4H),6.14(dd,J=7.1,3.0Hz,1H),4.10(q,J=7.1Hz,1H),3.85(dt,J=8.8,2.7Hz,1H),3.77 (s,6H),3.63-3.56(m,2H),3.39(dd,J=11.0,2.9Hz,1H),2.59(ddd,J=13.9,7.6,2.9Hz,1H),2.35(ddd,J=13.9,10.2,7.1Hz,1H),1.55(d,J=6.9Hz,1H),1.48(d,J=1.2Hz,3H)ppm. The spectral data showed good agreement with literature values.

[0052] (d) Compound 5T Compound 4T (360 mg, 0.640 mmol) was dissolved in dichloromethane (5.00 ml), and then N,N-diisopropylethylamine (331 μL, 1.92 mmol) was added. Subsequently, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (185 μL, 0.830 mmol) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with dichloromethane and washed with saturated sodium bicarbonate aqueous solution. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. The solution was purified by column chromatography (neutral flash silica, developing solvent: hexane / ethyl acetate mixed solvent containing 1% triethylamine = 2 / 1 → 1 / 1.5) to obtain 312 mg (0.410 mmol) of compound 5T as a white solid (yield 64%). 1H NMR(400MHz,CD3CN)δ9.41(s,1H),7.55-7.40(m,3H),7.38-7.24(m,6H),7.24-7.14(m, 1H),6.83(dd,J=8.7,5.5Hz,4H),6.18-5.99(m,1H),4.10-3.93(m,2H),3.86-3.75(m,1 H),3.73(s,6H),3.71-3.44(m,4H),3.44-3.28(m,1H),2.66-2.53(m,2H),2.53-2.39(m ,1H),1.95(s,1H),1.55-1.42(m,3H),1.31-1.05(m,9H),1.03(s,1H),1.01(s,1H)ppm. 31 The 1P NMR (162MHz, CD3CN) spectrum data showed good agreement with literature values, with δ values ​​of 161.96 and 158.44 ppm.

[0053] (e) Compound 2C N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine (compound 1C) (1.00 g, 1.57 mmol) was azeotropically mixed three times with toluene (20.0 mL). Triphenylphosphine (0.456 g, 1.74 mmol) was added, and the mixture was dissolved in dichloromethane (6.00 mL). Diisopropyl azodicarboxylic acid (0.456 mL, 1.74 mmol) was slowly added dropwise under ice. The mixture was then stirred at room temperature for 16 hours. The reaction solution was concentrated using a rotary evaporator and purified by column chromatography (neutral flash silica, developing solvent: methanol / ethyl acetate mixed solvent containing 1% triethylamine = 0 / 1 → 1 / 4), resulting in a white solution. solid 0.780 g (1.26 mmol) of compound 2C was obtained (yield 78%). 1H-NMR (600 MHz, DMSO-d6) δ 7.81 (d, J = 7.1 Hz, 2H), 7.69 (d, J = 7.5 Hz, 1H), 7.51 (t, J = 7.3 Hz, 1H), 7.18 - 7.39 (m, 12H), 6.84 (dd, J = 9.0, 3.6 Hz, 4H), 6.42 (d, J = 7.5 Hz, 1H), 5.97 (d, J = 4.1 Hz, 1H), 5.33 (s, 1H), 4.40 (s, 1H), 3.70 (d, J = 2.4 Hz, 7H), 3.04 - 3.13 (m, 2H), 2.63 (d, J = 11.9 Hz, 1H) ppm. The spectral data showed good agreement with the literature values.

[0054] (f) Compound 3C Compound 2C (2.8 g, 4.8 mmol) and cesium thiobenzoate (6.0 g, 24 mmol) were dissolved in 1,4-dioxane (60 mL) and stirred at room temperature for 5 hours. Then, the reaction solution was diluted with ethyl acetate (100 mL) and washed twice with a saturated aqueous sodium hydrogen carbonate solution (100 mL) and twice with saturated brine. The organic phase was dried over anhydrous sodium sulfate and then the solvent was evaporated off using a rotary evaporator. Purification by column chromatography (neutral flash silica, eluent: ethyl acetate / hexane = 1 / 1 → 7 / 1) gave 2.0 g (2.7 mmol) of Compound 3C as a brown solid (yield 56%). 1 H-NMR (600 MHz, DMSO-d6) δ 11.29 (s, 1H), 8.55 (d, J = 7.5 Hz, 1H), 8.00 (d, J = 7.5 Hz, 2H), 7.88 (d, J = 7.1 Hz, 2H), 7.50 - 7.73 (m, 4H), 7.38 (d, J = 7.5 Hz, 2H), 7.14 - 7.27 (m, 9H), 6.82 (q, J = 4.5 Hz, 5H), 6.12 (d, J = 9.2 Hz, 1H), 4.40 (t, J = 9.0 Hz, 1H), 4.23 (s, 1H), 3.69 (d, J = 5.4 Hz, 1H), 3.66 (s, 6H), 2.65 (s, 2H), 2.36 (s, 1H) ppm. The spectral data showed good agreement with the literature values.

[0055] (g) Compound 4C, Compound 5C Compound 3C (1.64 g, 2.2 mmol) was dissolved in a mixed solution of methanol (15 mL), THF (21 mL), and 0.5 M sodium hydroxide aqueous solution (22 mL, 11 mmol) that had been deoxygenated by argon bubbling for 30 minutes. The mixture was then stirred at -10°C for 30 minutes under argon bubbling. The reaction was stopped by slowly adding 1 M potassium dihydrogen phosphate aqueous solution (46.6 mL, 46.6 mmol) dropwise to the reaction solution. The mixture was diluted with ethyl acetate (100 mL) and washed twice with water (100 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. After purification by column chromatography (neutral flash silica, developing solvent: methanol / dichloromethane mixed solvent containing 1% triethylamine = 1 / 50 → 1 / 10), 1.18 g of compound 4C was obtained as a white solid. 0.94 g (1.43 mmol) of the obtained compound 4C was weighed out and dissolved in dichloromethane (15.2 mL). Then, N,N-diisopropylethylamine (0.35 mL, 1.57 mmol) and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.35 mL, 1.57 mmol) were added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with dichloromethane (20 mL) and washed with saturated sodium bicarbonate aqueous solution (20 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. After purification by column chromatography (neutral flash silica, developing solvent: acetonitrile / dichloromethane mixed solvent containing 1% triethylamine = 3 / 7), 1.0 g (1.2 mmol) of compound 5C was obtained as a white solid (2-step total yield 68%). 1H-NMR(400MHz,CDCl3)δ9.19(s,1H),8.78(s,1H),8.30(d,J=19.5Hz,1H),8.02-8.05(m,2H),7.51-7.63(m,3H) ,7.37-7.42(m,2H),7.27-7.31(m,5H),7.18-7.25(m,3H),6.77-6.81(m,4H),6.42(dd,J=7.0,2.5Hz,1H),4.21 -4.25(m,1H),3.90(t,J=6.2Hz,1H),3.79-3.85(m,1H),3.77(d,J=4.3Hz,6H),3.57-3.67(m,5H),3.41-3.45(m ,1H),3.07-3.14(m,1H),2.60(t,J=6.2Hz,1H),2.44(t,J=6.3Hz,2H),1.18-1.22(m,6H),1.13-1.17(m,6H)ppm. 31 P-NMR (162 MHz, CDCl3) δ 165.57, 161.68 ppm. The spectral data showed good agreement with literature values.

[0056] (2) Synthesis of thiolated amidite reagent (adenosine derivative: A) The synthesis scheme for the thiolated amidite reagent (adenosine derivative: A) is shown below. [ka]

[0057] (a) Compound 2A, compound 3A N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (compound 1A) (10.0 g, 15.2 mmol), 4-nitrobenzoic acid (5.08 g, 30.4 mmol), and triphenylphosphine (16.0 g, 61.0 mmol) were dissolved in THF (800 mL), and diisopropyl azodicarboxylic acid (11.9 mL, 61.0 mmol) was slowly added dropwise while stirring at 0°C. After stirring at 0°C for 2 hours, the reaction solution was concentrated using a rotary evaporator and purified by column chromatography (neutral flash silica, developing solvent: ethyl acetate / hexane = 7 / 3) to obtain 18.5 g of compound 2A as a white solid. 10.0 g (12.6 mmol) of the obtained compound 2A was weighed out, potassium carbonate (1.71 g, 49.6 mmol) was added, and then it was dissolved in methanol (125 mL). After stirring in an ice bath for 2 hours, the reaction solution was concentrated. The residue was dissolved in ethyl acetate (75 mL) and washed twice with water (75 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. The compound was purified by column chromatography (neutral flash silica, developing solvent: ethyl acetate / hexane = 1 / 1) to obtain 1.40 g (2.10 mmol) of compound 3A as a white solid (2-step total yield 38%). 1 H-NMR(400MHz,DMSO-d6)δ11.18(s,1H),8.75(s,1H),8.47(s,1H),8.03(d,J=7.6Hz,2H ),7.53-7.66(m,3H),7.17-7.39(m,9H),6.75-6.83(m,4H),6.50(d,J=7.4Hz,1H),5.48 (d,J=4.3Hz,1H),4.31(d,J=44.2Hz,2H),3.66-3.74(m,6H),3.37(d,J=8.1Hz,1H),3.20(d,J=10.3Hz,1H),2.78(s,1H),2.38(d,J=14.6Hz,1H)ppm. The spectral data showed good agreement with literature values.

[0058] (b) Compound 4A, Compound 5A, Compound 6A Triphenylphosphine (2.4 g, 9.0 mmol) was dissolved in THF (53 mL), and diisopropyl azodicarboxylic acid (1.75 mL, 9.0 mmol) was slowly added dropwise while stirring under ice. After stirring under ice for 30 minutes, thiobenzoic acid (1.08 mL, 9.0 mmol) was added, and stirring continued under ice for another 30 minutes. Subsequently, compound 3A (2.0 g, 3.0 mmol) was added, and stirring continued under ice for 16 hours. The reaction solution was concentrated using a rotary evaporator, and the residue was purified by column chromatography (neutral flash silica, developing solvent: ethyl acetate / hexane = 1 / 1 → 2 / 1) to obtain 2.3 g of compound 4A as a white solid. 2.3 g (3.0 mmol) of the obtained compound 4A was weighed out and dissolved in a methanol (42 mL)-THF (28 mL)-0.5 M sodium hydroxide aqueous solution (18 mL, 9.0 mmol) mixed solution that had been deoxygenated by argon bubbling for 30 minutes. The mixture was then stirred at -10°C for 30 minutes under argon bubbling. The reaction was stopped by slowly adding 1 M potassium dihydrogen phosphate aqueous solution (38 mL, 19 mmol) dropwise to the reaction solution. The resulting precipitate was collected by suction filtration and washed three times with water (200 mL) on filter paper. The obtained solid was purified by column chromatography (neutral flash silica, developing solvent: methanol / dichloromethane mixed solvent containing 1% triethylamine = 1 / 50 → 1 / 20) to obtain 1.62 g of compound 5A as a white solid. 1.5 g (2.3 mmol) of the obtained compound 5A was weighed out, and 5-(ethylthio)-1H-tetrazole (0.30 g, 2.3 mmol) was added, followed by dissolution in dichloromethane (10 mL). Subsequently, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (1.1 mL, 3.5 mmol) was added, and the mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with dichloromethane (50 mL) and then washed with saturated sodium bicarbonate aqueous solution (50 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The compound was purified by column chromatography (neutral flash silica, developing solvent: ethyl acetate / hexane = 2 / 1 → 8 / 1) to obtain 0.46 g (0.54 mmol) of compound 6A as a white solid (total yield 17% in 3 steps). 1 H-NMR(400MHz,CDCl3)δ9.19(s,1H),8.78(s,1H),8.30(d,J=19.5Hz,1H),8.02-8.05(m,2H),7.51-7.63 (m,3H),7.37-7.42(m,2H),7.27-7.31(m,5H),7.18-7.25(m,3H),6.77-6.81(m,4H),6.42(dd,J=7.0,2.5 Hz,1H),4.21-4.25(m,1H),3.90(t,J=6.2Hz,1H),3.79-3.85(m,1H),3.76-3.77(m,7H),3.57-3.67(m,5H) ),3.41-3.45(m,1H),3.07-3.14(m,1H),2.60(t,J=6.2Hz,1H),1.18-1.22(m,6H),1.13-1.17(m,6H)ppm. 31 P-NMR (162 MHz, CDCl3) δ 165.22 ppm. The spectral data showed good agreement with literature values.

