WO2024101294A1 - Method for obtaining sequence for non-cyclic artificial nucleic acid - Google Patents

Method for obtaining sequence for non-cyclic artificial nucleic acid Download PDF

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WO2024101294A1
WO2024101294A1 PCT/JP2023/039831 JP2023039831W WO2024101294A1 WO 2024101294 A1 WO2024101294 A1 WO 2024101294A1 JP 2023039831 W JP2023039831 W JP 2023039831W WO 2024101294 A1 WO2024101294 A1 WO 2024101294A1
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polynucleotide
sequence
dna
nucleic acid
artificial nucleic
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Japanese (ja)
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直人 根本
佐恵 瀧澤
浩之 浅沼
恵司 村山
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国立大学法人埼玉大学
国立大学法人東海国立大学機構
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    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing

Definitions

  • the present invention relates to a method for obtaining the sequence of an artificial nucleic acid. More specifically, the present invention relates to a method for amplifying a DNA sequence complementary to the sequence of a non-circular artificial nucleic acid and decoding the sequence.
  • Nucleic acids whose physical properties have been changed by chemically modifying the nucleic acid base, sugar, or phosphate diester moiety are called artificial nucleic acids.
  • Artificial nucleic acids have artificial functions not found in natural nucleic acids, such as resistance to nucleases and being difficult to decompose.
  • the base moiety of artificial nucleic acids can be adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), etc. Therefore, in addition to genome sequencing, gene detection, and sequence-selective gene suppression, they have a wide range of usefulness as a base material for creating compound libraries and as pharmaceutical materials, and are expected to become a new modality.
  • L-acyclic Threoninol Nucleic Acid L-aTNA
  • Serinol Nucleic Acid SNA
  • Locked Nucleic Acid LNA
  • Peptide Nucleic Acid PNA
  • Glycol Nucleic Acid Glycol Nucleic Acid
  • L-aTNA and SNA which are artificial nucleic acids in which the main chain structure of natural nucleic acids has been modified to an acyclic backbone, can form double strands with natural DNA and RNA (Non-Patent Document 1), while being highly resistant to nucleases compared to conventional nucleic acid drugs that are easily degraded in the blood, and even when degraded in the body, they are reduced to amino acids and phosphates, meaning that they have no or very few side effects. For this reason, they are expected to be applicable to technologies such as siRNA, anti-miRNA oligo (AMO), and antisense oligo (ASO).
  • siRNA siRNA
  • AMO anti-miRNA oligo
  • ASO antisense oligo
  • ligases used to link natural nucleic acids cannot be used to link the end to another polynucleotide.
  • the artificial nucleic acid is L-aTNA or SNA
  • the two can be efficiently linked by reacting a polynucleotide containing an artificial nucleic acid with another polynucleotide in the presence of imidazole and cyanogen halide, and/or N-cyanoimidazole (Patent Document 2).
  • the sequence of a polynucleotide made of an artificial nucleic acid cannot be analyzed with a commercially available sequencer, unlike a DNA polynucleotide.
  • a polynucleotide made of a non-cyclic artificial nucleic acid such as L-aTNA
  • a polynucleotide made of a non-cyclic artificial nucleic acid having a complementary sequence such as L-aTNA
  • a DNA polynucleotide having amplification primer regions at both ends is hybridized to a polynucleotide made of a non-cyclic artificial nucleic acid, it tends not to form a double strand.
  • sequence information cannot be obtained because a method for converting the sequence into a DNA sequence has not been established. Therefore, a method for determining the sequence of a polynucleotide consisting of an acyclic artificial nucleic acid is desired.
  • the inventors have therefore conducted extensive research and found that the efficiency of chemical linkage between a DNA polynucleotide and a primer region can be improved by adding a DNA fragment of several bases to both ends of a polynucleotide consisting of a non-cyclic artificial nucleic acid.
  • This DNA fragment of several bases is complementary to a portion of the primer sequence for DNA amplification at both ends of the DNA polynucleotide to be hybridized with the polynucleotide consisting of a non-cyclic artificial nucleic acid, or to the sequences at both ends of the DNA polynucleotide to be hybridized and a portion of the primer sequence for DNA amplification.
  • the inventors discovered that the efficiency of chemically linking the DNA polynucleotide to the primer region can be improved by hybridizing a polynucleotide consisting of a non-cyclic artificial nucleic acid with a DNA polynucleotide and then linking primer regions to both ends of the DNA polynucleotide.
  • the present invention relates to a method for producing a polynucleotide sequence comprising the steps of: hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences; wherein the DNA polynucleotide has a sequence common to the DNA polynucleotides contained in the pool added to both ends of a random sequence,
  • the polynucleotide consisting of the non-cyclic artificial nucleic acid has DNA fragments added to both ends, and the DNA fragments are 1) a sequence complementary to the 3' side of a primer region that is linked to the 5' end of the DNA polynucleotide or to the DNA polynucleotide in a later step on the 3' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, and a sequence complementary to the 5' side of a primer region that is linked to the 3' end of the DNA polyn
  • the present invention provides a method for producing a nucleic acid sequence comprising the steps of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences; ligating primer regions to both ends of the DNA polynucleotide; amplifying DNA from said DNA polynucleotide; determining the sequence of the DNA polynucleotide; determining the sequence of a polynucleotide consisting of a non-cyclic artificial nucleic acid from the sequence of the DNA polynucleotide.
  • the present invention provides a method for producing a nucleic acid sequence comprising the steps of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences; wherein the DNA polynucleotide has a sequence common to the DNA polynucleotides contained in the pool added to both ends of a random sequence,
  • the polynucleotide consisting of the non-cyclic artificial nucleic acid has DNA fragments added to both ends, and the DNA fragments are 1) a sequence complementary to the 3' side of the primer region linked to the 5' end of the DNA polynucleotide on the 3' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, and a sequence complementary to the 5' side of the primer region linked to the 3' end of the DNA polynucleotide on the 5' side of the polynucleotide consisting of the non-cyclic artificial
  • L-aTNA, SNA, or PNA can be used as the acyclic artificial nucleic acid.
  • the primer regions are preferably linked to both ends of the DNA polynucleotide by chemical linkage in the presence of N-cyanoimidazole and a divalent cation through a phosphoric acid reaction. Furthermore, it is preferable to use cadmium ions or manganese ions as the divalent cation.
  • N-cyanoimidazole imidazole and cyanogen bromide (Br-CN) can also be used as an activator.
  • the sequence of a polynucleotide consisting of a desired non-cyclic artificial nucleic acid can be transferred to the sequence of a DNA polynucleotide as a complementary sequence, and then amplified by a known method such as PCR, thereby enabling efficient sequence determination.
  • a known method such as PCR
  • FIG. 1 is a diagram illustrating an embodiment of the present invention in which DNA fragments are added to both ends of an L-aTNA polynucleotide.
  • FIG. 1 is a diagram illustrating one embodiment of the present invention in which a DNA polynucleotide having no primer regions at either end is used to hybridize with an L-aTNA polynucleotide, and a primer region is chemically linked to the DNA polynucleotide after hybridization.
  • FIG. 1 illustrates yet another embodiment of the present invention showing a method for sequencing an L-aTNA polynucleotide using a DNA polynucleotide containing a random sequence.
  • FIG. 1 shows the results of Example 1.
  • FIG. 13 shows the results of Example 2.
  • FIG. 13 shows the results of Example 3.
  • non-cyclic artificial nucleic acid refers to an artificial nucleic acid in which the main chain structure of a natural nucleic acid is modified to an acyclic backbone, such as D-aTNA, L-aTNA, and SNA.
  • acyclic backbone such as D-aTNA, L-aTNA, and SNA.
  • L-aTNA, SNA, and PNA are preferred, L-aTNA and SNA are particularly preferred, and L-aTNA is the most preferred.
  • L-aTNA and SNA can form a double strand with DNA or RNA, and, without being bound by a specific theory, they share a flexible structure due to being a chain structure that does not contain a cyclic structure, and both can be linked to other polynucleotides using the same method.
  • PNA can also be linked to other polynucleotides using the same method as in the case of L-aTNA and SNA.
  • the structures of L-aTNA and SNA are shown below.
  • Base represents a base such as adenine, thymine, cytosine, guanine, or uracil.
  • Base represents a base such as adenine, thymine, cytosine, guanine, or uracil.
  • Polynucleotide refers to a polymer in which multiple nucleotide monomers are covalently bonded.
  • polynucleotides are not limited to those whose monomers are natural nucleotides consisting of phosphate, pentose, and bases, but also include those containing nucleotides whose main chain structure has been modified to an acyclic backbone, as described above.
  • DNA polynucleotide refers to a polynucleotide composed of deoxyribonucleic acid as a monomer.
  • Polynucleotide composed of acyclic artificial nucleic acid refers to a polynucleotide composed of acyclic artificial nucleic acid as a monomer.
  • L-aTNA polynucleotide refers to a polynucleotide composed of L-aTNA as a monomer.
  • Hybridization refers to the bonding of two polynucleotides through hydrogen bonds based on the complementarity of nucleotide bases.
  • “hybridization” includes not only the bonding of complementary strands between DNA polynucleotides or between a DNA polynucleotide and an RNA polynucleotide, but also the bonding of complementary strands between polynucleotides made of artificial nucleic acids, and the bonding of complementary strands between a DNA polynucleotide or an RNA polynucleotide and a polynucleotide made of artificial nucleic acid.
  • the term “hybridization” is sometimes also referred to as "annealing.”
  • a pool of DNA polynucleotides containing random sequences refers to a mixture of an infinite number of DNA polynucleotides with various sequence combinations.
  • the DNA polynucleotide has a sequence common to all DNA polynucleotides contained in the pool added to both ends of a random sequence.
  • the length and sequence of this common sequence can be determined arbitrarily.
  • the common sequence can be any sequence having a length of about 2 to 5 mers.
  • the 3'-corresponding side that binds to the phosphate group of the adjacent nucleotide is the "1' side”
  • the 5'-corresponding side of a DNA polynucleotide that binds to the phosphate is the "3' side”.
  • the 3'-corresponding side is the (S) end
  • the 5'-corresponding side is the (R) end.
  • the "primer region” is a short oligonucleotide that is linked to both ends of the polynucleotide to be amplified and serves as the origin of DNA replication.
  • amplification is usually performed using a pair of short oligonucleotides (forward primer and reverse primer) that have the same sequence as the primer region linked to both ends of the polynucleotide.
  • the primer sequence and the sequence complementary to the primer sequence may be collectively referred to as the "primer region”.
  • the length of the primer region sequence is not particularly limited as long as it is long enough to amplify the target polynucleotide, but can be, for example, about 5 to 22 mer.
  • Linking refers to the joining of the 5' phosphate group of a DNA polynucleotide (e.g., the 3' phosphate group in the case of an L-aTNA polynucleotide) to the 3' hydroxyl group of a polynucleotide (e.g., the 1' hydroxyl group in the case of an L-aTNA polynucleotide), or the 5' hydroxyl group of a DNA polynucleotide (e.g., the 1' hydroxyl group in the case of an L-aTNA polynucleotide) to the 5' phosphate group of a polynucleotide (e.g., the 3' phosphate group in the case of an L-aTNA polynucleotide).
  • a DNA polynucleotide e.g., the 3' phosphate group in the case of an L-aTNA polynucleotide
  • linking refers to both enzymatic linking using DNA ligase, etc., as well as chemical linking in which a phosphorylation reaction is carried out in the presence of N-cyanoimidazole and a divalent cation.
  • Polynucleotide amplification can be performed by known methods such as PCR and LAMP, and is not particularly limited.
  • DNA polynucleotide sequence determination can be performed by known methods such as the Maxam-Gilbert method, the Sanger method, pyrosequencing, and bridge PCR, and is not particularly limited.
  • DNA fragments to both ends of the polynucleotide consisting of a non-cyclic artificial nucleic acid. It is preferable that the portion of this DNA fragment located proximal to the polynucleotide consisting of a non-cyclic artificial nucleic acid has a sequence complementary to the sequence at both ends of the DNA polynucleotide.
  • the length of this sequence is not particularly limited, but it can be preferably 0-6 mer, more preferably 0-3 mer or 1-3 mer.
  • the portion distal to the polynucleotide consisting of a non-cyclic artificial nucleic acid has a sequence complementary to a portion of the primer sequence linked to the DNA polynucleotide that is adjacent to the DNA polynucleotide.
  • the length of the portion complementary to the primer sequence is not particularly limited, but it can be preferably 3-8 mer, more preferably 4-6 mer.
  • FIG. 1 is a diagram illustrating one embodiment of the present invention.
  • a DNA fragment (gray part in the figure) is added to both the 1' end (corresponding to the 3' end of a natural nucleic acid) and the 3' end (corresponding to the 5' end of a natural nucleic acid) of the L-aTNA polynucleotide.
