WO2025239314A1 - Oligonucleotide aptamer capable of inhibiting activity of strand-displacing dna polymerase - Google Patents

Abstract

The present disclosure provides an aptamer capable of inhibiting the activity of a strand-displacing DNA polymerase, the aptamer including: an oligonucleotide region 1 comprising a sequence in which a sequence X1a is linked to the 3'-end of a first sequence or the sequence X1a is linked to the 3'-end of a mutant sequence of the first sequence; and an oligonucleotide region 2 comprising a sequence in which a sequence X1b is linked to the 5'-end of a second sequence or the sequence X1b is linked to the 5'-end of a mutant sequence of the second sequence. The present disclosure also provides: a composition and a kit each containing the aptamer; and a method for amplifying a nucleic acid using the composition or the kit.

Description

Oligonucleotide aptamers that inhibit the activity of strand-displacing DNA polymerases

The present invention relates to oligonucleotide aptamers capable of inhibiting the activity of strand-displacing DNA polymerases, compositions and kits containing the aptamers, and methods for amplifying nucleic acids using them.

Isothermal nucleic acid amplification technology has the advantage of allowing reactions to proceed at a constant temperature without the need for repeated temperature cycling, and is used in a variety of situations where nucleic acid amplification is required. For example, in the LAMP (Loop-Mediated Isothermal Amplification) method, target nucleic acids are amplified at a constant temperature of 60-65°C through the action of strand-displacing DNA polymerases such as Bst DNA polymerase.

Because strand displacement DNA polymerases retain a certain degree of activity even at ambient temperatures below the reaction temperature, isothermal nucleic acid amplification poses the problem of inhibiting the amplification of target nucleic acids due to the generation of non-specific amplification products and the formation of primer dimers. Therefore, there is a need to develop a means of inhibiting the activity of strand displacement DNA polymerases at ambient temperatures and restoring their activity during the reaction. For example, Patent Document 1 discloses an aptamer that specifically binds to the active site of the large fragment of Bst DNA polymerase.

China Patent Application Publication No. 114990126

The present invention provides a new means for controlling the activity of strand-displacing DNA polymerases.

The inventors have found that when X1a and X1b are sequences that hybridize with each other to form a double-stranded structure, an oligonucleotide having a secondary structure formed from a sequence represented by TTTCCAG-X1a or a mutated sequence thereof and a sequence represented by X1b-AGTGTGAACGCGCAGCC (SEQ ID NO: 1) or a mutated sequence thereof has the ability to inhibit the activity of strand-displacing DNA polymerase.