[0059] (3) Synthesis of thiolated amidite reagent (guanosine derivative: G) The synthesis scheme for the thiolated amidite reagent (guanosine derivative: G) is shown below. [ka]

[0060] (a) Compound 2G 1 g (7.3 g, 16 mmol) of the compound was dissolved in dichloromethane (370 mL), and Dess-Martin periodinane (34 g, 80 mmol) and sodium bicarbonate (21 g) were added. The mixture was stirred under ice for 2 hours, followed by 3 hours at room temperature. After the reaction was complete, saturated sodium bicarbonate aqueous solution (370 mL) and sodium thiosulfate (370 mL) were added, and the mixture was extracted twice with dichloromethane (730 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. The resulting 2'-keto compound was dissolved in tetrahydrofuran (260 mL) and stirred at -78 °C for 30 minutes. Subsequently, 1 M L-selectride / tetrahydrofuran solution (40 mL, 40 mmol) was added dropwise, and the mixture was stirred at -78 °C for 16 hours. The reaction was terminated by adding saturated ammonium chloride aqueous solution (50 mL) to the reaction solution, and the mixture was extracted three times with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. The compound was purified by column chromatography (neutral flash silica, developing solvent: methanol / dichloromethane = 1 / 15) to obtain 3.6 g (8.0 mmol) of compound 2G as a white solid (yield 50%). 1 H-NMR(400MHz,DMSO-d6)δ12.06(s,1H),11.70(s,1H),8.12-8.19(m,1H),6.12(dd,J=8.2,1.9Hz,1H),5.35-5.39(m,1H),4.29-4 .37(m,1H),3.92-4.11(m,2H),3.73-3.79(m,1H),2.65-2.81(m,2H),2.21-2.32(m,1H),1.08-1.15(m,6H),0.83-0.86(m,9H),0.1 6- The spectral data of 0.14 (m, 6H) ppm showed good agreement with literature values.

[0061] (b) Compound 3G, compound 4G Triphenylphosphine (11 g, 40 mmol) was dissolved in tetrahydrofuran (140 mL), and diisopropyl azodicarboxylic acid (7.8 mL, 40 mmol) was slowly added dropwise while stirring under ice. The mixture was stirred under ice for 30 minutes, thiobenzoic acid (4.7 mL, 40 mmol) was added, and the mixture was stirred at 0°C for another 30 minutes. Then, 2 G of compound (3.0 g, 6.7 mmol) was added, and the mixture was stirred under ice for 3 hours, and then at room temperature for 16 hours. The reaction solution was concentrated using a rotary evaporator, and then purified by column chromatography (neutral flash silica, developing solvent: ethyl acetate / hexane = 1 / 1) to obtain 25 g of compound 3 G as a white solid. The obtained compound 3 G (25 g, 6.7 mol) was dissolved in tetrahydrofuran (100 mL), and triethylamine trifluoride (16 ml, 100 mmol) was added, and the mixture was stirred at room temperature for 16 hours. The reaction solution was concentrated using a rotary evaporator and then purified by column chromatography (neutral flash silica, developing solvent: methanol / dichloromethane = 1 / 15). Further recrystallization with dichloromethane yielded 2.4 g (5.2 mmol) of compound 4G as a white solid (78% total yield in two steps). 1 H-NMR(600MHz,DMSO-d6)δ12.09(s,1H),11.69(s,1H),8.34(d,J=15.6Hz,1H),7.93(dd,J=8 .3,0.8Hz,2H),7.67-7.74(m,1H),7.54-7.63(m,2H),6.24(t,J=5.9Hz,1H),5.20(t,J=5.4H The spectral data showed good agreement with literature values. (z, 1H), 4.34 (q, J=7.1Hz, 1H), 4.09-4.14 (m, 1H), 3.60-3.74 (m, 2H), 3.01-3.05 (m, 1H), 2.72-2.79 (m, 1H), 2.58-2.63 (m, 1H), 1.11 (dd, J=7.0, 2.2Hz, 6H) ppm.

[0062] (c) Compound 5G Compound 4G (1.5 g, 3.3 mmol) and 4,4'-dimethyltrityl chloride (2.2 g, 6.6 mmol) were dissolved in pyridine (30 mL) and stirred at room temperature for 16 hours. The reaction solution was concentrated using a rotary evaporator, and the residue was dissolved in dichloromethane (50 mL) and washed with saturated sodium bicarbonate aqueous solution (50 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed using a rotary evaporator. After purification by column chromatography (neutral flash silica, developing solvent: methanol / dichloromethane = 1 / 20), 2.41 g (3.2 mmol) of compound 5G was obtained as a white solid (yield 97%). 1 H-NMR(600MHz,DMSO-d6)δ12.11(s,1H),11.70(d,J=17.3Hz,1H),8.17-8.26(m,1H),7.89(dd, J=8.3,1.2Hz,2H),7.52-7.79(m,3H),7.14-7.40(m,9H),6.73-6.82(m,4H),6.30(q,J=3.5Hz, The spectral data showed good agreement with literature values. (1H), 4.45-4.57 (m,1H), 4.20-4.23 (m,1H), 3.61-3.79 (m,6H), 3.22-3.31 (m,2H), 3.17 (dq,J=13.7,3.9Hz,1H), 2.61-2.82 (m,2H), 1.13 (dd,J=6.8,1.4Hz,6H) ppm.

[0063] (d) Compound 6G, Compound 7G Compound 5G (2.0g, 2.6 mmol) was dissolved in methanol / tetrahydrofuran mixed solvent (3 / 2, 90 mL) and stirred under ice bath for 30 minutes. Then, 0.5 M sodium hydroxide aqueous solution (18 mL, 9.0 mmol) was added, and the mixture was treated under deoxygenation conditions by argon bubbling at 0°C for 2 hours and at room temperature for 30 minutes. The reaction was stopped by slowly adding 1 M potassium dihydrogen phosphate aqueous solution (33 mL, 33 mmol) dropwise to the reaction solution. Subsequently, the reaction solution was diluted with ethyl acetate (300 mL) and washed with saturated sodium bicarbonate aqueous solution (300 mL) and saturated brine (300 mL). After drying the organic phase with anhydrous sodium sulfate, the solvent was removed using a rotary evaporator, and 1.7 g of compound 6G was obtained as a white solution. solid The compound was obtained as a solid. 5-(ethylthio)-1H-tetrazole (0.34 g, 2.6 mmol) was added to the obtained compound 6G (1.7 g, 2.6 mmol) and dissolved in dichloromethane (40 mL). Subsequently, 2-cyanoethyl N,N,N',N'-tetraisopropylphosphodiamidite (1.3 mL, 4.0 mmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with dichloromethane (300 mL) and washed twice with saturated sodium bicarbonate aqueous solution (300 mL). The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporator. The compound was purified by column chromatography (neutral flash silica, developing solvent: acetonitrile / dichloromethane mixed solvent containing 1% triethylamine = 1 / 4) to obtain 1.2 g (1.4 mmol) of compound 7G as a white solid (yield 53%). 1 H-NMR(600MHz,CDCl3)δ12.00(d,J=10.2Hz,1H),8.45(d,J=34.0Hz,1H),7. 83-7.87(m,1H),7.41-7.43(m,2H),7.29-7.31(m,4H),7.17-7.24(m,3H),6. 73-6.82(m,4H),6.14-6.17(m,1H),4.19-4.25(m,1H),3.87-3.55(11H),3. 54-3.27(2H),3.00-3.12(m,1H),2.32-2.68(m,4H),1.09-1.21(m,18H)ppm. 31 P-NMR (243 MHz, CDCl3) δ 160.81, 159.97 ppm. The spectral data showed good agreement with literature values.

[0064] (4) Synthesis of oligonucleotides having a 3'-thiophosphate bond using a thioamide reagent (compound 5T) and analysis by reverse-phase HPLC and MALDI-TOF-MS

[0065] (a) DNA synthesis The oligodeoxyribonucleotide 5'-TAACTsCACATTAATTGCGTT-FAM-3' was synthesized using compound 5T and a commercially available phosphoramidite reagent (ChemGenes) according to a standard procedure on an automated nucleic acid synthesizer NR-2A 7MX (Nippon Techno Service Co., Ltd.). Ts represents a thymidine base that has been 3' thiolated. Fluorescein (FAM) was introduced at the 3' end. All amidite reagents were prepared as 70 mM acetonitrile solutions, except for compound 5T, which was prepared as a 150 mM acetonitrile solution. 0.3 M BTT was used as the activator, and 0.05 M iodine (pyridine / water; v / v:9 / 1) solution was used as the oxidizing agent. Furthermore, a 6-FAM-glycerol support 500 Å (ChemGenes) was used as the solid support to ensure that FAM was introduced at the 3' end. The synthesis was performed using the Final DMTr-ON setting, and the DMTr group was removed while retaining the hydroxyl group at the 5'-terminus. After the synthesis was complete, concentrated ammonia water (500 μL) and 40% methylamine aqueous solution (500 μL) were added to the solid support, and the mixture was cleaved from the solid support and deprotected by treatment at 65°C for 1 hour. The supernatant was filtered using a Millex LH filter (0.45 μm, Merck), and the filtrate was dried under reduced pressure using a centrifugal evaporator. The obtained oligonucleotide was redissolved in superdeionized water, and the concentration was calculated by measuring the absorption at 260 nm. MALDI-TOF molecular weight measurement of the oligonucleotide was performed using 3-hydroxypicolinic acid as the matrix and the linear-positive mode of UltrafleXtreme (Bruker).

[0066] (b) Analysis results Figure 2 shows the synthesis of oligonucleotides containing a 3'-thiophosphate bond using a thioamide reagent (compound 5T), and the results of analysis by reverse-phase HPLC and MALDI-TOF-MS. Figure (a) shows the results of synthesizing the 20-base oligodeoxyribonucleotide 5'-TAACTsCACATTAATTGCGTT-FAM-3', which has a 3'-thiophosphate bond between the 4th and 5th bases from the 5' end, using compound 5T, and analyzing it by reverse-phase HPLC. (a,b) The HPLC analysis conditions are as follows. <System Used> • Column: YMC Hydrosphere C18 (Column size: 250 x 10.0 mm) • Eluent A: 50 mM triethylammonium acetate (pH 7.0) containing 5% acetonitrile. • Eluent B: Acetonitrile, <Gradient Conditions> ·Flow rate: 3mL / min, Column temperature: 50°C • Detection wavelength: 260 nm.

[0067] Reverse-phase HPLC analysis revealed that the main peak around 18.8 minutes of retention represents the 20-mer target DNA, with the DMTr group remaining as the 5'-hydroxyl protecting group. In contrast, some products where synthesis stopped at the compound 5T introduction stage were observed around 12.7–13.5 minutes of retention, but these were easily separated from the 20-mer target oligodeoxyribonucleotide (DNA). By separating the peak around 18.8 minutes of retention, the 5'-DMTr protected form of the target DNA was obtained.

[0068] Figure (b) shows the results of deprotecting the DMTr group from the 5'-DMTr protected compound, which was preparatively purified by reverse-phase HPLC, by adding a 10% aqueous acetic acid solution and treating it at room temperature for one hour. The resulting DNA was then analyzed by reverse-phase HPLC. As a result, a single peak was observed around the retention time of 12.3 minutes, confirming that the target DNA was obtained with a high purity of over 99%.

[0069] Figure (c) shows the results of MALDI-TOF-MS molecular weight analysis of the DNA obtained above. As a result, a peak at m / z 6650.7, originating from the target substance, was observed.

[0070] 2. DNA strand cleavage reaction of thiolated oligonucleotides using metal nanoparticles (1) Silver nanoparticle size of the DNA strand cleavage reaction by silver nanoparticle treatment (a) Silver nanoparticle treatment 10 μL of 3 μM PS-modified DNA (5'-TAACTsCACATTAATTGCGTT-FAM-3') was measured out, and 50 μL of silver nanoparticle dispersion (10 nm, 0.02 mg / mL) was added. The mixture was incubated at 37°C for 31 hours. 20 μL of the reaction solution was measured out and 20 μL of 2x loading buffer was added. After heating at 95°C for 5 minutes, the mixture was analyzed by 15% denatured acrylamide electrophoresis (containing 7.5 M urea, 10 x 12 cm, 30 mA, 20 min, 6 μL apply). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM.

[0071] (b) Dependence of reaction time and reaction temperature of DNA strand cleavage reaction by silver nanoparticle treatment 10 μL of 3 μM PS-modified DNA (5'-TAACTsCACATTAATTGCGTT-FAM-3') was measured out, and 50 μL of silver nanoparticle dispersion (10 nm, 0.02 mg / mL) was added. The mixture was incubated at 70°C or 95°C for the specified time. 30 μL of the reaction solution was measured out and 30 μL of 2x loading buffer was added. After heating at 95°C for 5 minutes, the mixture was analyzed by 15% denatured acrylamide electrophoresis (containing 7.5 M urea, 10 x 12 cm, 30 mA, 20 min, 6 μL apply). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM.