  • This DNA fragment is a part of the primer region linked to both ends of the DNA polynucleotide to be hybridized with the L-aTNA polynucleotide, specifically, the 1' side of the L-aTNA polynucleotide has a sequence complementary to the 3' side sequence of the primer region linked to the 5' end of the DNA polynucleotide, and the 3' side of the L-aTNA polynucleotide has a sequence complementary to the 5' side sequence of the primer region linked to the 3' end of the DNA polynucleotide.
  • the length of the DNA fragment sequence is not particularly limited, but can be preferably 3 to 8 mer, more preferably 4 to 6 mer.
  • the DNA fragment added to the L-aTNA polynucleotide can be labeled for detection, for example with FITC.
  • Hybridization with an L-aTNA polynucleotide is performed using a DNA polynucleotide that does not have primer regions at either end.
  • the DNA fragment added to the L-aTNA polynucleotide specifically consists of sequences complementary to the sequences at either end of the DNA polynucleotide (light gray parts in the figure), a 3'-side sequence of the primer region linked to the 5'-end of the DNA polynucleotide to be hybridized with the L-aTNA polynucleotide, and sequences complementary to the 5'-side sequence of the primer region linked to the 3'-end of the DNA polynucleotide (dark gray parts in the figure).
  • the DNA fragment added to the L-aTNA polynucleotide can be labeled for detection, for example with FITC.
  • the L-aTNA polynucleotide to which the DNA fragment is added as described above is hybridized to a DNA polynucleotide, and then primer regions are linked to both ends of the DNA polynucleotide.
  • the 3' end of the primer region to be linked to the 5' side of the DNA polynucleotide and the 3' end of the DNA polynucleotide are phosphorylated, and the 5' end of the primer region to be linked to the 5' side of the DNA polynucleotide is blocked with 5'DMT (4,4'-dimethoxytrityl).
  • the DNA polynucleotide and the primer region can be chemically linked in the presence of N-cyanoimidazole and divalent cations (e.g., CdCl 2 , MnCl 2 , etc.).
  • N-cyanoimidazole and divalent cations e.g., CdCl 2 , MnCl 2 , etc.
  • ligation of DNA fragments containing primer regions for PCR, etc. can be performed more efficiently.
  • FIG. 3 is a diagram illustrating yet another embodiment of the present invention.
  • a specific sequence common to other DNA polynucleotides containing random sequences is added to both ends of a DNA polynucleotide containing a random sequence (black part in the figure).
  • a DNA fragment is added to both ends of an L-aTNA polynucleotide for ligation.
  • the DNA fragment is composed of a sequence (light gray part in the figure) complementary to the specific sequence (black part in the figure) common to other DNA polynucleotides containing random sequences added to both ends of the DNA polynucleotide containing a random sequence, and a sequence (dark gray part in the figure) complementary to the 3'-side sequence of the primer region linked to the 5'-end of the DNA polynucleotide to be hybridized to the L-aTNA polynucleotide and the 5'-side sequence of the primer region linked to the 3'-end of the DNA polynucleotide.
  • the DNA fragment added to the L-aTNA polynucleotide can be labeled for detection, for example with FITC.
  • a primer region is chemically linked to both ends of a double-stranded DNA polynucleotide formed by hybridization of an L-aTNA polynucleotide and a DNA polynucleotide.
  • a chemical linking method the method described in Nat. Commun., 2021, 12, 804. can be used.
  • the 3' end of the primer region to be linked to the 5' side of the DNA polynucleotide and the 3' end of the DNA polynucleotide are phosphorylated, and the 5' end of the primer region to be linked to the 5' side of the DNA polynucleotide is blocked with 5'DMT (4,4'-dimethoxytrityl).
  • the DNA polynucleotide and the primer region can be chemically linked in the presence of N-cyanoimidazole and a divalent cation (e.g., CdCl 2 , MnCl 2 , etc.). Ligation can be performed more efficiently by linking the primer region to the DNA polynucleotide after hybridization between the L-aTNA polynucleotide and the DNA polynucleotide.
  • a divalent cation e.g., CdCl 2 , MnCl 2 , etc.
  • the DNA polynucleotide contained in the double strand contains a sequence complementary to the L-aTNA polynucleotide.
  • the sequence of the DNA polynucleotide can be obtained by amplifying it by PCR or the like and then determining the sequence of the L-aTNA polynucleotide by a known method.
  • an L-aTNA polynucleotide that binds to a target is selected from a pool of L-aTNA polynucleotides having random sequences. If the sequence of the selected L-aTNA polynucleotide can be non-enzymatically transcribed into a DNA sequence using the method of the present invention, then the desired L-aTNA that binds to the target can be selected by a general nucleic acid amplification technique, such as PCR.
  • the L-aTNA polynucleotide was annealed with a randomized DNA polynucleotide, and then primer regions were ligated to both ends of the DNA polynucleotide, followed by PCR and sequencing.
  • L-aTNA polynucleotide (TNA20 + 3 + 5, final concentration 2.0 nM) was mixed with a DNA polynucleotide in which the portion that hybridizes with the L-aTNA polynucleotide was randomized (DNArandom, final concentration 50 ⁇ M), and annealed to a DNA polynucleotide having a complementary sequence by heating to 95°C for 1 minute, cooling to 56°C for 30 seconds, and then cooling to 4°C.
  • the sequence of TNA20 + 3 + 5 (manufacturer: Hokkaido System Science) is as follows.
  • the italicized parts are the L-aTNA polynucleotide parts, and the non-italicized parts are the DNA polynucleotide parts.
  • the underlined parts of the DNA polynucleotide parts are the sequences complementary to the randomized DNA.
  • DNArandom manufactured by Eurofins Genomics
  • DNArandom 26mer
  • SEQ ID NO: 2 ATT NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN TCA [PHO]-3' (SEQ ID NO: 2) (wherein N represents any DNA nucleotide.
  • the 3mers (underlined) at both ends are complementary to the underlined portions of TNA20 + 3 + 5)
  • ⁇ Step 2 Ligation> The primer sequences for amplification (complement to pColdTF-F1 short and pCold-R shortened_5DMT_3P, each at a final concentration of 1.0 ⁇ M), an aqueous NaCl solution (Wako Pure Chemical Industries, for molecular biology) (final concentration 100 mM), and an aqueous CdCl 2 (Wako Pure Chemical Industries) or MnCl 2 (Wako Pure Chemical Industries) solution (final concentration 20 mM) were added to the annealing solution, and the solution was made up to 27.0 ⁇ l with MilliQ water. The solution was vortexed for a few seconds and incubated in a heat block at 25°C for at least 3 minutes.
  • PCR ⁇ Step 3 PCR>
  • the collected samples were buffer exchanged using Micro Bio-Spin 6 Columns (BioRad) and eluted in MilliQ water according to the manufacturer's protocol.
  • the eluate was diluted 10-3 times and subjected to PCR.
  • PCR was performed using 0.4 ⁇ M primers (pCold-R Primer shortened, pColdTF-F1 Primer shortened), 3.0 ⁇ l of 10-3 times eluate (total volume 25 ⁇ l), annealing temperature 55°C, and 40 cycles.
  • Rf a 63 nt full-length DNA (Reference DNA) was PCRed under the same conditions.
  • the PCR products were analyzed by 12% PAGE using 8M Urea (Wako Pure Chemical Industries, Ltd.).
  • a 10 bp DNA ladder (Promega, Ltd.) was used as a marker.
  • the sequences of primer1-DNA20-primer2 (manufacturer: Eurofins Genomics) and TNA-20 (manufacturer: Hokkaido System Science) are as follows:
  • Electrophoresis was performed at 20°C using 5% glycerol 20% polyacrylamide gels.
  • the lane configuration is as follows: 1. Sample annealed by mixing 0.5 ⁇ l of DNA20 (1 ⁇ M) and 2.0 ⁇ l of H2O 2. Sample annealed by mixing 0.5 ⁇ l of TNA-20 (1 ⁇ M) and 2.0 ⁇ l of H2O 3. Sample annealed at 50°C 4. Sample annealed at 52°C 5. Sample annealed at 54°C 6. Sample annealed at 56°C 7. Sample annealed at 58°C 8. Sample annealed at 60°C 9. Sample annealed at 62°C 10. Sample annealed at 64°C
  • DNA polynucleotides having a sequence complementary to L-aTNA were mixed with L-aTNA polynucleotides (TNA20 + 0 + 5, TNA20 + 1 + 5, or TNA20 + 3 + 5, final concentration 2.0 nM) or DNA polynucleotides (TNA20+0 (all DNA)), heated to 95°C for 1 minute, cooled to 56°C for 30 seconds, and then cooled to 4°C.
  • L-aTNA polynucleotide manufactured by Hokkaido System Science
  • DNA polynucleotide manufactured by Eurofins Genomics
  • the italics indicate the L-aTNA polynucleotide portion, and the non-italicized portion indicates the DNA polynucleotide portion.
  • the underlined portion of the DNA polynucleotide portion is the sequence complementary to the DNA polynucleotide.
  • the DNA polynucleotide used was either DNA20+0_3P, DNA20+1_3P, or DNA20+3_3P (manufacturer: Eurofins Genomics), which has a sequence complementary to the DNA added to the end of the L-aTNA polynucleotide (0 mer, 1 mer, or 3 mer at each end).
  • annealing solution was added with the same primer region as used in Example 1 (complement to pColdTF-F1 short and pCold-R shortened_5DMT_3P, each final concentration 1.0 ⁇ M), NaCl aqueous solution (final concentration 100 mM), and CdCl2 or MnCl2 aqueous solution (final concentration 20 mM), and the solution was made up to 27.0 ⁇ l with MilliQ water. The mixture was stirred with a vortex for a few seconds and incubated in a heat block at 25 ° C for 3 minutes or more.
  • PCR PCR was performed under the reaction conditions of the same primers as in Example 1 (pCold-R Primer shortened, pColdTF-F1 Primer shortened) at 0.4 ⁇ M each, 3.0 ⁇ l of 10-3 times eluate (total volume 25 ⁇ l), annealing temperature 55°C, and 40 cycles.
  • the PCR product was analyzed by 8M urea 12% PAGE. A 10 bp DNA ladder was used as a marker.
  • Rf a reference

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Abstract

[Problem] To provide a method for obtaining a sequence for a nucleic acid including an artificial nucleic acid, more specifically a method for amplifying a sequence for a nucleic acid including a non-cyclic artificial nucleic acid to amplify the sequence. [Solution] Provided is a method for obtaining a sequence for a polynucleotide comprising a non-cyclic artificial nucleic acid, the method comprising: a step of hybridizing a polynucleotide comprising a non-cyclic artificial nucleic acid having DNA fragments respectively attached to both ends thereof to a DNA polynucleotide pool including random sequences; a step of chemically linking a primer region to each of the both ends of the DNA polynucleotide; a step of determining the sequence for the DNA polynucleotide; and a step of determining the sequence for the polynucleotide comprising the non-cyclic artificial nucleic acid on the basis of the sequence for the DNA polynucleotide.

Description

非環状型人工核酸の配列取得法Method for obtaining sequences of non-circular artificial nucleic acids
　本発明は、人工核酸の配列の取得法に関する。より詳しくは、非環状型の人工核酸の配列に相補なDNA配列を増幅させてその配列を解読する方法に関する。 The present invention relates to a method for obtaining the sequence of an artificial nucleic acid. More specifically, the present invention relates to a method for amplifying a DNA sequence complementary to the sequence of a non-circular artificial nucleic acid and decoding the sequence.
　核酸塩基・糖・リン酸ジエステル部に化学修飾を加えて物性を変化させた核酸は、人工核酸と称される。人工核酸は、核酸分解酵素に対する耐性をもち、分解されにくい等の天然の核酸には無い人工的な機能を有する。人工核酸は、天然核酸であるDNAやRNAと同様に、塩基部分をアデニン（A)、グアニン（G)、シトシン（C)、チミン（T)、ウラシル（U)などとすることが可能である。そのため、ゲノム配列決定や遺伝子検出、配列選択的な遺伝子抑制に加え、化合物ライブラリーの作成基盤材料や医薬品素材として利用価値が広く、新たなモダリティとして期待されている。 Nucleic acids whose physical properties have been changed by chemically modifying the nucleic acid base, sugar, or phosphate diester moiety are called artificial nucleic acids. Artificial nucleic acids have artificial functions not found in natural nucleic acids, such as resistance to nucleases and being difficult to decompose. Like natural nucleic acids such as DNA and RNA, the base moiety of artificial nucleic acids can be adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), etc. Therefore, in addition to genome sequencing, gene detection, and sequence-selective gene suppression, they have a wide range of usefulness as a base material for creating compound libraries and as pharmaceutical materials, and are expected to become a new modality.