[0072] (c) Results Figure 3 shows the results of an investigation into the DNA strand cleavage reaction induced by silver nanoparticle treatment, and the dependence of silver nanoparticle size on reaction time and reaction temperature. Figure (a) shows the results of the size dependence of silver nanoparticles on DNA strand cleavage by silver nanoparticles. Using 10 nm, 20 nm, and 100 nm silver nanoparticles, the generation of 15-mer DNA 5'-pCACATTAATTGCGTT-FAM-3' by strand cleavage of 20-mer DNA 5'-TAACTsCACATTAATTGCGTT-FAM-3' was analyzed by denaturing gel electrophoresis. Ts represents a thymidine base that has undergone 3' thiolation. FAM was introduced as a fluorescent dye at the 3' end. The design was such that a phosphate group (p) remained at the 5' end of the 15-mer DNA after cleavage. The cleavage reaction was carried out at 37°C for 31 hours. From the band intensity analysis of the gel using FAM-derived fluorescence detection, the cleavage efficiencies for 10 nm, 20 nm, and 100 nm silver nanoparticles were 35.9%, 21.9%, and 13.1%, respectively, revealing that the cleavage efficiency decreased as the nanoparticle size increased.

[0073] Figure (b) shows the reaction time and temperature dependence of DNA strand cleavage by silver nanoparticles. 10 nm silver nanoparticles were used to cleave 20-mer DNA strands at 70°C or 95°C. The reaction was carried out for 15, 30, 60, and 120 minutes. Each reaction solution was analyzed by denaturing gel electrophoresis, and the cleavage efficiency at each temperature and time was calculated from the band intensity analysis of the gel using FAM-derived fluorescence detection. The reaction time was plotted on the x-axis, and the cleavage efficiency on the y-axis. The results showed a reaction time-dependent improvement in strand cleavage efficiency; approximately 50% cleavage was observed at 70°C and over 90% at 95°C after 2 hours from the start of the reaction, clearly indicating that cleavage activity improved with increasing temperature. This suggests that high-temperature conditions are required to induce highly efficient DNA strand cleavage with commercially available nanoparticles.

[0074] (2) DNA strand breaks using surface PEG-modified silver nanoparticles (a) Polymer modification of the surface of silver nanoparticles 9 μL of 3.4 μM PS-modified DNA (5'-TAACTsCACATTAATTGCGTT-FAM-3') was weighed out, and 51 μL of surface PEG-modified silver nanoparticle dispersion (10 nm) was added, and the mixture was incubated for a specified time and temperature. The surface PEG-modified silver nanoparticle dispersion was prepared by adding 1 μL of 23.9 g / L of a thiolated terminal PEG (O-[2-(3-mercaptopropionylamino)ethyl]-O'-methyl polyethylene glycol, average molecular weight 5,000, Sigma-Aldrich) aqueous solution to 50 μL of a commercially available silver nanoparticle dispersion (Sigma-Aldrich, 10 nm, 0.02 mg / mL). 20 μL of the reaction solution was weighed out and 20 μL of 2x loading buffer was added. After heat treatment at 95°C for 5 minutes, the samples were analyzed by 15% denatured acrylamide electrophoresis (containing 7.5M urea, 10 x 12 cm, 30 mA, 20 minutes, 6 μL application). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM.

[0075] (b) Results Figure 4 shows the results of improving DNA strand cleavage activity by polymer modification of the silver nanoparticle surface. Figure (a) is a schematic diagram representing the surface of polymer-modified silver nanoparticles. The nanoparticles were modified with polyethylene glycol with an average molecular weight of 5,000 and terminal thiols. Surface-modified nanoparticles can be easily prepared by mixing any amount of polymer with a commercially available silver nanoparticle dispersion. The degree of modification can also be controlled by adjusting the amount of polymer added.

[0076] Figure (b) shows the results of a comparison of DNA strand cleavage activity with and without surface modification of 1 nm silver nanoparticles. PEG-SH in the figure refers to polyethylene glycol with terminal thiols. The reaction was carried out at 37°C for 31 hours, and the reaction solution was analyzed by denatured gel electrophoresis. The cleavage efficiency was calculated by analyzing the band intensity of the gel using FAM-derived fluorescence detection. The results showed that the cleavage efficiency was 35.9% without PEG-SH modification, while the PEG-modified silver nanoparticles showed a cleavage activity of 91.8%, indicating a significant improvement in cleavage activity due to surface PEG modification. This improvement in cleavage activity can be explained by the prevention of surface oxidation of silver nanoparticles and improved dispersibility in aqueous solution due to PEG modification.

[0077] Figure (c) shows the reaction time dependence of DNA strand cleavage using 1 nm surface-PEG modified silver nanoparticles. The reaction was carried out at 50°C for 15, 30, and 60 minutes, and each reaction solution was analyzed by denatured gel electrophoresis. The cleavage efficiency at each reaction time was calculated by analyzing the band intensity of the gel using FAM-derived fluorescence detection. As a result, an improvement in strand cleavage efficiency was observed in a reaction time dependent manner, with over 90% cleavage observed after 60 minutes from the start of the reaction. From these results, it was found that PEG modification of the surface of commercially available silver nanoparticles enables highly efficient DNA strand cleavage under relatively mild temperature conditions.

[0078] (3) Preparation of 3' overhang adhesive ends of double-strand short-strand DNA by silver nanoparticle treatment (a) Preparation of the 3' overhang adhesive end PS-modified DNA (5'-TAACTsCACATTAATTGCGTT-FAM-3') and complementary DNA (5'-AACGCAATTAATGTGAGTTA-3') were mixed to a concentration of 3 μM each, treated at 95°C for 3 minutes, and then annealed by cooling on an ice bath for at least 10 minutes. 9 μL of the annealed solution was measured out, and 51 μL of surface PEG-modified silver nanoparticle dispersion (10 nm) was added. The mixture was incubated at 50°C for a specified time. The surface PEG-modified silver nanoparticle dispersion was prepared by adding 1 μL of terminal thiol PEG aqueous solution (23.9 g / L) to 50 μL of commercially available silver nanoparticle dispersion (Sigma-Aldrich, 10 nm, 0.02 mg / mL). 30 μL of the reaction solution was measured out, and 30 μL of 2x loading buffer was added. After heat treatment at 95°C for 5 minutes, the samples were analyzed by 15% denatured acrylamide electrophoresis (containing 7.5M urea, 10x12cm, 30mA, 20 minutes, 6μL application). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM.

[0079] (b) Results Figure 5 shows the results of preparing 3' overhanging adhesive ends of double-stranded short-chain DNA using silver nanoparticle treatment. A 20-mer double-strand DNA with a 3'-SP bond was cleaved using 1 nm surface-PEG-modified silver nanoparticles. The SP bond was located between the 5th and 6th base pairs from the 5' end, and the design ensured that a 5-base overhanging adhesive end was prepared upon cleavage. The reaction was carried out at 50°C for 15, 30, and 60 minutes. Each reaction solution was analyzed by denatured gel electrophoresis, and the cleavage efficiency at each reaction time was calculated by analyzing the gel band intensity using FAM-derived fluorescence detection. A comparison of cleavage efficiency with and without a complementary strand was performed, plotting the cleavage efficiency at each reaction time on the vertical axis and the reaction time on the horizontal axis. The results showed that, although the cleavage activity was slightly lower with a complementary strand compared to without, it was almost the same after 2 hours from the start of the reaction, with over 90% of the DNA being cleaved. This demonstrates that DNA strand cleavage using surface-PEG-modified silver nanoparticles can be applied to the preparation of adhesive end DNA.

[0080] 3. Application of thiolated oligonucleotides as primers for polymerase chain elongation (PCR) to oligonucleotide ligation. (1) Experimental section (a) Examination of PCR using thiolated DNA as a template 0.37 μL of 110 μM thiolated template DNA and 1.34 μL of 15 μL of primer DNA were mixed. Following the manufacturer's recommended conditions for each polymerase, a dNTP mixture, various polymerases (KOD-Pkus-Neo, PrimeSTAR HS, Phusion High Fidelity, Q5 High Fidelity, Deep Vent, Taq DNA polymerase), and polymerase buffer were added to a total volume of 20 μL. Subsequently, denaturation (95°C, 1 min) - annealing (50°C, 30 sec) - chain extension (72°C, 30 min) were performed, and the reaction solution was diluted with 2x loading buffer (20 μL). Analysis was performed by 20% denatured gel electrophoresis (containing 7.5 M urea, 20 x 22 cm, 20 W, 2 h). The gel was stained by shaking it with 1x SYBR Green II solution for 30 minutes, and a gel electrophoresis image was obtained using a gel image analyzer (BioRad).

[0081] (b) PCR using thiolated DNA primers A mixture of 20 μM thiolated forward primer (1.25 μL), 20 μM thiolated reverse primer (1.25 μL), 10 ng / μL template DNA (2.5 μL), 2 mM dNTP mixture (5 μL), 25 mM magnesium sulfate (3 μL), 10x PCR Buffer for KOD-Plus-Neo (5 μL), and superdeionized water (31 μL) was added. 1 U / μL KOD-Plus-Neo (1 μL) was then added, and the mixture was treated at 95°C for 2 minutes using a thermal cycler (BioRad). Subsequently, DNA amplification was performed by 30 cycles of denaturation (95°C, 15 sec) - annealing (55°C, 15 sec) - strand extension (68°C, 30 sec). The PCR product was purified using a wizard column (Promega) according to the manufacturer's recommended protocol to obtain thiolated DNA.

[0082] (c) Preparation of adhesive fragments by metal nanoparticle treatment 7.5 nM PCR product thiolated DNA (225 fmol / sample) was dissolved in superdeionized water to prepare a 4.5 μL aqueous solution. PEG-modified silver nanoparticles were prepared by mixing 10 nm silver nanoparticles (Sigma-Aldrich, 200 μg / mL citrate buffer suspension, 25 μL) with terminally thiol-modified PEG (Sigma-Aldrich, 23.9 g / L aqueous solution, 0.5 μL). The prepared PCR product thiolated DNA aqueous solution (4.5 μL) and the PEG-modified silver nanoparticle dispersion (25.5 μL) were mixed and treated at 50°C for 2-4 hours to cleave SP bonds and prepare adherent ends.

[0083] (d) Ligation and gel analysis using DNA ligase 7.5nM silver nanoparticle-treated DNA_1 (3.6 μL), 7.5nM silver nanoparticle-treated DNA_2 (3.6 μL), 10x T4 DNA Ligase Reaction Buffer (New England BioLabs, 0.90 μL), and superdeionized water (0.45 μL) were mixed, and then T4 DNA ligase (New England BioLabs, 2000 U / μL, 0.45 μL) was added and incubated at 25°C for 3 hours. 1 μL of 10x loading buffer was added to the reaction solution (9 μL), and the mixture was analyzed by 1% agarol gel electrophoresis (electrophoresis buffer: 1x TBE, 100 V, 30 minutes). The gel was stained by shaking with 10,000x SYBR green I solution for 30 minutes, and the gel electrophoresis image was obtained using a gel image analyzer (BioRad).

[0084] (2) Results Figure 6 shows the results of PCR studies when thiolated oligonucleotides were introduced into a DNA template. A 22-mer 5'-FAM-modified DNA primer, 5'-FAM-AACGCAATTAATGTGAGTTAGC-3', was annealed to a template DNA, 5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3', which had a 3'-SP bond introduced between the 14th and 15th base pairs from the 5' end. Polymerase extension reactions were then performed using various polymerases (KOD-Pkus-Neo, PrimeSTAR HS, Phusion High Fidelity, Q5 High Fidelity, Deep Vent, Taq DNA polymerase). When extension stopped at the 3'-SP modification site, a product with 4 or 5 base pairs extended from the 22-mer primer was produced. On the other hand, when the 3'-SP modification site was extended without issue, a full-length 41-mer DNA was generated. The reaction solution was analyzed by 20% denatured gel electrophoresis (containing 7.5 M urea, 20 x 22 cm, 20 W, 2 hours), and the bands were visualized using a gel image analyzer (BioRad) with fluorescence detection derived from 5'-FAM. As a result, bands derived from full-length 41-mer DNA were observed in all polymerases used, and no extension termination products at the 3'-SP modification site were observed. This indicates that DNA with 3'-SP modification can function as a primer.

[0085] Figure 7 shows a schematic diagram of DNA amplification by PCR and preparation of adherent fragments by BsaI or silver nanoparticle treatment. Figure (a) shows the results of preparing 4-base overhang adherent ends using the restriction enzyme BsaI. Figure (b) shows the results of preparing 8-base overhang adherent ends using 3'-SP bond cleavage by silver nanoparticle treatment. 10 nm sized silver nanoparticles with surface PEG modification were used.

[0086] Figure 8 shows the results of a linking experiment of adherent fragments using T4 DNA ligase. Figure (a) shows the results of ligation of two adherent DNA fragments, prepared by silver nanoparticle treatment or restriction enzyme BsaI treatment as a control, using T4 DNA ligase, followed by analysis by 1% agarol gel electrophoresis (electrophoresis buffer: 1x TBE, 100V, 30 min). The reaction was carried out at 25°C for 3 hours, and the PCR product DNA was treated with silver nanoparticles for 60, 120, 180, and 240 minutes. The cleavage reaction products at each reaction time were used to compare the ligation efficiency by T4 DNA ligase. From the gel results, the formation of an 838 bp ligated product was confirmed under all ligation reaction conditions. Figure (b) shows a graph illustrating the ligation efficiency of the product and the product treated with BsaI at different silver nanoparticle treatment times. The amount of ligated product increased with increasing silver nanoparticle treatment time. When DNA fragments prepared by 240 minutes of silver nanoparticle treatment were ligated, a higher ligation efficiency was observed compared to the ligation of DNA fragments prepared by BsaI treatment.

[0087] 4. Comparison of silver nitrate treatment (existing technology) and silver nanoparticle treatment (technology of the present invention) in thiolated oligonucleotide chain cleavage. (1) Thiodate oligonucleotide chain cleavage by silver nitrate treatment 30 μM PS-modified DNA 20mer (5'-TAACTsCACATTAATTGCGTT-FAM-3') was used. Ts represents a thymidine base that has undergone 3' thiolation. FAM was introduced as a fluorescent dye at the 3' end. 1 μL of this PS-modified DNA was measured out and mixed with 29 μL of 50 mM silver nitrate aqueous solution. The mixture was incubated at room temperature for 5–40 minutes to allow the cleavage reaction to occur. After the specified time, 5 μL of each reaction solution was measured out and the reaction was stopped by adding 5 μL of 60 mM ethanethiol aqueous solution. At this point, the formation of a white precipitate derived from silver ions was confirmed. 2x loading buffer (10 μL) was added to this mixture (10 μL). After heat treatment at 95°C for 5 minutes, the mixture was analyzed by 15% denatured acrylamide gel electrophoresis (containing 7.5 M urea, 10 x 12 cm, 30 mA, 20 min, 6 μL apply). Gel electrophoresis images were acquired using a gel image analyzer (BioRad) by detecting fluorescence originating from FAM. The results are shown in Figure 9.

[0088] Figure 9 shows the results of thiolated oligonucleotide strand cleavage using the existing technique of silver nitrate treatment. The generation of 15-mer DNA 5'-pCACATTAATTGCGTT-FAM-3', a strand cleavage product of 20-mer PS-DNA 5'-TAACTsCACATTAATTGCGTT-FAM-3', was analyzed by denaturing gel electrophoresis. The 15-mer DNA is designed to retain a phosphate (p) group at the 5' end after cleavage. Band intensity analysis of the gel using FAM-derived fluorescence detection showed that more than 90% of the DNA was cleaved within 5 minutes of the reaction starting.

[0089] (2) Molecular weight analysis of cleavage products of thiolated oligonucleotide chains cleaved by silver nitrate treatment by MALDI-TOF-MS 3 μL of 110 μM PS-modified DNA 41mer (5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3') was weighed out and mixed with 67 μL of 50 mM silver nitrate aqueous solution, then incubated at room temperature for 22 hours. The reaction solution was removed using an ultrafiltration centrifugal filter (Amicon Ultra 3K, Merck) according to the manufacturer's recommended protocol, by cutting off molecules smaller than 3K molecular weight. This procedure roughly removes silver ions and other contaminants from the reaction system. The solution remaining after ultrafiltration was collected and subjected to MALDI-TOF-MS molecular weight analysis. MALDI-TOF-MS molecular weight analysis was performed using 3-hydroxypicolinic acid as the matrix and measured in linear-negative mode on an UltraFleXtreme (Bruker). The results are shown in Figure 10.

[0090] Figure 10 shows the results of molecular weight analysis of cleavage products of thiolated oligonucleotide chains using silver nitrate treatment, an existing technology, by MALDI-TOF-MS. Figure (a) shows the sequence of the cleavage target DNA (41mer) 5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3' and the cleavage product DNA fragment. The design is that cleavage occurs at the 3'-SP binding site indicated by the arrow in the figure, with the 5'-fragment (15mer) side becoming 3'-SH (5'-AGGGGTGCCTAATGTs-3') and the 3'-fragment (26mer) becoming the 5'-phosphated form (5'-pGTGAGCTAACTCACATTAATTGCGTT-3', where p indicates a phosphate group). Figure (b) shows the MALDI-TOF-MS molecular weight analysis of the cleavage reaction solution after silver nitrate treatment. The molecular weight of the 5'-fragment (15mer) and 3'-fragment (26mer) produced by cleavage is 466. 4 The observation of peaks corresponding to .2 and 8040.2 suggests that the target DNA fragment was generated. Figure (c) is a magnified view of the MALDI-TOF-MS spectral peak region derived from the 5'-fragment (15mer) cleavage product obtained by silver nitrate treatment. Figure (d) is a magnified view of the MALDI-TOF-MS spectral peak region derived from the 3'-fragment (26mer) cleavage product by silver nitrate treatment.

[0091] Figures (c) and (d) show that the cleavage product exists with multiple silver ions attached to the phosphate-binding site. On the MALDI-TOF-MS spectrum, it was detected as peaks with cluster-like molecular weight distributions having different numbers of attached silver ions. These results indicate that in thiolated oligonucleotide chain cleavage by silver nitrate treatment, it is difficult to remove excess added silver ions with simple ultrafiltration. Silver ions are present in chloride ions (Cl) contained in buffers used in physiological and biochemical experiments. - ) binds firmly, forming poorly water-soluble AgCl and causing a precipitate to form (AgCl water solubility is 192 μg / 100m: J. Phys. Chem. C., 2015, 119, 20632-20641). Therefore, even if the DNA cleavage reaction occurs with high efficiency, silver ions remain in the solution, making silver ion-based cleavage systems difficult to apply to ligation reactions after DNA cleavage.

[0092] (3) Comparison of the amount of DNA recovered as cleavage product after thiolated oligonucleotide strand cleavage by silver nitrate treatment and after removal of silver ions by thiol addition. It has been reported that silver ions can be removed as a precipitate due to their strong interaction with thiols. Therefore, the reaction was stopped by adding dithiothreitol (DTT) or ethanethiol (EtSH) to the cleavage reaction solution, and the resulting silver-derived precipitate was removed by centrifugation. The supernatant was collected and analyzed by denatured gel electrophoresis.

[0093] 10 μL (1.10 nmol) of 110 μM PS-modified DNA 41mer (5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3') was measured out and mixed with 90 μL of 50 mM silver nitrate aqueous solution. The mixture was incubated at room temperature for 1 day to perform the cleavage reaction. 20 μL (220 pmol) of the reaction solution was measured out and the reaction was stopped by adding 20 μL of 60 mM dithiothreitol (DTT) aqueous solution or 20 μL of 60 mM ethanethiol (EtSH) aqueous solution. At this time, the formation of a white precipitate derived from silver ions was confirmed. This mixture was diluted with superdeionized water (160 μL) and incubated at room temperature for 20 minutes. Subsequently, the precipitate derived from silver ions was removed by centrifugation (15,000 rpm, 15 minutes). The supernatant was collected and concentrated using an ultrafiltration centrifugal filter (Amicon Ultra 3K, Merck) according to the manufacturer's recommended protocol.

[0094] The amount of cleavage products recovered was calculated by quantifying the cleavage products from the concentrated sample solution by measuring the absorbance at 260 nm derived from nucleic acids using NanoDrop. The sum of the molar extinction coefficients at 260 nm of both cleavage products, ε, was used to calculate the amount of cleavage products. 260 = 396,900 L·mol -1 ·cm -1 Alternatively, the molar extinction coefficient at 260 nm of the 3'-side cleavage product (5'-pGTGAGCTAACTCACATTAATTGCGTT-3') is ε 260 = 250,000 L·mol -1 ·cm -1DNA was quantified using the Lambert-Beer law. 5 μL of the concentrated sample solution was measured and mixed with 2x loading buffer (5 μL). After processing at 95°C for 10 minutes, the mixture was analyzed by 15% denatured acrylamide gel electrophoresis (containing 7.5 M urea, 10 x 12 cm, 30 mA, 20 min, 6 μL applied). The gel was removed after electrophoresis and immersed in 1x SYBR Green II aqueous solution, and the DNA bands present in the gel were stained by shaking for 20 minutes. Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from SYBR Green II bound to DNA. The results are shown in Figure 11.

[0095] Figure 11 shows the results of investigating existing technologies: thiolated oligonucleotide strand cleavage by silver nitrate treatment and silver ion removal by thiol addition. The target DNA for cleavage was a 41-mer 5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3'. As shown in the figure, the design was such that cleavage occurred at the 3'-SP binding site, generating a 5'-fragment (15mer) 5'-AGGGGTGCCTAATGTs-3' and a 3'-fragment (26mer) 5'-pGTGAGCTAACTCACATTAATTGCGTT-3'.

[0096] For DNA staining using gel analysis, SYBR Green II was used, and the bands on the gel were visualized by detecting fluorescence derived from SYBR Green II bound to the DNA. As a result, in the system where DTT was used to stop the reaction, a band was observed that originated from the cleavage products 5'-fragment and 3'-fragment, as well as a band derived from the dimerization of the thiol moiety of the 5'-fragment via a disulfide bond. The band derived from dimerization had almost the same mobility as the 5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3' of the 41-mer, which was the target DNA for cleavage in the starting material, making identification difficult. However, after cutting out the band from the gel and extracting DNA from the gel fragment, molecular weight analysis of the extract using MALDI-TOF-MS revealed that it was a dimerized 5'-fragment.

[0097] In the system where EtSH was used to terminate the reaction, the band derived from the 41-mer 5'-AGGGGTGCCTAATGTsGTGAGCTAACTCACATTAATTGCGTT-3', which is the target DNA for cleavage, completely disappeared, and only the cleavage product 3'-fragment(26mer)5'-pGTGAGCTAACTCACATTAATTGCGTT-3' was observed on the gel. This is thought to be because the 5'-side cleavage product, 5'-fragment(15mer)5'-AGGGGTGCCTAATGTs-3', strongly bound to EtSH and formed a precipitate with silver ions. After removing the silver ion precipitate, only the 3'-side cleavage product, 3'-fragment(26mer)5'-pGTGAGCTAACTCACATTAATTGCGTT-3', was recovered as the supernatant.

[0098] Furthermore, the amount of DNA in the supernatant after removing silver ions by adding thiols was quantified by measuring the absorbance of DNA at 260 nm, and the results are shown in the table in the figure. The total amount of DNA per reaction solution was 220 pmol, but the amount of cleavage product DNA recovered was 92 pmol in the DTT-added system and 88 pmol in the EtSH-added system, with a recovery rate of approximately 40% in both systems. These results indicate that the silver ion precipitation removal method using thiols results in the co-precipitation of approximately 60% of the cleavage product DNA along with the silver ions, reducing the yield of the cleavage product. This also makes it clear that the existing technology of thiolated oligonucleotide cleavage using silver nitrate is difficult to put into practical use.

[0099] (4) Demonstration of removal of silver nanoparticles by centrifugation Silver nanoparticles are known to exhibit absorption in the 350-700 nm range, dependent on particle size, due to surface plasmon resonance effects. Therefore, by obtaining the absorption spectrum of the dispersion, the amount of nanoparticles present in the dispersion can be estimated from the absorbance.

[0100] 700 μL of a dispersion of silver nanoparticles with particle sizes of 10, 20, and 100 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution) was measured and diluted with superdeionized water (700 μL). The absorption spectrum was then measured using a quartz cell (optical path length: 1 cm, optical path width: 1 cm). The instrument used for the measurement was a JASCO V-650 UV-Vis spectrometer. The measurement conditions were set to data interval: 0.5 nm, bandwidth: 1.0 nm, response: medium, and scanning speed: 100 nm / min. After measuring the absorption spectrum, the silver nanoparticle dispersion diluted with superdeionized water (700 μL) was transferred to an Eppendorf tube, and the nanoparticles were precipitated by centrifugation at 15,000 rpm for 1 hour. The supernatant was collected and transferred again to a quartz cell (optical path length: 1 cm, optical path width: 1 cm), and the absorption spectrum was measured again. The results are shown in Figure 12.

[0101] Figure 12 shows the removal of silver nanoparticles by centrifugation (dependence on nanoparticle size). Figure (a) shows the results when using silver nanoparticles with a particle size of 10 nm. Figure (b) shows the results when using silver nanoparticles with a particle size of 20 nm. Figure (c) shows the results when using silver nanoparticles with a particle size of 100 nm. The blue spectrum in the figure originates from a 0.01 mg / mL silver nanoparticle dispersion. The orange spectrum in the figure is the result of measuring the supernatant obtained by centrifugating the nanoparticle dispersion at 15,000 rpm for 1 hour to precipitate the nanoparticles.