　人工核酸の中でもDNA又はRNAと二重鎖を形成することができる人工核酸として、L-acyclic Threoninol Nucleic Acid (L-aTNA)、Serinol Nucleic Acid(SNA)、Locked Nucleic Acid(LNA)、Peptide Nucleic Acid (PNA)、Glycol Nucleic Acid (GNA）等が知られている（特許文献１）。 Among the artificial nucleic acids, L-acyclic Threoninol Nucleic Acid (L-aTNA), Serinol Nucleic Acid (SNA), Locked Nucleic Acid (LNA), Peptide Nucleic Acid (PNA), Glycol Nucleic Acid (GNA), etc. are known as artificial nucleic acids that can form double strands with DNA or RNA (Patent Document 1).
　特に、天然核酸の主鎖構造を非環状骨格に改変した人工核酸であるL-aTNA及びSNAは、天然のDNAやRNAと二重鎖を形成することができる一方（非特許文献１）、血中で分解され易い従来の核酸医薬と比べ、ヌクレアーゼに対する耐性が高く、体内で分解されてもアミノ酸とリン酸になるため、副作用がないか、極めて少ない。そのため、siRNA、anti-miRNA oligo(AMO)、アンチセンスオリゴ(ASO)等の技術に応用可能であると期待されている。 In particular, L-aTNA and SNA, which are artificial nucleic acids in which the main chain structure of natural nucleic acids has been modified to an acyclic backbone, can form double strands with natural DNA and RNA (Non-Patent Document 1), while being highly resistant to nucleases compared to conventional nucleic acid drugs that are easily degraded in the blood, and even when degraded in the body, they are reduced to amino acids and phosphates, meaning that they have no or very few side effects. For this reason, they are expected to be applicable to technologies such as siRNA, anti-miRNA oligo (AMO), and antisense oligo (ASO).
　ポリヌクレオチドの末端が人工核酸である場合、その末端を他のポリヌクレオチドと連結させるためには、天然核酸の連結に用いるリガーゼのような酵素を用いることができない。そのため、例えば、人工核酸がL-aTNA又はSNAのときには、イミダゾール及びハロゲン化シアン、及び／又はN－シアノイミダゾールの存在下で、人工核酸を含むポリヌクレオチドと他のポリヌクレオチドを反応させることにより、両者を効率的に連結させることができる（特許文献２）。 When the end of a polynucleotide is an artificial nucleic acid, enzymes such as ligases used to link natural nucleic acids cannot be used to link the end to another polynucleotide. For this reason, for example, when the artificial nucleic acid is L-aTNA or SNA, the two can be efficiently linked by reacting a polynucleotide containing an artificial nucleic acid with another polynucleotide in the presence of imidazole and cyanogen halide, and/or N-cyanoimidazole (Patent Document 2).
　上述のとおり、天然核酸同様に、人工核酸も塩基部分をA、G、C、T又はUとして、DNAやRNAとハイブリダイゼーションすることも理論的には可能である。また、L-aTNAと L-aTNAとの間の相補鎖におけるハイブリダイゼーションも可能である。 As mentioned above, like natural nucleic acids, it is theoretically possible for artificial nucleic acids to hybridize with DNA or RNA when the base moiety is A, G, C, T, or U. Hybridization between complementary strands of L-aTNA and L-aTNA is also possible.
特開２０１６－１３０２３２号公報JP 2016-130232 A 特開２０１９－１３７６２８号公報JP 2019-137628 A
　人工核酸からなるポリヌクレオチドは、DNAポリヌクレオチドのように市販のシーケンサーで配列を解析することができない。上述のとおり、L-aTNAのような非環状型人工核酸からなるポリヌクレオチドを、相補的な配列を有する非環状型人工核酸からなるポリヌクレオチドと二重鎖を形成させることは可能であるが、両末端に増幅用のプライマー領域を有するDNAポリヌクレオチドを、非環状型人工核酸からなるポリヌクレオチドにハイブリダイゼーションさせた場合には、二重鎖が形成されにくい傾向がある。そのため、非環状型人工核酸からなるポリヌクレオチドの塩基配列を、当該非環状型人工核酸からなるポリヌクレオチドと相補的なDNAポリヌクレオチドを用いて配列決定することが困難であった。 The sequence of a polynucleotide made of an artificial nucleic acid cannot be analyzed with a commercially available sequencer, unlike a DNA polynucleotide. As mentioned above, it is possible to form a double strand with a polynucleotide made of a non-cyclic artificial nucleic acid such as L-aTNA and a polynucleotide made of a non-cyclic artificial nucleic acid having a complementary sequence. However, when a DNA polynucleotide having amplification primer regions at both ends is hybridized to a polynucleotide made of a non-cyclic artificial nucleic acid, it tends not to form a double strand. Therefore, it has been difficult to determine the base sequence of a polynucleotide made of a non-cyclic artificial nucleic acid using a DNA polynucleotide complementary to the polynucleotide made of the non-cyclic artificial nucleic acid.
　例えば、特定の標的に結合するアプタマーとして使用可能な非環状型人工核酸からなるポリヌクレオチドを選択しても、その配列をDNA配列に変換する方法が確立されていないため、その配列情報を得ることができなかった。そのため、非環状型人工核酸からなるポリヌクレオチドの配列決定を行う方法が望まれていた。 For example, even if a polynucleotide consisting of an acyclic artificial nucleic acid that can be used as an aptamer that binds to a specific target is selected, sequence information cannot be obtained because a method for converting the sequence into a DNA sequence has not been established. Therefore, a method for determining the sequence of a polynucleotide consisting of an acyclic artificial nucleic acid is desired.
　そこで、本発明者らは鋭意検討し、非環状型人工核酸からなるポリヌクレオチドの両末端に数塩基のDNA断片を付加させることによって、DNAポリヌクレオチドとプライマー領域との化学的な連結の効率が向上することを見出した。この数塩基のDNA断片は、非環状型人工核酸からなるポリヌクレオチドとハイブリダイゼーションさせるDNAポリヌクレオチドの両末端におけるDNA増幅用のプライマー配列の一部、又は、ハイブリダイゼーションさせるDNAポリヌクレオチドの両末端の配列及びDNA増幅用のプライマー配列の一部に対して相補的である。 The inventors have therefore conducted extensive research and found that the efficiency of chemical linkage between a DNA polynucleotide and a primer region can be improved by adding a DNA fragment of several bases to both ends of a polynucleotide consisting of a non-cyclic artificial nucleic acid. This DNA fragment of several bases is complementary to a portion of the primer sequence for DNA amplification at both ends of the DNA polynucleotide to be hybridized with the polynucleotide consisting of a non-cyclic artificial nucleic acid, or to the sequences at both ends of the DNA polynucleotide to be hybridized and a portion of the primer sequence for DNA amplification.
　さらに、本発明者らは、非環状型人工核酸からなるポリヌクレオチドとDNAポリヌクレオチドをハイブリダイゼーションした後に、DNAポリヌクレオチドの両末端にプライマー領域を連結させることによって、DNAポリヌクレオチドとプライマー領域の化学的な連結の効率が向上することを見出した。 Furthermore, the inventors discovered that the efficiency of chemically linking the DNA polynucleotide to the primer region can be improved by hybridizing a polynucleotide consisting of a non-cyclic artificial nucleic acid with a DNA polynucleotide and then linking primer regions to both ends of the DNA polynucleotide.
　本発明は、非環状型人工核酸からなるポリヌクレオチドと、ランダム配列を含むDNAポリヌクレオチドのプールとをハイブリダイゼーションさせる工程と、
　ここで、前記DNAポリヌクレオチドは、ランダム配列の両末端に、前記プールに含まれるDNAポリヌクレオチドに共通する配列が付加されており、
　前記非環状型人工核酸からなるポリヌクレオチドは、両末端にDNA断片が付加されており、前記DNA断片は、
　１）前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に、前記DNAポリヌクレオチドの5'末端に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の3'側の配列、及び、前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に、前記DNAポリヌクレオチドの3'末端側に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の5'側の配列にそれぞれ相補的な配列、又は、
　２）i)前記非環状型人工核酸からなるポリヌクレオチドの3'相当側及び5'相当側に、前記DNAポリヌクレオチドの前記ランダム配列の両末端に付加された前記共通する配列にそれぞれ相補的な配列、並びに、
　ii) 前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に付加された前記共通する配列に相補的な配列の3'側に、前記DNAポリヌクレオチドの5'末端に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の3'側の配列に相補的な配列、及び、
　前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に付加された前記共通する配列に相補的な配列の5'側に、前記DNAポリヌクレオチドの3'末端側に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の5'側の配列に相補的な配列、
を含み、
　前記DNAポリヌクレオチドからDNAを増幅する工程と、
　前記DNAポリヌクレオチドの配列を決定する工程と、
　前記DNAポリヌクレオチドの配列から非環状型人工核酸からなるポリヌクレオチドの配列を決定する工程と
を含む、非環状型人工核酸からなるポリヌクレオチドの配列を取得する方法を提供する。 The present invention relates to a method for producing a polynucleotide sequence comprising the steps of: hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences;
wherein the DNA polynucleotide has a sequence common to the DNA polynucleotides contained in the pool added to both ends of a random sequence,
The polynucleotide consisting of the non-cyclic artificial nucleic acid has DNA fragments added to both ends, and the DNA fragments are
1) a sequence complementary to the 3' side of a primer region that is linked to the 5' end of the DNA polynucleotide or to the DNA polynucleotide in a later step on the 3' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, and a sequence complementary to the 5' side of a primer region that is linked to the 3' end of the DNA polynucleotide or to the DNA polynucleotide in a later step on the 5' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, or
2) i) sequences complementary to the common sequences added to both ends of the random sequence of the DNA polynucleotide on the 3'-side and 5'-side of the polynucleotide consisting of the non-circular artificial nucleic acid, respectively; and
ii) a sequence complementary to the 3' side sequence of a primer region that is linked to the 5' end of the DNA polynucleotide or to the DNA polynucleotide in a later step, on the 3' side of the sequence complementary to the common sequence added to the 3' corresponding side of the polynucleotide consisting of the non-circular artificial nucleic acid; and
a sequence complementary to the 5'-side sequence of a primer region that is linked to the 3'-end of the DNA polynucleotide or that is linked to the DNA polynucleotide in a later step, on the 5'-side of the sequence complementary to the common sequence added to the 5'-side of the polynucleotide consisting of the non-circular artificial nucleic acid;
Including,
amplifying DNA from said DNA polynucleotide;
determining the sequence of the DNA polynucleotide;
determining the sequence of a polynucleotide consisting of a non-cyclic artificial nucleic acid from the sequence of the DNA polynucleotide.
　さらに、本発明は、非環状型人工核酸からなるポリヌクレオチドとランダム配列を含むDNAポリヌクレオチドのプールとをハイブリダイゼーションさせる工程と、
　前記DNAポリヌクレオチドの両端にプライマー領域を連結させる工程と、
　前記DNAポリヌクレオチドからDNAを増幅する工程と、
　前記DNAポリヌクレオチドの配列を決定する工程と、
　前記DNAポリヌクレオチドの配列から非環状型人工核酸からなるポリヌクレオチドの配列を決定する工程と
を含む、非環状型人工核酸からなるポリヌクレオチドの配列を取得する方法を提供する。 Furthermore, the present invention provides a method for producing a nucleic acid sequence comprising the steps of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences;
ligating primer regions to both ends of the DNA polynucleotide;
amplifying DNA from said DNA polynucleotide;
determining the sequence of the DNA polynucleotide;
determining the sequence of a polynucleotide consisting of a non-cyclic artificial nucleic acid from the sequence of the DNA polynucleotide.