[0102] As a result, a decrease in absorbance around 400-500 nm was observed by centrifugation in the silver nanoparticle dispersion with a particle size of 10 nm (29.9%), the silver nanoparticle dispersion with a particle size of 20 nm (82.4%), and the silver nanoparticle dispersion with a particle size of 100 nm (almost 100%). This result indicates that the nanoparticles that were suspended in the dispersion precipitated due to the centrifugation operation. Therefore, it was found that silver nanoparticles with particle sizes of 10-100 nm can be removed from the reaction solution as precipitate by centrifugation. Furthermore, it was shown that the larger the size of the silver nanoparticles, the easier they are to remove from the reaction system by centrifugation, and that nanoparticles with a particle size of 20 nm or larger can be removed by centrifugation.

[0103] (5) Comparison of silver nitrate treatment and silver nanoparticle treatment in thiolated oligonucleotide chain cleavage (calculation of oligonucleotide recovery amount) (a) Silver nitrate treatment 2 μL of 122 μM PS-modified DNA 20mer (5'-TAACTsCACATTAATTGCGTT-FAM-3') was measured out and mixed with 38 μL of 50 mM silver nitrate aqueous solution, then incubated at room temperature for 1.5 hours. After the specified time, the reaction was stopped by adding 40 μL of 60 mM DTT aqueous solution to the reaction solution (40 μL). At this time, the formation of a white precipitate derived from silver ions was confirmed. This mixture (80 μL) was diluted with 20 μL of superdeionized water and mixed thoroughly, and the precipitate was removed by centrifugation at 15,000 rpm for 1 hour. The supernatant was collected and concentrated using an ultrafiltration centrifugal filter (Amicon Ultra 3K, Merck) according to the manufacturer's recommended protocol. The amount of recovered product was calculated by quantifying the cleavage products of the concentrated sample solution by measuring the absorbance at 260 nm derived from nucleic acids using NanoDrop. The concentrated sample solution was diluted with superdeionized water to prepare a 0.50 μM aqueous solution. 5 μL of the 0.50 μM cleavage product solution was measured out and mixed with 2x loading buffer (5 μL). After processing at 95°C for 5 minutes, the mixture was analyzed by 15% denatured acrylamide gel electrophoresis (containing 7.5 M urea, 10 x 12 cm, 30 mA, 20 min, 5 μL applied). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM.

[0104] (b) Silver nanoparticle treatment 2 μL of 122 μM PS-modified DNA 20mer (5'-TAACTsCACATTAATTGCGTT-FAM-3') was measured and mixed with 98 μL of a dispersion of silver nanoparticles with a particle size of 100 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), and incubated at 90°C for 25 hours. The reaction solution was centrifuged at 15,000 rpm for 1 hour to remove the silver nanoparticles as precipitate. The supernatant was collected and concentrated using an ultrafiltration centrifugal filter (Amicon Ultra 3K, Merck) according to the manufacturer's recommended protocol. The amount of recovered product was calculated by quantifying the cleavage products of the concentrated sample solution by measuring the absorbance at 260 nm derived from nucleic acids using NanoDrop. The concentrated sample solution was diluted with superdeionized water to prepare a 0.50 μM aqueous solution. 5 μL of a 0.50 μM cleavage product solution was measured and mixed with 2x loading buffer (5 μL). After processing at 95°C for 5 minutes, the mixture was analyzed by 15% denatured acrylamide gel electrophoresis (containing 7.5 M urea, 10 x 12 cm, 30 mA, 20 min, 5 μL applied). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM. The results are shown in Figure 13.

[0105] Figure 13 compares silver nitrate treatment and silver nanoparticle treatment in thiolated oligonucleotide strand cleavage. In the gel image, Lane 1 shows the electrophoresis results of DNA (15mer) 5'-CACATTAATTGCGTT-FAM-3', which has the same base length as the DNA produced after cleavage. Lane 2 shows the electrophoresis results of the cleavage target DNA (20mer) 5'-TAACTsCACATTAATTGCGTT-FAM-3'. Ts in the sequence indicates a thymidine base that has undergone 3' thiolation. FAM has also been introduced as a fluorescent dye at the 3' end. Lane 3 shows the electrophoresis results of the reaction product when thiolated oligonucleotide strand cleavage was performed using silver nanoparticles with a particle size of 100 nm. The reaction was carried out by adding silver nanoparticles and incubating at 90°C for 25 hours. The silver nanoparticles in the reaction solution were removed as precipitate by centrifugation at 15,000 rpm for 1 hour, and then electrophoresis was performed. The results showed that almost all of the target DNA (20mer) was cleaved and converted into cleaved product DNA (15mer) (89.6%). Lane 4 shows the electrophoresis results of the reaction products when thiolated oligonucleotide strand cleavage was performed using silver nitrate. The reaction was carried out by adding silver nitrate and incubating at room temperature for 1.5 hours. Silver ions in the reaction solution were removed as precipitate by adding DTT, and then electrophoresis was performed. The results showed that the target DNA (20mer) was cleaved and converted into cleaved product DNA (15mer) (98.7%).

[0106] [Table 1]

[0107] The table above compares the amount of DNA cleavage product recovered in silver nitrate treatment and silver nanoparticle treatment. In the case of silver nitrate treatment, the amount of DNA in the supernatant after removal of silver ion precipitate by DTT addition was quantified by measuring the absorbance of DNA at 260 nm. In the case of silver nanoparticle treatment, after removing the silver nanoparticles as precipitate by centrifugation, the amount of DNA in the supernatant was quantified by measuring the absorbance of DNA at 260 nm. As a result, while the total amount of DNA per reaction solution was 244 pmol, the amount of DNA cleavage product recovered in the case of silver nitrate treatment was 35.1 pmol, a recovery rate of only 14.4%, suggesting that most of the cleavage product co-precipitated during the removal of silver ion precipitate by DTT addition. On the other hand, in the case of silver nanoparticle treatment, the amount was 240 pmol, and the DNA cleavage product was recovered almost quantitatively. From these results, it can be concluded that the existing method of thiolated oligonucleotide cleavage using silver nitrate is difficult to commercialize due to the significantly reduced yield, but the cleavage method using silver nanoparticles is a practical technology that can recover cleavage products with high efficiency.

[0108] (6) Evaluation of dispersibility associated with surface PEG modification of silver nanoparticles Surface modification of silver nanoparticles can be easily prepared by mixing terminally thiolated polyethylene glycol (PEG-SH) with a commercially available silver nanoparticle dispersion (containing citric acid as an antioxidant, particle size 20 nm). PEG-SH with an average molecular weight of 5,000 was used. The dispersibility of the nanoparticles was evaluated by measuring the absorption spectrum based on the surface plasmon resonance effect of the nanoparticle dispersion.

[0109] The absorption spectrum of a dispersion of unmodified silver nanoparticles was measured using a 350 μL sample of a dispersion of silver nanoparticles with a particle size of 20 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), diluted with superdeionized water (1050 μL), and then measured using a quartz cell (path length: 1 cm, path width: 1 cm). The absorption spectrum of a dispersion of unmodified silver nanoparticles in the presence of buffers and salts was prepared by measuring 350 μL of a dispersion of silver nanoparticles with a particle size of 20 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), diluting it with superdeionized water (630 μL), and then adding 100 mM Tris-HCl buffer (pH 8.3) (140 μL), 500 mM potassium chloride aqueous solution (140 μL), and 15 mM magnesium chloride aqueous solution (140 μL). The sample was then measured using a quartz cell (path length: 1 cm, path width: 1 cm).

[0110] The absorption spectrum of the surface-PEG-modified silver nanoparticle dispersion was measured by measuring 350 μL of a dispersion of silver nanoparticles with a particle size of 20 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), adding 140 μL of a 23.9 g / L aqueous solution of terminally thiolated PEG (O-[2-(3-mercaptopropionylamino)ethyl]-O'-methyl polyethylene glycol, average molecular weight 5,000, Sigma-Aldrich), diluting with super-deionized water (910 μL), and measuring using a quartz cell (path length: 1 cm, path width: 1 cm). The absorption spectrum of a surface-PEG-modified silver nanoparticle dispersion in the presence of buffers and salts was measured by measuring 350 μL of a dispersion of silver nanoparticles with a particle size of 20 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), adding 140 μL of 23.9 g / L of terminally thiolated PEG (O-[2-(3-mercaptopropionylamino)ethyl]-O'-methyl polyethylene glycol, average molecular weight 5,000, Sigma-Aldrich) aqueous solution, diluting with superdeionized water (490 μL), and then adding 140 μL of 100 mM Tris-HCl buffer (pH 8.3), 140 μL of 500 mM potassium chloride aqueous solution, and 140 μL of 15 mM magnesium chloride aqueous solution. The sample was then measured using a quartz cell (path length: 1 cm, path width: 1 cm). The instrument used for the measurement was a V-650 UV-Vis spectrometer manufactured by JASCO Corporation. The measurement conditions were set to data interval: 0.5 nm, bandwidth: 1.0 nm, response: medium, and scanning speed: 100 nm / min. The results are shown in Figure 14.

[0111] Figure 14 shows that the dispersibility of silver nanoparticles was improved by surface PEG modification. The blue spectrum in the figure is the absorption spectrum derived from a commercially available silver nanoparticle dispersion, and a maximum absorption was observed at 396 nm. When Tris-HCl buffer (pH 8.3), commonly used in biochemical experiments, especially DNA ligation with ligases, and aqueous solutions of potassium chloride and magnesium chloride were added to this nanoparticle dispersion, the absorption around 396 nm disappeared, as shown in the red spectrum. This result indicates that the surface of the silver nanoparticles was coated with AgCl by the addition of buffers and salts, and the nanoparticles bound together through van der Waal's interactions and precipitated as aggregates. On the other hand, silver nanoparticles with surface PEG modification also have absorption characteristics similar to commercially available silver nanoparticles, as shown in the gray spectrum, but the surface PEG-modified silver nanoparticles showed a strong absorption around 396 nm even after the addition of buffers and salts, as shown in the orange spectrum. From this, it was found that the surface of surface PEG-modified silver nanoparticles is protected by the excluded volume effect of PEG, and exhibits high dispersibility even in the presence of buffers and salts.

[0112] (7) Evaluation of the dispersibility of silver nanoparticles in the presence of PEG4000 The absorption spectrum of a silver nanoparticle dispersion in the presence of PEG4000 (polyethylene glycol 4,000, average molecular weight 2,700-3,300, manufactured by Fujifilm-Wako Pure Chemical Industries) was prepared by measuring 350 μL of a dispersion of silver nanoparticles with a particle size of 20 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), diluting it with superdeionized water (910 μL), and then adding 140 μL of 15.8 g / L PEG4000 aqueous solution. The spectrum was then measured using a quartz cell (path length: 1 cm, path width: 1 cm). Absorption spectra in the presence of buffers and salts were prepared by measuring 350 μL of a dispersion of silver nanoparticles with a particle size of 20 nm (Sigma-Aldrich, 0.02 mg / mL sodium citrate aqueous solution), diluting it with superdeionized water (490 μL), and then adding 140 μL of 15.8 g / L PEG4000 aqueous solution, 140 μL of 100 mM Tris-HCl buffer (pH 8.3), 140 μL of 500 mM potassium chloride aqueous solution, and 140 μL of 15 mM magnesium chloride aqueous solution. The samples were then measured using a quartz cell (optical path length: 1 cm, optical path width: 1 cm). The instrument used for the measurement was a JASCO V-650 ultraviolet-visible spectrometer. The measurement conditions were set to data interval: 0.5 nm, bandwidth: 1.0 nm, response: medium, and scanning speed: 100 nm / min. The results are shown in Figure 15.

[0113] Figure 15 illustrates that the thiol groups of surface-modifying PEG are important for interaction with silver nanoparticles, and that surface PEG modification improved the dispersibility of silver nanoparticles. In this experiment, for comparison, the behavior of silver nanoparticles (particle size 20 nm) in a dispersion was analyzed by absorption spectroscopy in the presence of thiol-unmodified PEG4000 (average molecular weight 2700-3300, manufactured by Fujifilm-Wako Pure Chemical Industries), which is not thought to directly interact with silver nanoparticles. The blue spectrum in the figure shows the absorption behavior of a dispersion in which PEG4000 was added at 1.58 g / L to the silver nanoparticle dispersion. A spectrum showing a maximum absorption at 396 nm was observed, similar to that of the commercially available silver nanoparticle dispersion itself. However, when Tris-HCl buffer (pH 8.3) and aqueous solutions of potassium chloride and magnesium chloride were added to this dispersion, the absorption around 396 nm almost disappeared, as shown in the gray and red spectra. This indicates that PEG4000, which does not undergo surface modification of silver nanoparticles, does not provide a protective effect on the surface of the silver nanoparticles, and therefore improvement in dispersibility cannot be expected. From this, it is considered that surface modification with thiolated PEG is essential for improving and maintaining the activity of silver nanoparticles.