　さらに、本発明は、非環状型人工核酸からなるポリヌクレオチドとランダム配列を含むDNAポリヌクレオチドのプールとをハイブリダイゼーションさせる工程と、
　ここで、前記DNAポリヌクレオチドは、ランダム配列の両末端に、前記プールに含まれるDNAポリヌクレオチドに共通する配列が付加されており、
　前記非環状型人工核酸からなるポリヌクレオチドは、両末端にDNA断片が付加されており、前記DNA断片は、
　１）前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に、前記DNAポリヌクレオチドの5'末端に連結されるプライマー領域の3'側の配列、及び、前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に、前記DNAポリヌクレオチドの3'末端に連結されるプライマー領域の5'側の配列にそれぞれ相補的な配列、又は、
　２）i) 前記非環状型人工核酸からなるポリヌクレオチドの3'相当側及び5'相当側に、前記DNAポリヌクレオチドの前記ランダム配列の両末端に付加された前記共通する配列にそれぞれ相補的な配列、並びに、
　ii) 前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に付加された前記共通する配列に相補的な配列の3'側に、前記DNAポリヌクレオチドの5'末端に連結されるプライマー領域の3'側の配列、及び、
　前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に付加された前記共通する配列に相補的な配列の5'側に、前記DNAポリヌクレオチドの3'末端側に連結されるプライマー領域の5'側の配列にそれぞれ相補的な配列、
を含み、
　DNAポリヌクレオチドの両端にプライマー領域を連結させる工程と、
　前記DNAポリヌクレオチドの配列を決定する工程と、
　前記DNAポリヌクレオチドの配列から非環状型人工核酸からなるポリヌクレオチドの配列を決定する工程と
を含む、非環状型人工核酸からなるポリヌクレオチドの配列を取得する方法を提供する。 Furthermore, the present invention provides a method for producing a nucleic acid sequence comprising the steps of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences;
wherein the DNA polynucleotide has a sequence common to the DNA polynucleotides contained in the pool added to both ends of a random sequence,
The polynucleotide consisting of the non-cyclic artificial nucleic acid has DNA fragments added to both ends, and the DNA fragments are
1) a sequence complementary to the 3' side of the primer region linked to the 5' end of the DNA polynucleotide on the 3' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, and a sequence complementary to the 5' side of the primer region linked to the 3' end of the DNA polynucleotide on the 5' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, or
2) i) sequences complementary to the common sequences added to both ends of the random sequence of the DNA polynucleotide on the 3'-side and 5'-side of the polynucleotide consisting of the non-circular artificial nucleic acid, respectively; and
ii) a sequence on the 3' side of a sequence complementary to the common sequence added to the 3' corresponding side of the polynucleotide consisting of the non-circular artificial nucleic acid, the sequence being a primer region linked to the 5' end of the DNA polynucleotide; and
a sequence complementary to the 5' side of a sequence complementary to the common sequence added to the 5' corresponding side of the polynucleotide consisting of the non-circular artificial nucleic acid, and a sequence complementary to the 5' side sequence of a primer region linked to the 3' end of the DNA polynucleotide;
Including,
ligating primer regions to both ends of a DNA polynucleotide;
determining the sequence of the DNA polynucleotide;
determining the sequence of a polynucleotide consisting of a non-cyclic artificial nucleic acid from the sequence of the DNA polynucleotide.
　本発明において、非環状型人工核酸として、L-aTNA、SNA又はPNAを用いることができる。 In the present invention, L-aTNA, SNA, or PNA can be used as the acyclic artificial nucleic acid.
　また、本発明におけるDNAポリヌクレオチドの両端へのプライマー領域の連結は、Nーシアノイミダゾール及び２価陽イオンの存在下にてリン酸反応を行う化学的な連結が望ましい。さらに、２価陽イオンとして、カドミウムイオン又はマンガンイオンを用いるのが望ましい。またNーシアノイミダゾール以外に、イミダゾールとブロモシアン（Br-CN）を活性化剤として使用することもできる。 In addition, in the present invention, the primer regions are preferably linked to both ends of the DNA polynucleotide by chemical linkage in the presence of N-cyanoimidazole and a divalent cation through a phosphoric acid reaction. Furthermore, it is preferable to use cadmium ions or manganese ions as the divalent cation. In addition to N-cyanoimidazole, imidazole and cyanogen bromide (Br-CN) can also be used as an activator.
　本発明により、所望の非環状型人工核酸からなるポリヌクレオチドの配列をDNAポリヌクレオチドの配列に相補的な配列として移し、PCR等の公知の方法により増幅して、効率よく配列を決定することができる。特に、非環状型人工核酸からなるポリヌクレオチドの配列決定を直接行うのではなく、配列決定法が確立されているために容易に扱うことが可能なDNAとして配列決定することができるため、例えばアプタマー等として結合性の高い等、有用な非環状型人工核酸からなるポリヌクレオチドを、より効率良く、迅速に配列決定することができる。 　According to the present invention, the sequence of a polynucleotide consisting of a desired non-cyclic artificial nucleic acid can be transferred to the sequence of a DNA polynucleotide as a complementary sequence, and then amplified by a known method such as PCR, thereby enabling efficient sequence determination. In particular, since the sequence of a polynucleotide consisting of a non-cyclic artificial nucleic acid is not determined directly, but can be determined as DNA, which can be easily handled because a sequencing method has been established, it is possible to more efficiently and quickly sequence a polynucleotide consisting of a useful non-cyclic artificial nucleic acid, for example, one that has high binding properties as an aptamer, etc.
L-aTNAポリヌクレオチドの両末端にDNA断片が付加されている本発明の一態様を説明する図である。FIG. 1 is a diagram illustrating an embodiment of the present invention in which DNA fragments are added to both ends of an L-aTNA polynucleotide. 両末端にプライマー領域を持たないDNAポリヌクレオチドを用いてL-aTNAポリヌクレオチドとのハイブリダイゼーションを行い、ハイブリダイゼーションの後にDNAポリヌクレオチドにプライマー領域を化学的に連結させる本発明の一態様を説明する図である。FIG. 1 is a diagram illustrating one embodiment of the present invention in which a DNA polynucleotide having no primer regions at either end is used to hybridize with an L-aTNA polynucleotide, and a primer region is chemically linked to the DNA polynucleotide after hybridization. ランダム配列を含むDNAポリヌクレオチドを用いた、L-aTNAポリヌクレオチドの配列決定方法を示す本発明のさらに別の態様を説明する図である。FIG. 1 illustrates yet another embodiment of the present invention showing a method for sequencing an L-aTNA polynucleotide using a DNA polynucleotide containing a random sequence. 実施例１の結果を示す図である。FIG. 1 shows the results of Example 1. 実施例２の結果を示す図である。FIG. 13 shows the results of Example 2. 実施例３の結果を示す図である。FIG. 13 shows the results of Example 3.
　以下に本発明を、必要に応じて、添付の図面を参照して説明する。本明細書において使用される用語は、特に言及しない限り、当該分野で通常用いられる意味で用いられることが理解されるべきである。したがって、他に定義されない限り、本明細書中で使用される全ての専門用語および科学技術用語は、本発明の属する分野の当業者によって一般的に理解されるのと同じ意味を有する。本明細書では、単数形の表現は、特に言及しない限り、複数の場合をも含み得る。 The present invention will now be described with reference to the accompanying drawings, if necessary. It should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In this specification, the singular form may also include the plural unless otherwise specified.
　本明細書では、「非環状型人工核酸」とは、D-aTNA、L-aTNA及びSNAのような、天然核酸の主鎖構造を非環状骨格に改変した人工核酸をいう。本発明では、非環状型人工核酸として、L-aTNA、SNA及びPNAが好ましく、L-aTNA及びSNAが特に好ましく、L-aTNAが最も好ましい。L-aTNAとSNAは、DNA又はRNAと二重鎖を形成することができ、特定の理論にとらわれるわけではないが、環状構造を含まない鎖状構造であることに起因する柔軟な構造を共通して有し、両者は同様の方法を用いて他のポリヌクレオチドと連結することができる。また、L-aTNAやSNAの場合と同様な方法を用いて、PNAも他のポリヌクレオチドと連結することが可能である。下記にL-aTNA及びSNAの構造を示す。 In this specification, "non-cyclic artificial nucleic acid" refers to an artificial nucleic acid in which the main chain structure of a natural nucleic acid is modified to an acyclic backbone, such as D-aTNA, L-aTNA, and SNA. In the present invention, as the non-cyclic artificial nucleic acid, L-aTNA, SNA, and PNA are preferred, L-aTNA and SNA are particularly preferred, and L-aTNA is the most preferred. L-aTNA and SNA can form a double strand with DNA or RNA, and, without being bound by a specific theory, they share a flexible structure due to being a chain structure that does not contain a cyclic structure, and both can be linked to other polynucleotides using the same method. In addition, PNA can also be linked to other polynucleotides using the same method as in the case of L-aTNA and SNA. The structures of L-aTNA and SNA are shown below.
Figure JPOXMLDOC01-appb-C000001
 （式中、Baseは、アデニン、チミン、シトシン、グアニン、ウラシル等の塩基を表す。）
Figure JPOXMLDOC01-appb-C000001
 (In the formula, Base represents a base such as adenine, thymine, cytosine, guanine, or uracil.)
Figure JPOXMLDOC01-appb-C000002
 （式中、Baseは、アデニン、チミン、シトシン、グアニン、ウラシル等の塩基を表す。）
Figure JPOXMLDOC01-appb-C000002
 (In the formula, Base represents a base such as adenine, thymine, cytosine, guanine, or uracil.)
　「ポリヌクレオチド」とは、複数のヌクレオチドモノマーが共有結合した高分子をいう。本明細書では、ポリヌクレオチドは、リン酸、五炭糖、塩基からなる天然のヌクレオチドをモノマーとするものだけでなく、上述のとおり主鎖構造を非環状骨格に改変したヌクレオチドを含むものもポリヌクレオチドと称する。「DNAポリヌクレオチド」とは、デオキシリボ核酸をモノマーとして構成されるポリヌクレオチドをいう。「非環状型人工核酸からなるポリヌクレオチド」とは、非環状型人工核酸をモノマーとして構成されるポリヌクレオチドをいう。例えば、「L-aTNAポリヌクレオチド」とは、L-aTNAをモノマーとして構成されるポリヌクレオチドをいう。 "Polynucleotide" refers to a polymer in which multiple nucleotide monomers are covalently bonded. In this specification, polynucleotides are not limited to those whose monomers are natural nucleotides consisting of phosphate, pentose, and bases, but also include those containing nucleotides whose main chain structure has been modified to an acyclic backbone, as described above. "DNA polynucleotide" refers to a polynucleotide composed of deoxyribonucleic acid as a monomer. "Polynucleotide composed of acyclic artificial nucleic acid" refers to a polynucleotide composed of acyclic artificial nucleic acid as a monomer. For example, "L-aTNA polynucleotide" refers to a polynucleotide composed of L-aTNA as a monomer.
　「ハイブリダイゼーション」とは、ヌクレオチドの塩基の相補性に基づき、２つのポリヌクレオチドが水素結合により結合することをいう。本明細書における「ハイブリダイゼーション」は、DNAポリヌクレオチド同士の相補鎖の結合や、DNAポリヌクレオチドとRNAポリヌクレオチドの間の相補鎖の結合のみならず、人工核酸からなるポリヌクレオチド同士の間の相補鎖の結合や、DNAポリヌクレオチド又はRNAポリヌクレオチドと人工核酸からなるポリヌクレオチドとの間の相補鎖の結合も含む。本明細書では、用語「ハイブリダイゼーション」は、場合により「アニーリング」とも称する。 "Hybridization" refers to the bonding of two polynucleotides through hydrogen bonds based on the complementarity of nucleotide bases. In this specification, "hybridization" includes not only the bonding of complementary strands between DNA polynucleotides or between a DNA polynucleotide and an RNA polynucleotide, but also the bonding of complementary strands between polynucleotides made of artificial nucleic acids, and the bonding of complementary strands between a DNA polynucleotide or an RNA polynucleotide and a polynucleotide made of artificial nucleic acid. In this specification, the term "hybridization" is sometimes also referred to as "annealing."
　ランダム配列を含むDNAポリヌクレオチドのプールとは、様々な配列の組み合わせを持つ無数のDNAポリヌクレオチドの混合物をいう。 A pool of DNA polynucleotides containing random sequences refers to a mixture of an infinite number of DNA polynucleotides with various sequence combinations.
　本発明の一態様によれば、DNAポリヌクレオチドは、ランダム配列の両末端に、前記プールに含まれる全DNAポリヌクレオチドに共通する配列が付加されている。この共通する配列は、長さ及び配列ともに任意に決定することができる。例えば、2～5mer程度の長さの任意の配列を前記共通する配列とすることができる。
　この前記共通する配列に相補的な配列を、非環状型人工核酸からなるポリヌクレオチドの両末端にそれぞれ付加させることにより、DNAポリヌクレオチドと非環状型人工核酸からなるポリヌクレオチドとの間でより効率よくハイブリダイゼーションを行うことができる。 According to one aspect of the present invention, the DNA polynucleotide has a sequence common to all DNA polynucleotides contained in the pool added to both ends of a random sequence. The length and sequence of this common sequence can be determined arbitrarily. For example, the common sequence can be any sequence having a length of about 2 to 5 mers.
By adding a sequence complementary to the common sequence to each end of a polynucleotide consisting of a non-cyclic artificial nucleic acid, hybridization can be achieved more efficiently between a DNA polynucleotide and a polynucleotide consisting of a non-cyclic artificial nucleic acid.