[0114] Figure 16 shows the sequence design for a DNA ligation reaction utilizing the generation of adherent ends by DNA strand cleavage using silver nanoparticles. The total length of the ligated product DNA is 848 base pairs. By using two types of DNA primers having 3'-SP bonds, indicated by Ts, Rev for F1: 5'-AGCGCCATsTCGCCATTCAGG-3' and Fw for F1: 5'-ATGGCGCTsTTGCCTGGTTTC-3', DNA fragments of 298 base pairs and 558 base pairs, respectively, are amplified by PCR. The design allows for the preparation of adherent ends with an 8-base overhang on the 5'-side by treating the resulting PCR-amplified products with silver nanoparticles. The red underlined portion (ATGGCGCT) in the ligated product sequence corresponds to the sequence of the 8-base overhang. After treating the 298-base pair and 558-base pair DNA after PCR amplification with silver nanoparticles, ligation with T4 DNA ligase allows for the synthesis of a full-length 848-base-pair ligated product DNA. Two types of DNA primers, Rev for F1: 5'-AGCGCCATsTCGCCATTCAGG-3' and Fw for F1: 5'-ATGGCGCTsTTGCCTGGTTTC-3', were synthesized using an automated nucleic acid synthesizer.

[0115] (8) Optimization of coupling conditions in dimer model synthesis aimed at improving reaction conditions of 3'-thiolated amidites Reaction tracking experiment: Compound 5T (100 mg, 0.13 mmol, 1.0 eq.), 3,5-Bis[3,5-bis(trifluoromethyl)phenyl]-1H-1,2,4-triazole (533 mg, 1.69 mmol, 13 eq.), and d4T (88 mg, 0.39 mmol, 3.0 eq.) were dissolved in acetonitrile (6.76 ml) and stirred at room temperature for 2 to 120 minutes. 650 μL of the reaction solution was sampled at the start of the reaction and at 2, 5, 10, 30, 45, 60, and 120 minutes, transferred to an NMR sample tube, and the phosphorus NMR was measured.

[0116] Synthesis and isolation of dimeric model compounds [ka] Compound 5T (100 mg, 0.13 mmol, 1.0 eq.), 3,5-Bis[3,5-bis(trifluoromethyl)phenyl]-1H-1,2,4-triazole (82 mg, 0.26 mmol, 2.0 eq.), and d4T (88 mg, 0.39 mmol, 3.0 eq.) were dissolved in acetonitrile (13 ml) and stirred at room temperature for 1 hour. Then, tetrabutylammonium periodic acid (74 mg, 0.17 mmol, 1.3 eq.) was added, and the mixture was stirred at room temperature for another 7 minutes. Saturated aqueous solution of sodium thiosulfate (10 ml) was added, and the mixture was extracted twice with dichloromethane (20 ml). The reaction solution was concentrated using a rotary evaporator and purified by chromatography (neutral flash silica, methanol / dichloromethane = 1 / 7, 1% triethylamine) to obtain 50 mg (0.065 mmol, 50%) of a white solid. 1 1H-NMR (600MHz, CHLOROFORM-D)δ 9.15-9.29(m,1H),9.01(s,0H),6.98-7.65(m,15H),6.75-6.85(m,4H),6.19- 6.31(m,2H),5.90-5.93(m,1H),4.95(d,J=40.5Hz,1H),4.01-4.36(m,6H),3.6 7-3.91(m,6H),3.48-3.66(m,1H),3.38-3.46(m,1H),2.55-2.80(m,4H),1.87- 2.00(m,3H),1.78(d,J=32.0Hz,3H),1.34-1.56(m,3H),1.15-1.33(m,1H)ppm. 31 P-NMR (243MHz, CHLOROFORM-D)27.509,26.278ppm.

[0117] Synthesis and isolation of dimeric model compounds [ka] Compound 7G (30 mg, 0.035 mmol, 1.0 eq.), 3,5-Bis[3,5-bis(trifluoromethyl)phenyl]-1H-1,2,4-triazole (14 mg, 0.50 mmol, 13 eq.), and d4T (24 mg, 0.12 mmol, 3.0 eq.) were dissolved in acetonitrile (2.0 ml) and stirred at room temperature for 1 hour. Then, 0.01 M I2 (4.0 ml, 0.040 mmol, 1.1 eq.) was added, and the mixture was stirred at room temperature for another 7 minutes. A saturated aqueous solution of sodium thiosulfate (10 ml) was added, and the mixture was extracted twice with dichloromethane (20 ml). The reaction solution was concentrated using a rotary evaporator and purified by chromatography (neutral flash silica, methanol / dichloromethane = 1 / 7, 1% triethylamine) to obtain 31 mg (0.032 mmol, 89%) of a white solid. 1 H-NMR(600MHz,CHLOROFORM-D)δ 11.92(d,J=23.1Hz,1H),10.30-10.13(1H),8.60-8.68(m,2H),7.60-7.72(m,2H),7.11-7.30(m,15H),6 .92-6.98(m,1H),6.67-6.83(m,4H),6.31(dtd,J=58.3,3.8,2.0Hz,1H),5.94-6.04(m,2H),5.04-5.11( m,1H),4.80-4.93(m,1H),4.31-4.43(m,2H),4.19-4.24(m,1H),3.97-4.12(m,2H),3.75(d,J=5.4Hz,6H ),3.26-3.40(m,3H),2.79-2.87(m,1H),2.49-2.68(m,3H),1.87(t,J=1.5Hz,3H),1.14-1.25(m,7H)ppm. 31 P-NMR (243MHz, CHLOROFORM-D)29.983,28.438ppm.

[0118] As shown in Figure 2(a), in the synthesis of a 19-base DNA (sequence: 5'-TAACTsCACATTAATTGCGTT-FAM-3') with a 3'-thiolation modification introduced at the 4th base from the 5' end, a 15-base elongation termination product (approximately 50% of the HPLC peak area) was observed. The elongation termination product appeared as two peaks around 13-14 minutes on the HPLC chart. These were a mixture of 15-base 5' phosphorylated products formed after the uncoupled product (a failed 3'-thiolated amidite reaction) reacted with the 3'-thiolated amidite and then under iodine-pyridine oxidation conditions, resulting in chain cleavage. The formation of such byproducts is considered to be the cause of the reduced yield in oligomer synthesis. Therefore, we explored improved conditions for oligomer synthesis by optimizing the reaction conditions in a dimer model synthesis. Figure 17 shows the results of analyzing the yield of dimers of the target product after investigating various activators, their concentrations, and coupling times in dimer model synthesis. The activators used were 1H-tetrazole (1H-tet), 5-benzylthio-1H-tetrazole (BTT), 4,5-dicyanoimidazole (DCI), and 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (BTFTH). The yield of the target product was calculated by comparing the integral ratios of signals from the starting material 3'-thiolated amidite (around 160 ppm), the target product (190 ppm), and by-products (dithiolate: around 140 ppm) using phosphorus NMR analysis. The results showed that the yield of the target product was 1.4% when using 1H-tet (Entry 1), while it improved to 19% when using the more acidic BTT (Entry 2). When DCI, a more nucleophilic activator, was used, no change was observed in BTT or yield (Entry 3). Furthermore, using BTFTH, which has high acidity, improved the yield to 66% (Entry 4). Considering BTFTH to be the optimal activator, the concentration of BTFTH was increased from 0.02 M to 0.25 M, and the reaction proceeded almost quantitatively in 2 minutes (Entry 5). However, when the reaction time was extended to 1 hour under conditions using 0.25 M BTFTH, the yield of the target product decreased, and the formation of the by-product dithiolate was confirmed (Entry 6). From these results, it was shown that using a high concentration of BTFTH and completing the reaction in a short time is important to maximize the yield of the target product, and that increasing the reaction time causes the target product to react further and be converted into a by-product, thus decreasing the yield. After the coupling reaction of the 3'-thioamide, the phosphorus atom was oxidized from trivalent to pentavalent by adding 0.01 M iodine / pyridine / aqueous solution or tetrabutylammonium periodic acid as an oxidizing agent, thereby synthesizing a dimer model having a 3'-thiophosphate bond.

[0119] (9) Effect of reaction time when using 0.25 M BTFTH (13 equivalents) in dimer model synthesis Compound 5T (100 mg, 0.13 mmol, 1.0 eq.), 3,5-Bis[3,5-bis(trifluoromethyl)phenyl]-1H-1,2,4-triazole (533 mg, 1.69 mmol, 13 eq.), and d4T (88 mg, 0.39 mmol, 3.0 eq.) were dissolved in acetonitrile (6.76 ml) and stirred at room temperature for 2 to 120 minutes. 650 μL of the reaction solution was sampled at the start of the reaction and at 2, 5, 10, 30, 45, 60, and 120 minutes, transferred to an NMR sample tube, and the phosphorus NMR was measured.

[0120] Figure 18 shows experimental results illustrating the effect of reaction time when using 0.25 M BTFTH (13 equivalents) in a dimer model synthesis. Figure 18(A) plots the relative abundance of the compound against the reaction time, showing that the desired reaction was completed within 2 minutes of the start of the reaction, and that extending the reaction time further decomposed the target product and converted it to the dithiolate. Figure 18(B) shows the phosphorus NMR spectrum of the reaction product (2 hours after the start of the reaction). A signal from the target product was observed around 190 ppm, and a signal from the by-product, the dithiolate, was observed around 140 ppm.

[0121] Figure 19 shows the presumed mechanism of the side reaction. After the target dimer is formed, it is further activated by an activator, and after chain cleavage, the dithiolate is produced.

[0122] (10) Apply the optimized reaction conditions used in dimer synthesis to oligoDNA synthesis and improve the conditions. The oligodeoxyribonucleotide 5'-TAA Ts CATTAATTGCGTT-FAM-3' was synthesized using compound 5T and a commercially available phosphoramidite reagent (ChemGenes) according to a standard procedure on an automated nucleic acid synthesizer NR-2A 7MX (manufactured by Nippon Techno Service Co., Ltd.). Ts represents a thymidine base that has been 3' thiolated. Fluorescein (FAM) was introduced at the 3' end. All amidite reagents were prepared as 50 mM acetonitrile solutions, except for compound 5T, which was prepared as a 150 mM acetonitrile solution. BTFTH was used as the activator, and a 0.05 M iodine (pyridine / water; v / v:9 / 1) solution was used as the oxidizing agent. To investigate coupling conditions, 2 to 5 coupling cycles were performed over 20 to 450 seconds using 0.25 M or 1.0 M BTFTH. Furthermore, by using a 6-FAM-glycerol support 500 Å (ChemGenes) as the solid phase support, the design incorporated FAM at the 3' end. The synthesis was set to Final DMTr-ON, and the oligonucleotide was extracted while retaining the DMTr group on the 5'-terminal hydroxyl group. After the synthesis was complete, concentrated ammonia water (500 μL) and 40% methylamine aqueous solution (500 μL) were added to the solid phase support, and the oligonucleotide was cleaved from the solid phase support and deprotected by treatment at 65°C for 1 hour. The supernatant was filtered using a Millex LH filter (0.45 μm, Merck), and the filtrate was dried under reduced pressure using a centrifugal evaporator. The obtained oligonucleotide was redissolved in superdeionized water, and the concentration was calculated by measuring the absorption at 260 nm. By analyzing the product using reverse-phase HPLC, the peak area ratio was calculated between the peak derived from the target product with the DMTr group remaining as a 5'-hydroxyl protecting group (retention time 17.2 minutes) and the peaks derived from the product where synthesis was stopped at the introduction of 3'-thioamide 5T and the cleaved product (retention time around 8.0-12.0 minutes). The HPLC analysis conditions were as follows. Column: Hydrosphere C18 (250 x 10.0 mml.D., S-5 μm, 12 nm, HS12S05-2510WT, Ser. No. 131EA90023), Eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile (pH 7.0), Eluent B: Acetonitrile, Gradient conditions: Flow rate: 3 mL / min, Column temperature: 50°C, Detection wavelength: 260 nm.