　本明細書では、DNA及びRNAポリヌクレオチドの5'側及び3'側のみならず、非環状型人工核酸からなるポリヌクレオチドにおけるDNA及びRNAポリヌクレオチドの5'側及び3'側に対応する部分も含め、特に断りがない限り、DNAポリヌクレオチドの表記に合わせて、それぞれ、ポリヌクレオチドの「5'相当側」及び「3'相当側」（又は単に「5'側」及び「3'側」）と称する。例えば、L-aTNAポリヌクレオチドの場合、隣接するヌクレオチドのリン酸基と結合する、3'相当側は「1'側」であり、リン酸と結合しているDNAポリヌクレオチドの5'相当側は「3'側」である。また、SNAポリヌクレオチドの場合、3'相当側は(S)端であり、5'相当側は(R)端である。 In this specification, unless otherwise specified, not only the 5' and 3' sides of DNA and RNA polynucleotides, but also the parts of polynucleotides consisting of non-cyclic artificial nucleic acids that correspond to the 5' and 3' sides of DNA and RNA polynucleotides are referred to as the "5'-corresponding side" and "3'-corresponding side" of the polynucleotide (or simply the "5' side" and "3' side") in accordance with the notation of DNA polynucleotides. For example, in the case of an L-aTNA polynucleotide, the 3'-corresponding side that binds to the phosphate group of the adjacent nucleotide is the "1' side", and the 5'-corresponding side of a DNA polynucleotide that binds to the phosphate is the "3' side". In addition, in the case of an SNA polynucleotide, the 3'-corresponding side is the (S) end, and the 5'-corresponding side is the (R) end.
　「プライマー領域」は、増幅させるポリヌクレオチドの両末端に連結させた、DNA複製の起点となる短鎖オリゴヌクレオチドである。PCR法では、通常は、ポリヌクレオチドの両末端に連結させたプライマー領域と同じ配列を有する1組の短鎖オリゴヌクレオチド（フォワードプライマーとリバースプライマー）を用いて増幅を行う。本明細書では、プライマー配列と、プライマー配列に相補的な配列をまとめて「プライマー領域」と称する場合がある。プライマー領域の配列の長さは、目的とするポリヌクレオチドが増幅できる長さであれば特に限定されないが、例えば5～22mer程度とすることができる。 The "primer region" is a short oligonucleotide that is linked to both ends of the polynucleotide to be amplified and serves as the origin of DNA replication. In PCR, amplification is usually performed using a pair of short oligonucleotides (forward primer and reverse primer) that have the same sequence as the primer region linked to both ends of the polynucleotide. In this specification, the primer sequence and the sequence complementary to the primer sequence may be collectively referred to as the "primer region". The length of the primer region sequence is not particularly limited as long as it is long enough to amplify the target polynucleotide, but can be, for example, about 5 to 22 mer.
　「連結」、「ライゲーション」及び「付加」とは、DNAポリヌクレオチドの5' リン酸基（例えば、L-aTNAポリヌクレオチドの場合には3'リン酸基）と、ポリヌクレオチドの3' 水酸基（例えば、L-aTNAポリヌクレオチドの場合には1'水酸基）、あるいはDNAポリヌクレオチドの5' 水酸基（例えば、L-aTNAポリヌクレオチドの場合には1'水酸基）と、ポリヌクレオチドの5' リン酸基（例えば、L-aTNAポリヌクレオチドの場合には3'リン酸基）を結合させることをいう。本明細書では、「連結」とは、DNAリガーゼ等を用いる酵素的な連結に加え、Nーシアノイミダゾール及び２価陽イオンの存在下でリン酸化反応を行う化学的な連結の両方を指す。 "Linking," "ligation," and "addition" refer to the joining of the 5' phosphate group of a DNA polynucleotide (e.g., the 3' phosphate group in the case of an L-aTNA polynucleotide) to the 3' hydroxyl group of a polynucleotide (e.g., the 1' hydroxyl group in the case of an L-aTNA polynucleotide), or the 5' hydroxyl group of a DNA polynucleotide (e.g., the 1' hydroxyl group in the case of an L-aTNA polynucleotide) to the 5' phosphate group of a polynucleotide (e.g., the 3' phosphate group in the case of an L-aTNA polynucleotide). In this specification, "linking" refers to both enzymatic linking using DNA ligase, etc., as well as chemical linking in which a phosphorylation reaction is carried out in the presence of N-cyanoimidazole and a divalent cation.
　ポリヌクレオチドの増幅は、PCR法、LAMP法等の公知の方法で行うことができ、特に限定されない。DNAポリヌクレオチドの配列の決定は、特に限定されないが、マクサム・ギルバート法、サンガー法、パイロシークエンス法、ブリッジPCR法など、公知の方法により行うことができる。 Polynucleotide amplification can be performed by known methods such as PCR and LAMP, and is not particularly limited. DNA polynucleotide sequence determination can be performed by known methods such as the Maxam-Gilbert method, the Sanger method, pyrosequencing, and bridge PCR, and is not particularly limited.
　ここで、非環状型人工核酸からなるポリヌクレオチドの両末端には、DNA断片を付加するのが望ましい。このDNA断片のうち、非環状型人工核酸からなるポリヌクレオチドの近位に位置する部分は、DNAポリヌクレオチドの両末端の配列にそれぞれ相補的な配列とすることが望ましい。この配列の長さは、特に限定されないが、好ましくは0～6mer、より好ましくは0～3mer又は1～3merとすることができる。さらに、非環状型人工核酸からなるポリヌクレオチドから遠位の部分には、DNAポリヌクレオチドに連結させるプライマー配列のうち、前記DNAポリヌクレオチドに近接した部分に相補する配列とすることが望ましい。このプライマー配列に相補する部分の配列の長さは、特に限定されないが、好ましくは3～8mer、より好ましくは4～6merとすることができる。 Here, it is preferable to add DNA fragments to both ends of the polynucleotide consisting of a non-cyclic artificial nucleic acid. It is preferable that the portion of this DNA fragment located proximal to the polynucleotide consisting of a non-cyclic artificial nucleic acid has a sequence complementary to the sequence at both ends of the DNA polynucleotide. The length of this sequence is not particularly limited, but it can be preferably 0-6 mer, more preferably 0-3 mer or 1-3 mer. Furthermore, it is preferable that the portion distal to the polynucleotide consisting of a non-cyclic artificial nucleic acid has a sequence complementary to a portion of the primer sequence linked to the DNA polynucleotide that is adjacent to the DNA polynucleotide. The length of the portion complementary to the primer sequence is not particularly limited, but it can be preferably 3-8 mer, more preferably 4-6 mer.
　以下に図面を用いて、非環状型人工核酸としてL-aTNAを用いた例に関して本発明を説明するが、この説明は本発明を限定することを意図するものではない。例えば、例えばSNAやPNAのような他の非環状型核酸を用いた場合にも以下に説明するとおり同様に行うことができる。 The present invention will be described below with reference to the drawings, taking as an example the use of L-aTNA as the non-cyclic artificial nucleic acid, but this description is not intended to limit the present invention. For example, the same procedure can be carried out as described below when using other non-cyclic nucleic acids such as SNA or PNA.
　図１は、本発明の一態様を説明する図である。L-aTNAポリヌクレオチドには、DNAポリヌクレオチドとのハイブリダイゼーションの効率を改善するために、1'末端（天然核酸の3'末端に相当）及び3'末端（天然核酸の5'末端に相当）の両方にDNA断片（図中灰色の部分）が付加されている。このDNA断片は、L-aTNAポリヌクレオチドにハイブリダイゼーションさせるDNAポリヌクレオチドの両末端に連結されたプライマー領域の一部、具体的には、L-aTNAポリヌクレオチドの1'側には、当該DNAポリヌクレオチドの5'末端に連結されたプライマー領域の3'側の配列、及び、L-aTNAポリヌクレオチドの3'側には、DNAポリヌクレオチドの3'末端側に連結されたプライマー領域の5'側の配列にそれぞれ相補的な配列を有する。DNA断片の配列の長さは、特に限定されないが、好ましくは3～8mer、より好ましくは4～6merとすることができる。
　また、L-aTNAポリヌクレオチドに付加されたDNA断片に、例えばFITCのような検出用の標識を付けることができる。 FIG. 1 is a diagram illustrating one embodiment of the present invention. In order to improve the efficiency of hybridization with a DNA polynucleotide, a DNA fragment (gray part in the figure) is added to both the 1' end (corresponding to the 3' end of a natural nucleic acid) and the 3' end (corresponding to the 5' end of a natural nucleic acid) of the L-aTNA polynucleotide. This DNA fragment is a part of the primer region linked to both ends of the DNA polynucleotide to be hybridized with the L-aTNA polynucleotide, specifically, the 1' side of the L-aTNA polynucleotide has a sequence complementary to the 3' side sequence of the primer region linked to the 5' end of the DNA polynucleotide, and the 3' side of the L-aTNA polynucleotide has a sequence complementary to the 5' side sequence of the primer region linked to the 3' end of the DNA polynucleotide. The length of the DNA fragment sequence is not particularly limited, but can be preferably 3 to 8 mer, more preferably 4 to 6 mer.
Furthermore, the DNA fragment added to the L-aTNA polynucleotide can be labeled for detection, for example with FITC.
　図２は、本発明の別の態様を説明する図である。両末端にプライマー領域を持たないDNAポリヌクレオチドを用いてL-aTNAポリヌクレオチドとのハイブリダイゼーションを行う。L-aTNAポリヌクレオチドに付加されたDNA断片は、具体的には、DNAポリヌクレオチドの両末端の配列にそれぞれ相補的な配列（図中薄い灰色の部分）と、L-aTNAポリヌクレオチドにハイブリダイゼーションさせるDNAポリヌクレオチドの5'末端に連結されたプライマー領域の3'側の配列、及び、DNAポリヌクレオチドの3'末端側に連結されたプライマー領域の5'側の配列にそれぞれ相補的な配列（図中濃い灰色の部分）とからなる。
　また、L-aTNAポリヌクレオチドに付加されたDNA断片に、例えばFITCのような検出用の標識を付けることができる。 2 is a diagram illustrating another embodiment of the present invention. Hybridization with an L-aTNA polynucleotide is performed using a DNA polynucleotide that does not have primer regions at either end. The DNA fragment added to the L-aTNA polynucleotide specifically consists of sequences complementary to the sequences at either end of the DNA polynucleotide (light gray parts in the figure), a 3'-side sequence of the primer region linked to the 5'-end of the DNA polynucleotide to be hybridized with the L-aTNA polynucleotide, and sequences complementary to the 5'-side sequence of the primer region linked to the 3'-end of the DNA polynucleotide (dark gray parts in the figure).
Furthermore, the DNA fragment added to the L-aTNA polynucleotide can be labeled for detection, for example with FITC.
　上記のようなDNA断片が付加されたL-aTNAポリヌクレオチドを、DNAポリヌクレオチドにハイブリダイゼーションさせた後、DNAポリヌクレオチドの両末端に、プライマー領域を連結させる。その際に、DNAポリヌクレオチドの5'側に連結させるプライマー領域の3'末端、及び、DNAポリヌクレオチドの3'末端をリン酸化し、DNAポリヌクレオチドの5'側に連結させるプライマー領域の5'末端は5'DMT（4,4'-dimethoxytrityl）によりブロックする。N－シアノイミダゾールと２価陽イオン（例えば、CdCl2、MnCl2等）の存在下で、DNAポリヌクレオチドとプライマー領域とを化学的に連結することができる。DNAポリヌクレオチドへのプライマー領域の連結を、L-aTNAポリヌクレオチドと、DNAポリヌクレオチドとのハイブリダイゼーション後に行うことによって、より効率的にPCR等のためのプライマー領域を含むDNA断片のライゲーションを行うことができる。 The L-aTNA polynucleotide to which the DNA fragment is added as described above is hybridized to a DNA polynucleotide, and then primer regions are linked to both ends of the DNA polynucleotide. In this case, the 3' end of the primer region to be linked to the 5' side of the DNA polynucleotide and the 3' end of the DNA polynucleotide are phosphorylated, and the 5' end of the primer region to be linked to the 5' side of the DNA polynucleotide is blocked with 5'DMT (4,4'-dimethoxytrityl). The DNA polynucleotide and the primer region can be chemically linked in the presence of N-cyanoimidazole and divalent cations (e.g., CdCl 2 , MnCl 2 , etc.). By linking the primer region to the DNA polynucleotide after hybridization between the L-aTNA polynucleotide and the DNA polynucleotide, ligation of DNA fragments containing primer regions for PCR, etc. can be performed more efficiently.
　図３は、本発明のさらに別の態様を説明する図である。ランダム配列を含むDNAポリヌクレオチドの両末端には、他のランダム配列を含むDNAポリヌクレオチドに共通する特定の配列が付加されている（図中黒色の部分）。L-aTNAポリヌクレオチドには、ライゲーションのために両末端にDNA断片が付加されている。このDNA断片は、具体的には、ランダム配列を含むDNAポリヌクレオチドの両末端に付加された、他のランダム配列を含むDNAポリヌクレオチドに共通する特定の配列（図中黒色の部分）にそれぞれ相補的な配列（図中薄い灰色の部分）と、L-aTNAポリヌクレオチドにハイブリダイゼーションさせるDNAポリヌクレオチドの5'末端に連結されたプライマー領域の3'側の配列、及び、DNAポリヌクレオチドの3'末端側に連結されたプライマー領域の5'側の配列にそれぞれ相補的な配列（図中濃い灰色の部分）とからなる。
　また、L-aTNAポリヌクレオチドに付加されたDNA断片に、例えばFITCのような検出用の標識を付けることができる。 3 is a diagram illustrating yet another embodiment of the present invention. A specific sequence common to other DNA polynucleotides containing random sequences is added to both ends of a DNA polynucleotide containing a random sequence (black part in the figure). A DNA fragment is added to both ends of an L-aTNA polynucleotide for ligation. Specifically, the DNA fragment is composed of a sequence (light gray part in the figure) complementary to the specific sequence (black part in the figure) common to other DNA polynucleotides containing random sequences added to both ends of the DNA polynucleotide containing a random sequence, and a sequence (dark gray part in the figure) complementary to the 3'-side sequence of the primer region linked to the 5'-end of the DNA polynucleotide to be hybridized to the L-aTNA polynucleotide and the 5'-side sequence of the primer region linked to the 3'-end of the DNA polynucleotide.
Furthermore, the DNA fragment added to the L-aTNA polynucleotide can be labeled for detection, for example with FITC.
　L-aTNAポリヌクレオチドとDNAポリヌクレオチドのハイブリダイゼーションによって形成された二重鎖のDNAポリヌクレオチドの両末端に、プライマー領域を化学的に連結させる。この化学的な連結方法として、Nat. Commun., 2021, 12, 804.に記載の方法を用いることができる。また、その際に、DNAポリヌクレオチドの5'側に連結させるプライマー領域の3'末端、及び、DNAポリヌクレオチドの3'末端をリン酸化し、DNAポリヌクレオチドの5'側に連結させるプライマー領域の5'末端は5'DMT（4,4'-dimethoxytrityl）によりブロックする。N－シアノイミダゾールと２価陽イオン（例えば、CdCl2、MnCl2等）の存在下で、DNAポリヌクレオチドとプライマー領域とを化学的に連結することができる。DNAポリヌクレオチドへのプライマー領域の連結を、L-aTNAポリヌクレオチドと、DNAポリヌクレオチドとのハイブリダイゼーション後に行うことによって、より効率的にライゲーションを行うことができる。 A primer region is chemically linked to both ends of a double-stranded DNA polynucleotide formed by hybridization of an L-aTNA polynucleotide and a DNA polynucleotide. As a chemical linking method, the method described in Nat. Commun., 2021, 12, 804. can be used. In addition, at that time, the 3' end of the primer region to be linked to the 5' side of the DNA polynucleotide and the 3' end of the DNA polynucleotide are phosphorylated, and the 5' end of the primer region to be linked to the 5' side of the DNA polynucleotide is blocked with 5'DMT (4,4'-dimethoxytrityl). The DNA polynucleotide and the primer region can be chemically linked in the presence of N-cyanoimidazole and a divalent cation (e.g., CdCl 2 , MnCl 2 , etc.). Ligation can be performed more efficiently by linking the primer region to the DNA polynucleotide after hybridization between the L-aTNA polynucleotide and the DNA polynucleotide.
　二重鎖に含まれるDNAポリヌクレオチドは、L-aTNAポリヌクレオチドに相補的な配列を含むものである。DNAポリヌクレオチドの配列を、PCR等で増幅後、公知の方法で決定することによって、L-aTNAポリヌクレオチドの配列を取得することができる。 The DNA polynucleotide contained in the double strand contains a sequence complementary to the L-aTNA polynucleotide. The sequence of the DNA polynucleotide can be obtained by amplifying it by PCR or the like and then determining the sequence of the L-aTNA polynucleotide by a known method.
　例えば、ランダムな配列を有するL-aTNAポリヌクレオチドのプールから、標的に結合するL-aTNAポリヌクレオチドを選択する。選択されたL-aTNAポリヌクレオチドの配列を、本発明の方法を用いてDNAの配列に非酵素的に転写することができれば、その後一般的な核酸増幅技術、例えばＰＣＲにより標的に結合する所望のL-aTNAを選択することができる。 For example, an L-aTNA polynucleotide that binds to a target is selected from a pool of L-aTNA polynucleotides having random sequences. If the sequence of the selected L-aTNA polynucleotide can be non-enzymatically transcribed into a DNA sequence using the method of the present invention, then the desired L-aTNA that binds to the target can be selected by a general nucleic acid amplification technique, such as PCR.
　以下に実施例に基づいて本発明を説明するが、下記の実施例は本発明を説明するためのものであり、本発明を限定するためのものではない。 The present invention will be explained below based on examples. However, the following examples are intended to explain the present invention and are not intended to limit the present invention.
　L-aTNAポリヌクレオチドの配列を、ランダムなDNAライブラリーを用いて配列を決定することができることを確認するために、L-aTNAポリヌクレオチドとランダム化DNAポリヌクレオチドとをアニーリングさせた後にDNAポリヌクレオチドの両端にプライマー領域を連結し、PCR後、配列決定を行った。
＜ステップ1　アニーリング＞
　L-aTNAポリヌクレオチド（TNA20 + 3 + 5、終濃度2.0 nM）と、前記L-aTNAポリヌクレオチドとハイブリダイズする部分をランダム化したDNAポリヌクレオチド（DNArandom、終濃度50 μM）とを混合し、95℃まで加熱して1分間、56℃まで冷却して30秒間、4℃まで冷却することによって、相補的な配列を有するDNAポリヌクレオチドにアニーリングさせた。
　TNA20 + 3 + 5（製造元：北海道システムサイエンス）の配列は下記のとおりである。 To confirm that the sequence of the L-aTNA polynucleotide could be determined using a random DNA library, the L-aTNA polynucleotide was annealed with a randomized DNA polynucleotide, and then primer regions were ligated to both ends of the DNA polynucleotide, followed by PCR and sequencing.
<Step 1 Annealing>
L-aTNA polynucleotide (TNA20 + 3 + 5, final concentration 2.0 nM) was mixed with a DNA polynucleotide in which the portion that hybridizes with the L-aTNA polynucleotide was randomized (DNArandom, final concentration 50 μM), and annealed to a DNA polynucleotide having a complementary sequence by heating to 95°C for 1 minute, cooling to 56°C for 30 seconds, and then cooling to 4°C.
The sequence of TNA20 + 3 + 5 (manufacturer: Hokkaido System Science) is as follows.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
　イタリック体は、L-aTNAポリヌクレオチド部分であり、イタリック体でない部分はDNAポリヌクレオチド部分である。DNAポリヌクレオチド部分の下線部分はランダム化したDNAと相補する配列である。 The italicized parts are the L-aTNA polynucleotide parts, and the non-italicized parts are the DNA polynucleotide parts. The underlined parts of the DNA polynucleotide parts are the sequences complementary to the randomized DNA.
　DNArandom（製造元：ユーロフィンジェノミクス）の配列は下記の通りである。
DNArandom （26mer）5'-ATTNNNNNNNNNNNNNNNNNNNNTCA[PHO]-3'（配列番号２）（ここで、Nは任意のDNAヌクレオチドを示す。各両末端の3mer（下線部）は、TNA20 + 3 + 5の下線部分と相補する部分である） The sequence of DNArandom (manufacturer: Eurofins Genomics) is as follows:
DNArandom (26mer) 5'- ATT NNNNNNNNNNNNNNNNNNNN TCA [PHO]-3' (SEQ ID NO: 2) (wherein N represents any DNA nucleotide. The 3mers (underlined) at both ends are complementary to the underlined portions of TNA20 + 3 + 5)
＜ステップ2　ライゲーション＞
　増幅用のプライマー配列（complement to pColdTF-F1 short及びpCold-R shortened_5DMT_3P、各終濃度1.0μM）、NaCl水溶液（和光純薬製、分子生物学用）（終濃度100 mM）及びCdCl2（和光純薬製）又はMnCl2（和光純薬製）水溶液（終濃度20 mM）を、アニーリング溶液に加え、MilliQ水で27.0 μlにメスアップした。数秒間ボルテックスで撹拌し、25℃のヒートブロックで3分間以上インキュベートした。
　その後、直前に常温解凍したN-Cyanoimidazole（和光純薬製）水溶液3.0 μl（終濃度20 mM）を加え、数秒間ボルテックスで撹拌した。反応液を25℃のヒートブロックに戻し、48時間インキュベートした。回収したサンプルに等量のクエンチ溶液（12.5 mM NaOH（和光純薬製）、125 mM EDTA（和光純薬製、特級））を添加し反応を停止させた。 <Step 2 Ligation>
The primer sequences for amplification (complement to pColdTF-F1 short and pCold-R shortened_5DMT_3P, each at a final concentration of 1.0 μM), an aqueous NaCl solution (Wako Pure Chemical Industries, for molecular biology) (final concentration 100 mM), and an aqueous CdCl 2 (Wako Pure Chemical Industries) or MnCl 2 (Wako Pure Chemical Industries) solution (final concentration 20 mM) were added to the annealing solution, and the solution was made up to 27.0 μl with MilliQ water. The solution was vortexed for a few seconds and incubated in a heat block at 25°C for at least 3 minutes.
Then, 3.0 μl of N-cyanoimidazole (Wako Pure Chemical Industries, Ltd.) aqueous solution (final concentration 20 mM) that had been thawed at room temperature just before was added and vortexed for a few seconds. The reaction solution was returned to the heat block at 25°C and incubated for 48 hours. An equal volume of quenching solution (12.5 mM NaOH (Wako Pure Chemical Industries, Ltd.), 125 mM EDTA (Wako Pure Chemical Industries, Ltd., special grade)) was added to the collected sample to stop the reaction.
　使用したプライマーであるcomplement to pColdTF-F1 short及びpCold-R shortened_5DMT_3Pの配列は下記のとおりである。
complement to pColdTF-F1 short(19mer)：5’-CATCAGCTCGTTGAAAGTG-3’（配列番号３）
（製造元：ユーロフィンジェノミクス）
pCold-R shortened_5DMT_3P（18mer）：5'-[DMT]CATTCTCATTGCACCCAA[PHO]-3’（配列番号４）
[DMT]= 4,4'-ジメトキシトリチル（4,4'-Dimethoxytrityl）
（製造元：北海道システムサイエンス） The sequences of the primers used, complement to pColdTF-F1 short and pCold-R shortened_5DMT_3P, are as follows:
Complement to pColdTF-F1 short (19 mer): 5'-CATCAGCTCGTTGAAAGTG-3' (SEQ ID NO: 3)
(Manufacturer: Eurofins Genomics)
pCold-R shortened_5DMT_3P (18mer): 5'-[DMT]CATTCTCATTGCACCCAA[PHO]-3' (SEQ ID NO: 4)
[DMT] = 4,4'-Dimethoxytrityl
(Manufacturer: Hokkaido System Science)
＜ステップ3 PCR＞
　回収したサンプルをマイクロバイオスピン6 カラム（Micro Bio-Spin 6 Columns)（BioRad製）を使用してバッファー交換を行い、MilliQ水に溶出した。操作は製品のプロトコルに従った。
　溶出液を10-3倍に希釈し、PCRに供した。PCRは、プライマー（pCold-R Primer shortened、pColdTF-F1 Primer shortened）各0.4 μM、10－3倍溶出液3.0 μl（全量25 μl）、アニーリング温度55℃、40サイクルの反応条件で行った。リファレンス（Rf）として、63 ntの完全長DNA（Reference DNA）を同じ条件でPCRした。PCR産物を8M Urea（和光純薬製） 12％PAGEにて解析した。マーカーとして、10 bp DNA ladder（Promega製）を使用した。 <Step 3 PCR>
The collected samples were buffer exchanged using Micro Bio-Spin 6 Columns (BioRad) and eluted in MilliQ water according to the manufacturer's protocol.
The eluate was diluted 10-3 times and subjected to PCR. PCR was performed using 0.4 μM primers (pCold-R Primer shortened, pColdTF-F1 Primer shortened), 3.0 μl of 10-3 times eluate (total volume 25 μl), annealing temperature 55°C, and 40 cycles. As a reference (Rf), a 63 nt full-length DNA (Reference DNA) was PCRed under the same conditions. The PCR products were analyzed by 12% PAGE using 8M Urea (Wako Pure Chemical Industries, Ltd.). A 10 bp DNA ladder (Promega, Ltd.) was used as a marker.