[0123] Figure 20 shows the results of applying the optimized reaction conditions for dimer synthesis to oligoDNA synthesis and improving the conditions. BTFTH, which proved to be the most effective activator in dimer synthesis, was used. The activator concentration, coupling time, and number of coupling cycles were investigated, and the production ratio of the resulting 17-nucleotide oligoDNA (sequence: 5'-TAA Ts CATTAATTGCGTT-FAM-3') on the reverse-phase HPLC peak was calculated. The table in Figure 20 shows the peak ratio of the target product to the chain break product + unreacted product (13-nucleotide length: sequence: 5'-CATTAATTGCGTT-FAM-3'). The most common iodine pyridine solution was used as the oxidizing agent. As a control for condition optimization, when 0.25 M BTT was used and coupling was performed twice at 450 seconds, the target product ratio was 5% (Entry 1). When 0.25M BTFTH was used and coupling was performed twice over 450 seconds, the target product ratio was 46% (Entry 2). When the coupling time was reduced to 20 seconds, the target product ratio remained almost unchanged at 43% (Entry 3). Next, when the BTFTH concentration was increased to 1.0M and coupling was performed twice over 450 seconds, the target product ratio improved by about 20% (Entry 4). Further changes to the coupling time to 60 seconds and the number of coupling cycles to 5 cycles resulted in a slight improvement in the target product ratio to 69% (Entry 5). Subsequently, when 0.25M BTFTH was used and coupling was performed five times over 90 seconds, the target product ratio improved to 87% (Entry 6). These results indicate that in solid-phase synthesis of oligoDNA, the target product production ratio can be maximized by repeating the coupling cycle multiple times with short coupling times.

[0124] (11) Synthesis of oligoDNA (sequence: 5'-TAA Ts CATTAATTGCGTT-FAM-3') synthesized using optimized coupling conditions. The oligodeoxyribonucleotide 5'-TAA Ts CATTAATTGCGTT-FAM-3' was synthesized using compound 5T and a commercially available phosphoramidite reagent (ChemGenes) according to a standard procedure on an automated nucleic acid synthesizer NR-2A 7MX (Nippon Techno Service Co., Ltd.). Ts represents a thymidine base that has been 3' thiolated. Fluorescein (FAM) was introduced at the 3' end. All amidite reagents were prepared as 50 mM acetonitrile solutions, except for compound 5T, which was prepared as a 150 mM acetonitrile solution. 0.25 M BTFTH was used as the activator (5 coupling cycles in 90 seconds), and 0.05 M iodine (pyridine / water; v / v:9 / 1) solution was used as the oxidizing agent. Furthermore, a 6-FAM-glycerol support 500 Å (ChemGenes) was used as the solid support to ensure that FAM was introduced at the 3' end. The synthesis was performed using the Final DMTr-ON setting, and the DMTr group was removed while retaining the hydroxyl group at the 5'-terminus. After the synthesis was complete, concentrated ammonia water (500 μL) and 40% methylamine aqueous solution (500 μL) were added to the solid support, and the DNA was cleaved from the solid support and deprotected by treatment at 65°C for 1 hour. The supernatant was filtered using a Millex LH filter (0.45 μm, Merck), and the filtrate was dried under reduced pressure using a centrifugal evaporator. The obtained oligonucleotide was redissolved in superdeionized water, and the concentration was calculated by measuring the absorption at 260 nm. The 5'-DMTr protected DNA was separated by reverse-phase HPLC, and the DMTr group was deprotected by adding 10% acetic acid aqueous solution and treating at room temperature for 1 hour to obtain the target oligoDNA. The HPLC analysis conditions were as follows. Column: Hydrosphere C18 (250 x 10.0 mml.D., S-5 μm, 12 nm, HS12S05-2510WT, Ser. No. 131EA90023), Eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile (pH 7.0), Eluent B: Acetonitrile, Gradient conditions: Flow rate: 3 mL / min, Column temperature: 50°C, Detection wavelength: 260 nm.

[0125] Figure 21 shows the synthesis results of oligoDNA (sequence: 5'-TAA Ts CATTAATTGCGTT-FAM-3') synthesized using the coupling conditions (entry 6) optimized in the experiment shown in Figure 20. From the reverse-phase HPLC analysis results in Figure 21, the target product with a length of 17 base pairs containing a 3'-thiophosphate bond was obtained at a ratio of 87% of the peak area compared to the peak derived from by-products (strand breaks) (retention time 16.9 mins). After preparative purification of the target product peak with a retention time of 16.9 mins, the DMTr group was deprotected by adding a 10% aqueous acetic acid solution and treating at room temperature for one hour to obtain the target DNA (5'-TAA Ts CATTAATTGCGTT-FAM-3).

[0126] (12) Synthesis of a 51-nucleotide oligoDNA (5'-ATGAAACGCCGAGTTsAACGCCATCAAAAATsAATTCGCGTCTGGCCTTCCTsG-3') with three 3' thiophosphate bonds introduced. The oligodeoxyribonucleotide 5'-ATGAAACGCCGAGTTsAACGCCATCAAAAATsAATTCGCGTCTGGCCTTCCTsG-3' was synthesized using compound 5T and a commercially available phosphoramidite reagent (ChemGenes) according to a standard procedure on an automated nucleic acid synthesizer NR-2A 7MX (manufactured by Nippon Techno Service Co., Ltd.). Ts represents a thymidine base that has undergone 3'-thiolation. All amidite reagents were prepared as 50 mM acetonitrile solutions, except for compound 5T, which was prepared as a 150 mM acetonitrile solution. 0.25 M BTFTH (coupling 5 times in 90 seconds) was used as the activator, and 0.05 M iodine (pyridine / water; v / v:9 / 1) solution was used as the oxidizing agent. The synthesis was performed with the Final DMTr-ON setting, and the DMTr group on the 5'-terminal hydroxyl group was retained during extraction. After synthesis, concentrated ammonia water (500 μL) and 40% methylamine aqueous solution (500 μL) were added to the solid support, and the mixture was cleaved and deprotected by treatment at 65°C for 1 hour. The supernatant was filtered using a Millex LH filter (0.45 μm, Merck), and the filtrate was dried under reduced pressure using a centrifugal evaporator. The obtained oligonucleotides were redissolved in superdeionized water, and the concentration was calculated by measuring the absorption at 260 nm. The 5'-DMTr protected DNA was separated by reverse-phase HPLC, and the DMTr group was deprotected by adding 10% acetic acid aqueous solution and treating at room temperature for 1 hour to obtain the target oligoDNA. The HPLC analysis conditions were as follows. Column: Hydrosphere C18 (250 x 10.0 mml.D., S-5 μm, 12 nm, HS12S05-2510WT, Ser. No. 131EA90023), Eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile (pH 7.0), Eluent B: Acetonitrile, Gradient conditions: Flow rate: 3 mL / min, Column temperature: 50°C, Detection wavelength: 260 nm.

[0127] Figure 22 shows the results of analyzing a 51-base oligoDNA (5'-ATGAAACGCCGAGTTsAACGCCATCAAAAATsAATTCGCGTCTGGCCTTCCTsG-3') synthesized with three 3'-thiophosphate bonds, using reverse-phase HPLC. Ts represents the thymidine base that has undergone 3'-thiolation. The reverse-phase HPLC results show that the main peak observed around 17.2 minutes of retention is the 51-mer target DNA, with the DMTr group remaining as a 5'-hydroxyl group protecting group. In contrast, some products where synthesis stopped at the compound 5T introduction stage and cleaved products were observed around 8.0-12.0 minutes of retention, but these were easily separated from the 51-mer target DNA. By separating the peak around 17.2 minutes of retention, the 5'-DMTr protected form of the target DNA was obtained. The 5'-DMTr protected product, preparatively purified by HPLC, was analyzed by ESI-TOF-MS, confirming that the peaks at m / z 16 and 110.2 were derived from the target product. The DMTr group was deprotected by adding a 10% aqueous acetic acid solution and treating at room temperature for one hour to obtain the target DNA.

[0128] (13) Sequences and synthesis of PCR primer DNA with four and two 3'-thiophosphate bonds introduced. Oligodeoxyribonucleotides were synthesized using compound 5T and a commercially available phosphoramidite reagent (ChemGenes) according to a standard procedure on an automated nucleic acid synthesizer NR-2A 7MX (manufactured by Nippon Techno Service Co., Ltd.). Ts represents a thymidine base that has undergone 3'-thiolation. All amidite reagents were prepared as 50 mM acetonitrile solutions, except for compound 5T, which was prepared as a 150 mM acetonitrile solution. 0.25 M BTFTH (coupling 5 times in 90 seconds) was used as the activator, and 0.05 M iodine (pyridine / water; v / v:9 / 1) solution was used as the oxidizing agent. The synthesis was performed using the Final DMTr-ON setting, and the DMTr group was removed while retaining the hydroxyl group at the 5'-terminus. After synthesis, concentrated ammonia water (500 μL) and 40% methylamine aqueous solution (500 μL) were added to the solid support, and the compound was cleaved from the solid support and deprotected by treatment at 65°C for 1 hour. The supernatant was filtered using a Millex LH filter (0.45 μm, Merck), and the filtrate was dried under reduced pressure using a centrifugal evaporator. The obtained oligonucleotides were redissolved in superdeionized water, and their concentrations were calculated by measuring the absorption at 260 nm. The 5'-DMTr-protected DNA was separated by reverse-phase HPLC, and the DMTr group was deprotected by adding 10% aqueous acetic acid solution and treating at room temperature for one hour to obtain the target oligoDNA. The HPLC analysis conditions were as follows. Column: Hydrosphere C18 (250 x 10.0 mml.D., S-5 μm, 12 nm, HS12S05-2510WT, Ser. No. 131EA90023), Eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile (pH 7.0), Eluent B: Acetonitrile, Gradient conditions: Flow rate: 3 mL / min, Column temperature: 50°C, Detection wavelength: 260 nm.

[0129] Figure 23 shows the sequences of four types of PCR primer DNA with multiple 3'-thiophosphate bonds and their synthesis results. The synthesized primers are Rev_primer for F1_8 and Rev_primer for F2_8, which are cleaved at four locations by silver nanoparticle cleavage after PCR amplification, producing 34-base adherent ends, and Rev_primer for F1_16 and Rev_primer for F2_16, which are cleaved at two locations by silver nanoparticle cleavage after PCR amplification, producing 34-base adherent ends. The four synthesized oligoDNAs were analyzed by reverse-phase HPLC. The peaks of the target length oligoDNA with the DMTr group remaining as a 5'-hydroxyl protecting group (retention time around 15 minutes) were separated and purified, and then the DMTr group was deprotected by adding a 10% aqueous acetic acid solution and treating at room temperature for one hour to obtain the target DNA.

[0130] (14) Synthesis of 3'-thiophosphorylated oligoDNA (5'-TAACAs CACATTAATTGCGTT-FAM-3') using 3'-thiolated amidite adenosine derivatives The oligodeoxyribonucleotide 5'-TAACAs CACATTAATTGCGTT-FAM-3 was synthesized using compound 6A and a commercially available phosphoramidite reagent (ChemGenes) according to a standard procedure on an automated nucleic acid synthesizer NR-2A 7MX (manufactured by Nippon Techno Service Co., Ltd.). As was 3'-thiolated. AdenosineThe base is shown. Various amidite reagents were prepared as 50 mM acetonitrile solutions, except for compound 5T, which was prepared as a 150 mM acetonitrile solution. 0.25 M BTFTH was used as the activator (coupled twice in 450 seconds), and 0.05 M iodine (pyridine- / water; v / v:9 / 1) solution was used as the oxidizing agent. The synthesis was performed using the Final DMTr-ON setting, and the DMTr group was removed while retaining the hydroxyl group at the 5'-terminus. After the synthesis was complete, concentrated ammonia water (500 μL) and 40% methylamine aqueous solution (500 μL) were added to the solid support, and the mixture was cleaved from the solid support and deprotected by treatment at 65°C for 1 hour. The supernatant was filtered using a Millex LH filter (0.45 μm, Merck), and the filtrate was dried under reduced pressure using a centrifugal evaporator. The obtained oligonucleotide was redissolved in superdeionized water, and the concentration was calculated by measuring the absorption at 260 nm. The 5'-DMTr-protected DNA was isolated by reverse-phase HPLC, and then the DMTr group was deprotected by adding a 10% aqueous acetic acid solution and treating at room temperature for one hour to obtain the target oligoDNA. The HPLC analysis conditions were as follows: Column: Hydrosphere C18 (250 x 10.0 mml.D., S-5 μm, 12 nm, HS12S05-2510WT, Ser. No. 131EA90023), Eluent A: 50 mM triethylammonium acetate containing 5% acetonitrile (pH 7.0), Eluent B: Acetonitrile, Gradient conditions: Flow rate: 3 mL / min, Column temperature: 50°C, Detection wavelength: 260 nm.

[0131] Figure 24 shows the results of synthesizing 3'-thiophosphorylated oligoDNA (sequence: 5'-TAACAs CACATTAATTGCGTT-FAM-3') containing an adenosine derivative using a 3'-thiolated amidite adenosine derivative (compound 6A). The synthesized oligoDNA was analyzed by reverse-phase HPLC using BTFTH as an activator. As a result, the proportion of the 5'-DMTr protected target oligoDNA at a retention time of around 16.8 minutes was 40% based on the reverse-phase HPLC peak area ratio, and it was clearly separable from the uncoupled product and the strand-cleaved product (retention time around 11.5 minutes). After preparative purification of the target length oligoDNA peak, the DMTr group was deprotected by adding a 10% aqueous acetic acid solution and treating at room temperature for one hour to obtain the target DNA.