　使用したプライマー及びReference DNA（製造元：ユーロフィンジェノミクス）の配列を下記に示す。
pCold-R Primer shortened（18mer)：5’-CATTCTCATTGCACCCAA-3’ （配列番号５）
pColdTF-F1 Primer shortened(19mer)：5’-CACTTTCAACGAGCTGATG-3’ （配列番号６）
Reference DNA（63mer）：5'-CATTCTCATTGCACCCAAATTGAAACCATTGCGTTGTTCTTTCACATCAGCTCGTTGAAAGTG-3'（配列番号７） The sequences of the primers and reference DNA (manufacturer: Eurofins Genomics) used are shown below.
pCold-R Primer shortened (18mer): 5'-CATTCTCATTGCACCCAA-3' (SEQ ID NO: 5)
pColdTF-F1 Primer shortened (19mer): 5'-CACTTTCAACGAGCTGATG-3' (SEQ ID NO: 6)
Reference DNA (63mer): 5'-CATTCTCATTGCACCCAAATTGAAACCATTGCGTTGTTCTTTCACATCAGCTCGTTGAAAGTG-3' (SEQ ID NO: 7)
　結果を図４に示す。リファレンスサンプルと同じ泳動度のバンド（約60mer）が検出され、化学ライゲーションによる生成物はPCRにより増幅可能であることが確認できた。 The results are shown in Figure 4. A band (approximately 60 mer) with the same electrophoretic mobility as the reference sample was detected, confirming that the product of chemical ligation can be amplified by PCR.
　さらに、得られたコロニーの配列を解析したところ、目的DNAと同じ長さのインサートが挿入されたDNAのうち、ほぼ100％が目的配列と完全に一致し、L-aTNAポリヌクレオチドに相補的な配列を持つDNAポリヌクレオチドのみがPCRにより増幅されたことが確認された。すなわち、L-aTNAポリヌクレオチドの配列を、ランダムなDNAライブラリーを用いて配列を決定することができることが確認された。 Furthermore, analysis of the sequences of the colonies obtained confirmed that, of the DNAs containing inserts of the same length as the target DNA, nearly 100% matched the target sequence perfectly, and that only DNA polynucleotides with sequences complementary to the L-aTNA polynucleotide were amplified by PCR. In other words, it was confirmed that the sequence of the L-aTNA polynucleotide can be determined using a random DNA library.
　両末端にプライマーを予め連結させたDNAポリヌクレオチドと、L-aTNAポリヌクレオチドとをアニーリングさせて二重鎖が形成されることを確認した。 It was confirmed that a double strand was formed by annealing a DNA polynucleotide with primers pre-linked to both ends with an L-aTNA polynucleotide.
＜ステップ1　アニーリング＞
　DNAポリヌクレオチド（Primer1-DNA20-primer2 (1μM)） 2.0μlと、L-aTNAポリヌクレオチド（TNA-20 （1μM））0.5μlを混合し、95℃まで加熱して1分間、50～64℃まで冷却して30秒間、その後、4℃まで冷却した。
　primer1-DNA20-primer2（製造元：ユーロフィンジェノミクス）及びTNA-20（製造元：北海道システムサイエンス）の配列は、それぞれ下記のとおりである。 <Step 1 Annealing>
2.0 μl of DNA polynucleotide (Primer1-DNA20-primer2 (1 μM)) and 0.5 μl of L-aTNA polynucleotide (TNA-20 (1 μM)) were mixed, heated to 95°C for 1 minute, cooled to 50-64°C for 30 seconds, and then cooled to 4°C.
The sequences of primer1-DNA20-primer2 (manufacturer: Eurofins Genomics) and TNA-20 (manufacturer: Hokkaido System Science) are as follows:
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
＜ステップ2　非変性PAGE＞
　5％グリセロール20％ポリアクリルアミドゲルを使用して、20℃で電気泳動を行った。
レーン構成は以下である。１．DNA20（1μM）0.5μl、H2O 2.0μlを混合してアニーリングを行ったサンプル
２．TNA-20 （1μM）0.5μl、H2O 2.0μlを混合してアニーリングを行ったサンプル
３．50℃でアニーリングを行ったサンプル
４．52℃アニーリングを行ったサンプル
５．54℃アニーリングを行ったサンプル
６．56℃アニーリングを行ったサンプル
７．58℃アニーリングを行ったサンプル
８．60℃アニーリングを行ったサンプル
９．62℃アニーリングを行ったサンプル
10．64℃アニーリングを行ったサンプル <Step 2: Non-denaturing PAGE>
Electrophoresis was performed at 20°C using 5% glycerol 20% polyacrylamide gels.
The lane configuration is as follows: 1. Sample annealed by mixing 0.5 μl of DNA20 (1 μM) and 2.0 μl of H2O 2. Sample annealed by mixing 0.5 μl of TNA-20 (1 μM) and 2.0 μl of H2O 3. Sample annealed at 50°C 4. Sample annealed at 52°C 5. Sample annealed at 54°C 6. Sample annealed at 56°C 7. Sample annealed at 58°C 8. Sample annealed at 60°C 9. Sample annealed at 62°C
10. Sample annealed at 64℃
　FITCで検出した結果を図５に示す。薄くではあったがレーン3～10にDNA/TNA二重鎖のバンドが検出されることが確認された。 The results of detection with FITC are shown in Figure 5. Although faint, DNA/TNA double-stranded bands were detected in lanes 3 to 10.
　本実施例では、L-aTNAポリヌクレオチドの両端に数merのDNA部分を付加した影響を確認した。また、ライゲーション反応を行った後、精製せずにPCRに用いてもPCRが効率的に行われることを確認した。
＜ステップ1　アニーリング＞
　L-aTNAと相補的な配列を持つDNAポリヌクレオチド（DNA20+0_3P、DNA20+1_3P、又はDNA20+3_3P、終濃度50μM）と、L-aTNAポリヌクレオチド（TNA20 + 0 + 5またはTNA20 + 1 + 5またはTNA20 + 3 + 5、終濃度2.0 nM）又はDNAポリヌクレオチド（TNA20+0 (all DNA)）を混合し、95℃まで加熱して1分間、56℃まで冷却して30秒間、その後、4℃まで冷却した。 In this example, we confirmed the effect of adding a few-mer DNA moiety to both ends of the L-aTNA polynucleotide. We also confirmed that PCR was performed efficiently even if the L-aTNA polynucleotide was used in PCR without purification after the ligation reaction.
<Step 1 Annealing>
DNA polynucleotides having a sequence complementary to L-aTNA (DNA20+0_3P, DNA20+1_3P, or DNA20+3_3P, final concentration 50 μM) were mixed with L-aTNA polynucleotides (TNA20 + 0 + 5, TNA20 + 1 + 5, or TNA20 + 3 + 5, final concentration 2.0 nM) or DNA polynucleotides (TNA20+0 (all DNA)), heated to 95°C for 1 minute, cooled to 56°C for 30 seconds, and then cooled to 4°C.
　L-aTNAポリヌクレオチド（製造元：北海道システムサイエンス）又はDNAポリヌクレオチド（製造元：ユーロフィンジェノミクス）の配列は以下の通りである。 The sequences of the L-aTNA polynucleotide (manufactured by Hokkaido System Science) or DNA polynucleotide (manufactured by Eurofins Genomics) are as follows:
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
　イタリック体は、L-aTNAポリヌクレオチド部分であり、イタリック体でない部分はDNAポリヌクレオチド部分である。DNAポリヌクレオチド部分の下線部分はDNAポリヌクレオチドと相補する配列である。 The italics indicate the L-aTNA polynucleotide portion, and the non-italicized portion indicates the DNA polynucleotide portion. The underlined portion of the DNA polynucleotide portion is the sequence complementary to the DNA polynucleotide.
　DNAポリヌクレオチドは、L-aTNAポリヌクレオチドの末端に付加されたDNAに相補的な配列（両端にそれぞれ0mer、1mer、又は3mer）を有する、下記のDNA20+0_3P、DNA20+1_3P、又はDNA20+3_3P（製造元：ユーロフィンジェノミクス）のいずれかを用いた。 The DNA polynucleotide used was either DNA20+0_3P, DNA20+1_3P, or DNA20+3_3P (manufacturer: Eurofins Genomics), which has a sequence complementary to the DNA added to the end of the L-aTNA polynucleotide (0 mer, 1 mer, or 3 mer at each end).
DNA20+0_3P(20 mer)：5’-GAAACCATTGCGTTGTTCTT[PHO]-3’ （配列番号１４）
DNA20+1_3P(22 mer)：5’-TGAAACCATTGCGTTGTTCTTT[PHO]-3’ （配列番号１５）
DNA20+3_3P(26 mer)：5’-ATTGAAACCATTGCGTTGTTCTTTCA[PHO]-3’ （配列番号１６）
（各両末端の下線部は、L-aTNAポリヌクレオチドの下線部分と相補する部分である） DNA20+0_3P(20mer): 5'-GAAACCATTGCGTTGTTCTT[PHO]-3' (SEQ ID NO: 14)
DNA20+1_3P(22mer): 5'- T GAAACCATTGCGTTGTTCTT T [PHO]-3' (SEQ ID NO: 15)
DNA20+3_3P(26mer): 5'- ATT GAAACCATTGCGTTGTTCTT TCA [PHO]-3' (SEQ ID NO: 16)
(The underlined parts at both ends are complementary to the underlined parts of the L-aTNA polynucleotide.)
＜ステップ2　ライゲーション＞
　アニーリング溶液に、実施例１で用いたものと同じプライマー領域（complement to pColdTF-F1 shortおよびpCold-R shortened_5DMT_3P、各終濃度1.0μM）、NaCl水溶液（終濃度100 mM）、及びCdCl2又はMnCl2水溶液（終濃度20 mM）を加え、MilliQ水で27.0μlにメスアップした。数秒間ボルテックスで撹拌し、25℃のヒートブロックで3分間以上インキュベートした。 <Step 2 Ligation>
The annealing solution was added with the same primer region as used in Example 1 (complement to pColdTF-F1 short and pCold-R shortened_5DMT_3P, each final concentration 1.0 μM), NaCl aqueous solution (final concentration 100 mM), and CdCl2 or MnCl2 aqueous solution (final concentration 20 mM), and the solution was made up to 27.0 μl with MilliQ water. The mixture was stirred with a vortex for a few seconds and incubated in a heat block at 25 ° C for 3 minutes or more.
　その後、直前に常温解凍した1-Cyanoimidazole水溶液3.0μl（終濃度20 mM）を加え、数秒間ボルテックスで撹拌した。反応液を25℃のヒートブロックに戻し、0～48時間インキュベートした。回収したサンプルに等量のクエンチ溶液（12.5 mM NaOH、125 mM EDTA）を添加し反応を停止させた。 Then, 3.0 μl of 1-cyanoimidazole aqueous solution (final concentration 20 mM) that had been thawed at room temperature just before was added and vortexed for a few seconds. The reaction solution was returned to the heat block at 25°C and incubated for 0 to 48 hours. An equal volume of quench solution (12.5 mM NaOH, 125 mM EDTA) was added to the collected sample to stop the reaction.
＜ステップ3　PCR＞
　溶出液を10－3倍に希釈し、PCRに供した。PCRは、実施例１と同じプライマー（pCold-R Primer shortened、pColdTF-F1 Primer shortened）各0.4μM、10－3倍溶出液3.0μl（全量25μl）、アニーリング温度55℃、40サイクルの反応条件で行った。PCR産物を8M Urea 12％PAGEにて解析した。マーカーとして、10 bp DNA ladderを使用した。リファレンス（Rf）として、PCRする前のライゲーション反応液を同じゲル上で泳動した。
　その結果、L-aTNAの両端に3 ntDNAを付加した場合に最も濃いバンドが検出され、L-aTNAの両端にDNA断片を付加することで、ライゲーション効率が上昇することが示唆された（図６）。また、ライゲーション後、精製、脱塩を行わずにPCRを行うことが可能であることが確認された。 <Step 3 PCR>
The eluate was diluted 10-3 times and subjected to PCR. PCR was performed under the reaction conditions of the same primers as in Example 1 (pCold-R Primer shortened, pColdTF-F1 Primer shortened) at 0.4 μM each, 3.0 μl of 10-3 times eluate (total volume 25 μl), annealing temperature 55°C, and 40 cycles. The PCR product was analyzed by 8M urea 12% PAGE. A 10 bp DNA ladder was used as a marker. As a reference (Rf), the ligation reaction solution before PCR was electrophoresed on the same gel.
As a result, the strongest band was detected when 3 nt DNA was added to both ends of L-aTNA, suggesting that the ligation efficiency increases by adding DNA fragments to both ends of L-aTNA (Figure 6). It was also confirmed that PCR can be performed after ligation without purification or desalting.