[0132] (15) Adjustment of 3'-overhang adhesive ends by cleavage of 3'-thiophosphate-bound DNA containing adenosine analog with silver nanoparticles Made PS-modified DNA (5'-TAACAsCACATTAATTGCGTT-FAM-3') and complementary DNA (5'-AACGCAATTAATGTGTGTTA-3') were mixed to a concentration of 3 μM each, treated at 95°C for 3 minutes, and then annealed by cooling on an ice bath for at least 10 minutes. 9 μL of the annealed solution was measured out, and 51 μL of surface PEG-modified silver nanoparticle dispersion (10 nm) was added. The mixture was incubated at 50°C for a specified time. The surface PEG-modified silver nanoparticle dispersion was prepared by adding 1 μL of terminal thiol PEG aqueous solution (23.9 g / L) to 50 μL of commercially available silver nanoparticle dispersion (Sigma-Aldrich, 10 nm, 0.02 mg / mL). 30 μL of the reaction solution was measured out, and 30 μL of 2x loading buffer was added. After heat treatment at 95°C for 5 minutes, the samples were analyzed by 15% denatured acrylamide electrophoresis (containing 7.5M urea, 10x12cm, 30mA, 20 minutes, 6μL application). Gel electrophoresis images were obtained using a gel image analyzer (BioRad) by detecting fluorescence derived from FAM.

[0133] Figure 25 shows the results of preparing 3' overhanging adhesive ends of double-stranded short-chain DNA by silver nanoparticle treatment. Here, cleavage of a 3'-thioadenosine analog synthesized in the experiment shown in Figure 24 was performed using 1 nm surface-PEG-modified silver nanoparticles. The SP bond was positioned between the 5th and 6th base pairs from the 5' end, and the design ensured that a 5-base overhanging adhesive end was prepared upon cleavage. The reaction was carried out at 50°C for 15, 30, 60, and 120 minutes, and each reaction solution was analyzed by denatured gel electrophoresis. The cleavage efficiency at each reaction time was calculated by analyzing the band intensity of the gel using FAM-derived fluorescence detection. The cleavage efficiency was compared with and without a complementary strand, and the cleavage efficiency at each reaction time was plotted on the vertical axis and the reaction time on the horizontal axis. As a result, with a complementary strand, the cleavage activity was slightly lower compared to without a complementary strand, but after 2 hours from the start of the reaction, it showed almost the same level of cleavage activity, and more than 90% of the DNA was cleaved. Furthermore, this cleavage activity is shown in Figure 5 The results were almost identical to those of the thymidine analogs shown, indicating that changing the base species at the modification site does not affect the cleavage activity.

[0134] (16) Adhesion end treatment using multiple modification primers Made and linkage reaction Figure 26 shows the 34-nucleotide-length adherent end cleavage of PCR amplification products using the four primer DNAs synthesized in Figure 23 by silver nanoparticle cleavage. Made This is a schematic diagram of the experimental system showing the arrangement of the ligation products of those cleavage products.

[0135] (a) PCR using four types of primers A mixture of 20 μM thiolated forward primer (1.25 μL), 20 μM thiolated reverse primer (1.25 μL), 10 ng / μL template DNA (2.5 μL), 2 mM dNTP mixture (5 μL), 25 mM magnesium sulfate (3 μL), 10x PCR Buffer for KOD-Plus-Neo (5 μL), and superdeionized water (31 μL) was added. 1 U / μL KOD-Plus-Neo (1 μL) was then added, and the mixture was treated at 95°C for 2 minutes using a thermal cycler (BioRad). Subsequently, DNA amplification was performed by 30 cycles of denaturation (95°C, 15 sec) - annealing (55°C, 15 sec) - strand extension (68°C, 30 sec). The PCR product was purified using a wizard column (Promega) according to the manufacturer's recommended protocol to obtain thiolated DNA.

[0136] (b) Bonding end treatment by cutting with silver nanoparticles Made 7.5 nM PCR product thiolated DNA (225 fmol / sample) was dissolved in superdeionized water to prepare a 4.5 μL aqueous solution. PEG-modified silver nanoparticles were prepared by mixing 10 nm silver nanoparticles (Sigma-Aldrich, 200 μg / mL citrate buffer suspension, 25 μL) with terminally thiol-modified PEG (Sigma-Aldrich, 23.9 g / L aqueous solution, 0.5 μL). The prepared PCR product thiolated DNA aqueous solution (4.5 μL) and the PEG-modified silver nanoparticle dispersion (25.5 μL) were mixed and treated at 50°C for 4 hours to cleave SP bonds and prepare adherent ends.

[0137] Figure 27 shows the 34-nucleotide-length adherent end cleavage of PCR amplification products using the four primer DNAs synthesized in Figure 23 by silver nanoparticle treatment. Made The procedure for ligation experiments of these cleavage products and the agarose electrophoresis results of the PCR product DNA are shown. It was confirmed that PCR products of four desired lengths—529-bp, 307-bp, 529-bp, and 307-bp—were obtained.

[0138] (17) Comparison of linkage efficiency between 34-base-length adherent end DNA obtained by cleaving PCR amplification products at multiple sites and linkage of adherent end DNAs. 7.5nM silver nanoparticle-treated DNA_1 (3.6 μL), 7.5nM silver nanoparticle-treated DNA_2 (3.6 μL), 10x T4 DNA Ligase Reaction Buffer (New England BioLabs, 0.90 μL), and superdeionized water (0.45 μL) were mixed, and then T4 DNA ligase (New England BioLabs, 2000 U / μL, 0.45 μL) was added and incubated at 25°C for 3 hours. 1 μL of 10x loading buffer was added to the reaction solution (9 μL), and the mixture was analyzed by 1% agarol gel electrophoresis (electrophoresis buffer: 1x TBE, 100 V, 30 minutes). The gel was stained by shaking with 10,000x SYBR green I solution for 30 minutes, and the gel electrophoresis image was obtained using a gel image analyzer (BioRad).

[0139] Figure 28 shows a comparison of the ligation efficiency between 34-base-length adherent-end DNA fragments obtained by cleaving PCR-amplified products using Rev_primer for F1_8 and Rev_primer for F2_8 with silver nanoparticles, and 34-base-length adherent-end DNA fragments obtained by cleaving PCR-amplified products using Rev_primer for F1_16 and Rev_primer for F2_16 with silver nanoparticles. Products amplified using Rev_primer for F1_8 and Rev_primer for F2_8 have thiophosphate modifications introduced every 8 bases, while products amplified using Rev_primer for F1_16 and Rev_primer for F2_16 have thiophosphate modifications introduced every 16 bases. Therefore, by comparing these ligation efficiencies, the effect of the dissociation efficiency of the cleaved DNA fragments on ligation after cleavage with silver nanoparticles becomes clear. In other words, DNA fragments with 8 bases after silver nanoparticle cleavage are expected to dissociate rapidly after cleavage and show relatively high ligation efficiency, while DNA fragments with 16 bases after silver nanoparticle cleavage are expected to dissociate relatively slowly and show low ligation efficiency. Experimental results showed that the ligation efficiency was 29% in the system with two cleavage sites (Ts modification x2) and 44% in the system with four cleavage sites (Ts modification x4), indicating that cleavage at multiple sites promotes the dissociation of the DNA fragment after cleavage and shows high ligation efficiency. The scavenger strand in the figure has the function of separating products that are not ligated, even though the adhesive ends form complementary strands, and was used in gel analysis to clearly distinguish ligated products from unligated products.

Claims

1. The process includes a cleavage step of reacting a nucleic acid to be cleaved having the structure shown in formula (1) below with a cleavage agent to cleave the nucleic acid at the X portion of formula (1) to produce a nucleic acid having the structure shown in formula (2) below, A method for severing nucleic acid chains, characterized in that the severing agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. 【Chemistry 1】 (Here, B represents a base, and X represents sulfur or selenium.) NucA consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 5' end portion relative to X. NucB consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 3' end portion relative to X.

2. The nucleic acid chain cleavage method according to claim 1, characterized in that the cleavage agent is silver nanoparticles and X is sulfur.

3. The nucleic acid chain cleavage method according to claim 1, characterized in that the average particle size of the metal nanoparticles is in the range of 1 to 20 nm.

4. The nucleic acid chain cleavage method according to claim 1, characterized in that polyethylene glycol is bonded to the surface of the metal nanoparticles.

5. The further step is to prepare the nucleic acid to be cleaved, The nucleic acid chain cleavage method according to claim 1, characterized in that the nucleic acid preparation step involves synthesizing a part or all of the nucleic acid to be cleaved by the phosphoramidite method using amidite reagents represented by the following formulas (3) and (4). 【Chemistry 2】 (Here, B represents a base, X represents sulfur or selenium, and DMTr represents a dimethoxytrityl group.)

6. The nucleic acid chain cleavage method according to claim 5, characterized in that the nucleic acid preparation step involves synthesizing 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole as an activator by the phosphoramidite method.

7. The nucleic acid chain cleavage method according to claim 5, characterized in that the nucleic acid preparation step involves synthesizing a portion of the nucleic acid to be cleaved by a phosphoramidite method to form a primer, and using a template DNA having a sequence complementary to the nucleic acid to be cleaved as a template, multiple polymerase chain reaction cycles are performed to extend the primer along the template DNA and generate the nucleic acid to be cleaved.

8. A nucleic acid preparation means for preparing a nucleic acid to be cleaved having the structure shown in the following formula (1), A cleavage means that reacts the nucleic acid to be cleaved with a cleavage agent to cleave the nucleic acid at the X portion of formula (1) and generate a nucleic acid having the structure shown in formula (2) below, Equipped with, A nucleic acid chain severing device characterized in that the severing agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. 【Transformation 3】 (Here, B represents a base, and X represents sulfur or selenium.) NucA consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 5' end portion relative to X. NucB consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 3' end portion relative to X.

9. The nucleic acid chain cleavage apparatus according to claim 8, characterized in that the nucleic acid preparation means synthesizes part or all of the nucleic acid to be cleaved by the phosphoramidite method using amidite reagents represented by the following formulas (3) and (4). 【Chemistry 4】 (Here, B represents a base, X represents sulfur or selenium, and DMTr represents a dimethoxytrityl group.)

10. The nucleic acid chain cleavage apparatus according to claim 9, characterized in that the nucleic acid preparation means synthesizes 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole as an activator by the phosphoramidite method.

11. A method for producing double-stranded DNA having adherent ends, A double-stranded DNA to be cleaved, comprising a sense strand and an antisense strand having a sequence complementary to the sense strand, wherein at least one of the sense strand and the antisense strand has a structure represented by the following formula (1). The process includes a step of generating an adherent end, in which the sense strand and / or the antisense strand are cleaved at the X portion of formula (1) by reacting with a cleaving agent to produce a double-stranded DNA having the structure shown in formula (2) below and having adherent ends, A method for producing double-stranded DNA, characterized in that the cleavage agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. 【Transformation 5】 (Here, B represents a base, and X represents sulfur or selenium.) NucA consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 5' end portion relative to X. NucB consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 3' end portion relative to X.

12. The method for producing double-stranded DNA according to claim 11, characterized in that the double-stranded DNA to be cleaved has a plurality of structures represented by formula (1), and the number of nucleotides between each structure is 10 or less.

13. A double-stranded DNA manufacturing apparatus for producing double-stranded DNA having adherent ends, A double-stranded DNA preparation means for preparing a double-stranded DNA to be cleaved, comprising a sense strand and an antisense strand having a sequence complementary to the sense strand, wherein at least one of the sense strand and the antisense strand has a structure represented by the following formula (1); The system includes an adhesive end generating means for reacting the double-stranded DNA to be cleaved with a cleaving agent to cleave the sense strand and / or the antisense strand at the X portion of formula (1), thereby generating double-stranded DNA having the structure shown in formula (2) below and having adhesive ends, A double-stranded DNA production apparatus characterized in that the cleavage agent is a metal nanoparticle containing an atom selected from the group consisting of silver, mercury, and cadmium. 【Transformation 6】 (Here, B represents a base, and X represents sulfur or selenium.) NucA consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 5' end portion relative to X. NucB consists of at least one nucleotide and is part of the nucleic acid to be cleaved, representing the 3' end portion relative to X.

14. The apparatus for producing double-stranded DNA according to claim 13, characterized in that the double-stranded DNA to be cleaved has a plurality of structures represented by formula (1), and the number of nucleotides between each structure is 10 or less.

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