Claims (10)

  1. 　非環状型人工核酸からなるポリヌクレオチドの配列を取得する方法であって、
    　非環状型人工核酸からなるポリヌクレオチドと、ランダム配列を含むDNAポリヌクレオチドのプールとをハイブリダイゼーションさせる工程と、
    　ここで、前記DNAポリヌクレオチドは、ランダム配列の両末端に、前記プールに含まれるDNAポリヌクレオチドに共通する配列が付加されており、
    　前記非環状型人工核酸からなるポリヌクレオチドは、両末端にDNA断片が付加されており、前記DNA断片は、
    　１）前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に、前記DNAポリヌクレオチドの5'末端に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の3'側の配列、及び、前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に、前記DNAポリヌクレオチドの3'末端側に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の5'側の配列にそれぞれ相補的な配列、又は、
    　２）i)前記非環状型人工核酸からなるポリヌクレオチドの3'相当側及び5'相当側に、前記DNAポリヌクレオチドの前記ランダム配列の両末端に付加された前記共通する配列にそれぞれ相補的な配列、並びに、
    　ii) 前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に付加された前記共通する配列に相補的な配列の3'側に、前記DNAポリヌクレオチドの5'末端に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の3'側の配列に相補的な配列、及び、
    　前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に付加された前記共通する配列に相補的な配列の5'側に、前記DNAポリヌクレオチドの3'末端側に連結される、又は後の工程で前記DNAポリヌクレオチドに連結されるプライマー領域の5'側の配列に相補的な配列、
    を含み、
    　前記DNAポリヌクレオチドからDNAを増幅する工程と、
    　前記DNAポリヌクレオチドの配列を決定する工程と、
    　前記DNAポリヌクレオチドの配列から非環状型人工核酸からなるポリヌクレオチドの配列を決定する工程と
    を含む、方法。     A method for obtaining a polynucleotide sequence consisting of a non-circular artificial nucleic acid, comprising:
    A step of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences;
    wherein the DNA polynucleotide has a sequence common to the DNA polynucleotides contained in the pool added to both ends of a random sequence,
    The polynucleotide consisting of the non-cyclic artificial nucleic acid has DNA fragments added to both ends, and the DNA fragments are
    1) a sequence complementary to the 3' side of a primer region that is linked to the 5' end of the DNA polynucleotide or to the DNA polynucleotide in a later step on the 3' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, and a sequence complementary to the 5' side of a primer region that is linked to the 3' end of the DNA polynucleotide or to the DNA polynucleotide in a later step on the 5' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, or
    2) i) sequences complementary to the common sequences added to both ends of the random sequence of the DNA polynucleotide on the 3'-side and 5'-side of the polynucleotide consisting of the non-circular artificial nucleic acid, respectively; and
    ii) a sequence complementary to the 3' side sequence of a primer region that is linked to the 5' end of the DNA polynucleotide or to the DNA polynucleotide in a later step, on the 3' side of the sequence complementary to the common sequence added to the 3' corresponding side of the polynucleotide consisting of the non-circular artificial nucleic acid; and
    a sequence complementary to the 5'-side sequence of a primer region that is linked to the 3'-end of the DNA polynucleotide or that is linked to the DNA polynucleotide in a later step, on the 5'-side of the sequence complementary to the common sequence added to the 5'-side of the polynucleotide consisting of the non-circular artificial nucleic acid;
    Including,
    amplifying DNA from said DNA polynucleotide;
    determining the sequence of the DNA polynucleotide;
    determining the sequence of a polynucleotide consisting of a non-circular artificial nucleic acid from the sequence of the DNA polynucleotide.
  2. 　非環状型人工核酸は、L-aTNA又はSNAである、請求項１に記載の方法。 The method according to claim 1, wherein the non-circular artificial nucleic acid is L-aTNA or SNA.
  3. 　非環状型人工核酸からなるポリヌクレオチドの配列を取得する方法であって、
    　非環状型人工核酸からなるポリヌクレオチドとランダム配列を含むDNAポリヌクレオチドのプールとをハイブリダイゼーションさせる工程と、
    　前記DNAポリヌクレオチドの両端にプライマー領域を連結させる工程と、
    　前記DNAポリヌクレオチドからDNAを増幅する工程と、
    　前記DNAポリヌクレオチドの配列を決定する工程と、
    　前記DNAポリヌクレオチドの配列から非環状型人工核酸からなるポリヌクレオチドの配列を決定する工程と
    を含む、方法。     A method for obtaining a polynucleotide sequence consisting of a non-circular artificial nucleic acid, comprising:
    A step of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences;
    ligating primer regions to both ends of the DNA polynucleotide;
    amplifying DNA from said DNA polynucleotide;
    determining the sequence of the DNA polynucleotide;
    determining the sequence of a polynucleotide consisting of a non-circular artificial nucleic acid from the sequence of the DNA polynucleotide.
  4. 　前記DNAポリヌクレオチドの両端へのプライマー領域の連結は、Nーシアノイミダゾール及び２価陽イオンの存在下にてリン酸反応を行う化学的連結である、請求項３に記載の方法。 The method according to claim 3, wherein the primer regions are linked to both ends of the DNA polynucleotide by chemical linkage using a phosphorylation reaction in the presence of N-cyanoimidazole and a divalent cation.
  5. 　前記２価陽イオンはカドニウムイオン又はマンガンイオンである、請求項４に記載の方法。 The method according to claim 4, wherein the divalent cation is a cadmium ion or a manganese ion.
  6. 　非環状型人工核酸は、L-aTNA又はSNAである、請求項３～５のいずれか一項に記載の方法。 The method according to any one of claims 3 to 5, wherein the non-cyclic artificial nucleic acid is L-aTNA or SNA.
  7. 　非環状型人工核酸からなるポリヌクレオチドの配列を取得する方法であって、
    　非環状型人工核酸からなるポリヌクレオチドとランダム配列を含むDNAポリヌクレオチドのプールとをハイブリダイゼーションさせる工程と、
    　ここで、前記DNAポリヌクレオチドは、ランダム配列の両末端に、前記プールに含まれるDNAポリヌクレオチドに共通する配列が付加されており、
    　前記非環状型人工核酸からなるポリヌクレオチドは、両末端にDNA断片が付加されており、前記DNA断片は、
    　１）前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に、前記DNAポリヌクレオチドの5'末端に連結されるプライマー領域の3'側の配列、及び、前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に、前記DNAポリヌクレオチドの3'末端に連結されるプライマー領域の5'側の配列にそれぞれ相補的な配列、又は、
    　２）i) 前記非環状型人工核酸からなるポリヌクレオチドの3'相当側及び5'相当側に、前記DNAポリヌクレオチドの前記ランダム配列の両末端に付加された前記共通する配列にそれぞれ相補的な配列、並びに、
    　ii) 前記非環状型人工核酸からなるポリヌクレオチドの3'相当側に付加された前記共通する配列に相補的な配列の3'側に、前記DNAポリヌクレオチドの5'末端に連結されるプライマー領域の3'側の配列、及び、
    　前記非環状型人工核酸からなるポリヌクレオチドの5'相当側に付加された前記共通する配列に相補的な配列の5'側に、前記DNAポリヌクレオチドの3'末端側に連結されるプライマー領域の5'側の配列にそれぞれ相補的な配列、
    を含み、
    　DNAポリヌクレオチドの両端にプライマー領域を連結させる工程と、
    　前記DNAポリヌクレオチドからDNAを増幅する工程と、
    　前記DNAポリヌクレオチドの配列を決定する工程と、
    　前記DNAポリヌクレオチドの配列から非環状型人工核酸からなるポリヌクレオチドの配列を決定する工程と
    を含む、方法。     A method for obtaining a polynucleotide sequence consisting of a non-circular artificial nucleic acid, comprising:
    A step of hybridizing a polynucleotide consisting of a non-circular artificial nucleic acid with a pool of DNA polynucleotides containing random sequences;
    wherein the DNA polynucleotide has a sequence common to the DNA polynucleotides contained in the pool added to both ends of a random sequence,
    The polynucleotide consisting of the non-cyclic artificial nucleic acid has DNA fragments added to both ends, and the DNA fragments are
    1) a sequence complementary to the 3' side of the primer region linked to the 5' end of the DNA polynucleotide on the 3' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, and a sequence complementary to the 5' side of the primer region linked to the 3' end of the DNA polynucleotide on the 5' side of the polynucleotide consisting of the non-cyclic artificial nucleic acid, or
    2) i) sequences complementary to the common sequences added to both ends of the random sequence of the DNA polynucleotide on the 3'-side and 5'-side of the polynucleotide consisting of the non-circular artificial nucleic acid, respectively; and
    ii) a sequence on the 3' side of a sequence complementary to the common sequence added to the 3' corresponding side of the polynucleotide consisting of the non-circular artificial nucleic acid, the sequence being a primer region linked to the 5' end of the DNA polynucleotide; and
    a sequence complementary to the 5' side of a sequence complementary to the common sequence added to the 5' corresponding side of the polynucleotide consisting of the non-circular artificial nucleic acid, and a sequence complementary to the 5' side sequence of a primer region linked to the 3' end of the DNA polynucleotide;
    Including,
    ligating primer regions to both ends of a DNA polynucleotide;
    amplifying DNA from said DNA polynucleotide;
    determining the sequence of the DNA polynucleotide;
    determining the sequence of a polynucleotide consisting of a non-circular artificial nucleic acid from the sequence of the DNA polynucleotide.
  8. 　前記DNAポリヌクレオチドの両端へのプライマー領域の連結は、Nーシアノイミダゾール及び２価陽イオンの存在下にてリン酸反応を行う化学的連結である、請求項７に記載の方法。 The method according to claim 7, wherein the primer regions are linked to both ends of the DNA polynucleotide by chemical linkage using a phosphorylation reaction in the presence of N-cyanoimidazole and a divalent cation.
  9. 　前記２価陽イオンはカドニウムイオン又はマンガンイオンである、請求項８に記載の方法。 The method according to claim 8, wherein the divalent cation is a cadmium ion or a manganese ion.
  10. 　非環状型人工核酸は、L-aTNA又はSNAである、請求項７～９のいずれか一項に記載の方法。 The method according to any one of claims 7 to 9, wherein the non-cyclic artificial nucleic acid is L-aTNA or SNA.
PCT/JP2023/039831 2022-11-07 2023-11-06 Method for obtaining sequence for non-cyclic artificial nucleic acid WO2024101294A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100081801A1 (en) * 2005-10-17 2010-04-01 Rina Netzwerk Rna-Technologien Gmbh Method for determining an unknown PNA sequence and uses thereof
WO2013073602A1 (en) * 2011-11-18 2013-05-23 独立行政法人理化学研究所 Nucleic acid fragment binding to target protein
CN109295187A (en) * 2018-10-31 2019-02-01 南京大学 It is a kind of based on nano-pore to the dislocation sequencing approach of unnatural nucleic acids direct Sequencing
CN111500572A (en) * 2020-04-22 2020-08-07 南京大学 Method for screening non-natural aptamer based on capillary electrophoresis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100081801A1 (en) * 2005-10-17 2010-04-01 Rina Netzwerk Rna-Technologien Gmbh Method for determining an unknown PNA sequence and uses thereof
WO2013073602A1 (en) * 2011-11-18 2013-05-23 独立行政法人理化学研究所 Nucleic acid fragment binding to target protein
CN109295187A (en) * 2018-10-31 2019-02-01 南京大学 It is a kind of based on nano-pore to the dislocation sequencing approach of unnatural nucleic acids direct Sequencing
CN111500572A (en) * 2020-04-22 2020-08-07 南京大学 Method for screening non-natural aptamer based on capillary electrophoresis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KEIJI MURAYAMA, YOSHIHIRO TANAKA, TAKASUKE TODA, HIROMU KASHIDA, HIROYUKI ASANUMA: "Highly Stable Duplex Formation by Artificial Nucleic Acids Acyclic Threoninol Nucleic Acid ( a TNA) and Serinol Nucleic Acid (SNA) with Acyclic Scaffolds", CHEMISTRY - A EUROPEAN JOURNAL, JOHN WILEY & SONS, INC, DE, vol. 19, no. 42, 11 October 2013 (2013-10-11), DE, pages 14151 - 14158, XP055568099, ISSN: 0947-6539, DOI: 10.1002/chem.201301578 *
MURAYAMA KEIJI, ASANUMA HIROYUKI: "Design and Hybridization Properties of Acyclic Xeno Nucleic Acid Oligomers", CHEMBIOCHEM, JOHN WILEY & SONS, INC., HOBOKEN, USA, vol. 22, no. 15, 3 August 2021 (2021-08-03), Hoboken, USA, pages 2507 - 2515, XP093135127, ISSN: 1439-4227, DOI: 10.1002/cbic.202100184 *

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