The present disclosure provides the following:
Item 1. An oligonucleotide region 1 comprising a sequence in which the sequence X1a is linked to the 3' end of the first sequence TTTCCAG or to the 3' end of a mutant sequence of the first sequence, and an oligonucleotide region 2 comprising a sequence in which the sequence X1b is linked to the 5' end of the second sequence AGTGTGAACGCGCAGCC (SEQ ID NO: 1) or to the 5' end of a mutant sequence of the second sequence,
X1a and X1b are each independently a sequence of 4 nucleotides or more in length, and form a double-stranded structure by hybridizing with each other;
The mutant sequence of the first sequence is a sequence in which one or two nucleotides are deleted, substituted, or added in the first sequence,
The mutant sequence of the second sequence is a sequence in which 1 to 3 nucleotides are deleted, substituted, or added in the second sequence,
The 3' end of X1a and the 5' end of X1b may each have zero or one or more additional nucleotides and form the 3' and 5' ends of the same or different oligonucleotide molecules, or
The nucleotide at the 3'-terminal position of X1a and the nucleotide at the 5'-terminal position of X1b are linked via the sequence L1.
An aptamer capable of inhibiting the activity of strand-displacing DNA polymerase.
Item 2. The aptamer according to Item 1, wherein the mutant sequence of the first sequence is CTTCCAG, TTCCAG, TTTTCAG, TTTCTAG, TTTCCA, or any of these sequences with one nucleotide deleted or substituted.
Item 2A. The aptamer according to Item 1, wherein the mutant sequence of the first sequence is CTTCCAG, TTCCAG, TTTTCAG, TTTCTAG, TTTCCA, or any of these sequences in which one nucleotide has been deleted, substituted, or added.
Item 3. The aptamer of Item 1, 2, or 2A, wherein the mutant sequence of the second sequence is AGTGTGAACGCGCAGCT (SEQ ID NO: 2), AGTTGGAACCGCGCAGC (SEQ ID NO: 3), AGTGTGAACGCGCAGTC (SEQ ID NO: 4), AGTGTGAACGCGTAGCC (SEQ ID NO: 5), AGTGTGAATGCGCAGCC (SEQ ID NO: 6), GTGTGAACGCGCAGCC (SEQ ID NO: 7), AGCGTGAACGCGCAGCC (SEQ ID NO: 8), AGTGGGAACGCGCAGCC (SEQ ID NO: 9), or any of these sequences in which one or two nucleotides are deleted or substituted.
Item 3A. The aptamer of Item 1, 2, or 2A, wherein the mutant sequence of the second sequence is AGTGTGAACGCGCAGCT (SEQ ID NO: 2), AGTTGGAACCGCGCAGC (SEQ ID NO: 3), AGTGTGAACGCGCAGTC (SEQ ID NO: 4), AGTGTGAACGCGTAGCC (SEQ ID NO: 5), AGTGTGAATGCGCAGCC (SEQ ID NO: 6), GTGTGAACGCGCAGCC (SEQ ID NO: 7), AGCGTGAACGCGCAGCC (SEQ ID NO: 8), AGTGGGAACGCGCAGCC (SEQ ID NO: 9), or a sequence in which one or two nucleotides are deleted, substituted, or added in any of these sequences.
Item 4. The aptamer according to any one of Items 1 to 3, 2A, and 3A, wherein the double-stranded structure formed by X1a and X1b comprises 4 to 50 nucleotide pairs.
Item 5. The aptamer according to any one of Items 1 to 4, 2A and 3A, wherein the double-stranded structure formed by X1a and X1b contains a mismatch.
Item 6. The aptamer according to any one of Items 1 to 5, 2A, and 3A, wherein the double-stranded structure formed by X1a and X1b comprises a bulge structure.
Item 7. The aptamer according to any one of Items 1 to 6, 2A, and 3A, wherein the double-stranded structure formed by X1a and X1b contains 1 to 3 GC base pairs.
Item 8. The aptamer according to any one of Items 1 to 7, 2A, and 3A, wherein the double-stranded structure formed by X1a and X1b contains seven or more nucleotide pairs, provided that the nucleotide at the 5'-terminal position of X1a and the nucleotide at the 3'-terminal position of X1b do not form a GC base pair.
Item 8A: The aptamer according to any one of Items 1 to 8, 2A, and 3A, wherein L1 is 3 nucleotides or more in length.
Item 8B: The aptamer according to any one of Items 1 to 8, 2A, 3A and 8A, wherein L1 forms a stem-loop structure together with X1a and X1b.
Item 9. The 5'-terminal region of the oligonucleotide region 1 contains the sequence X2a, and the 3'-terminal region of the oligonucleotide region 2 contains the sequence X2b;
X2a and X2b hybridize to each other to form a double-stranded structure,
The 5' end of X2a and the 3' end of X2b may each have zero or one or more additional nucleotides and form the 5' and 3' ends of the same or different oligonucleotide molecules, or
The aptamer according to any one of Items 1 to 8, 2A, 3A, 8A and 8B, wherein the nucleotide at the 5'-end position of X2a and the nucleotide at the 3'-end position of X2b are linked via sequence L2.
Item 10. The aptamer according to Item 9, wherein the sequence X2a is linked to the 5' end of the oligonucleotide region 1, and the sequence X2b is linked to the 3' end of the oligonucleotide region 2.
Item 11. The aptamer according to Item 9 or 10, wherein the double-stranded structure formed by X2a and X2b contains a mismatch.
Item 12. The aptamer according to any one of Items 9 to 11, wherein the double-stranded structure formed by X2a and X2b comprises a bulge structure.
Item 13. The aptamer according to any one of Items 9 to 12, wherein the double-stranded structure formed by X2a and X2b comprises 1 to 8 nucleotide pairs.
Item 13A: The aptamer according to any one of Items 9 to 13, wherein L2 is 3 nucleotides or more in length.
Item 13B: The aptamer according to any one of Items 9 to 13 and 13A, wherein L2 forms a stem-loop structure together with X2a and X2b.
Item 14. A step of preparing a reaction mixture containing a target nucleic acid, at least one oligonucleotide primer, a strand-displacing DNA polymerase, and the aptamer according to any one of Items 1 to 13, 2A, 3A, 8A, 8B, 13A, and 13B;
A method for amplifying a nucleic acid comprising increasing the temperature of a reaction mixture and extending a primer with a strand-displacing DNA polymerase.
Item 15. The method according to Item 14, wherein the aptamer is present in the reaction mixture in an amount of 50 nmol or more per 1 Unit of the strand-displacing DNA polymerase.
Item 16. The method according to Item 14 or 15, wherein the temperature of the reaction mixture is increased from a temperature in the range of 0 to 30°C to a temperature in the range of 45 to 70°C.
Item 17. A composition comprising a strand-displacing DNA polymerase and the aptamer according to any one of Items 1 to 13, 2A, 3A, 8A, 8B, 13A, and 13B.
Item 18. A kit for nucleic acid amplification, comprising at least one oligonucleotide primer, a strand-displacing DNA polymerase, and the aptamer according to any one of Items 1 to 13, 2A, 3A, 8A, 8B, 13A, and 13B.

According to the present invention, non-specific amplification of target nucleic acids can be suppressed by controlling the activity of strand-displacing DNA polymerase.

FIG. 1 shows the secondary structure of oligonucleotide Bst-19-s2, which is an example of an aptamer of the present disclosure. This is an agarose gel electrophoresis photograph showing the inhibition of Bst DNA polymerase activity by an example of an aptamer of the present disclosure. This is an agarose gel electrophoresis photograph showing the inhibition of Bst DNA polymerase activity by an example of an aptamer of the present disclosure. This is an agarose gel electrophoresis photograph showing the inhibition of Bst DNA polymerase activity by an example of an aptamer of the present disclosure. This is an agarose gel electrophoresis photograph showing the inhibition of Bst DNA polymerase activity by an example of an aptamer of the present disclosure. This is an agarose gel electrophoresis photograph showing the inhibition of Bst DNA polymerase activity by an example of an aptamer of the present disclosure. This is an agarose gel electrophoresis photograph showing the inhibition of Bst DNA polymerase activity by an example of an aptamer of the present disclosure. Agarose gel electrophoresis photographs showing the inhibition of Bst DNA polymerase activity when the concentration of Bst-19-s2 is changed. This is an agarose gel electrophoresis photograph comparing the inhibitory activity of Bst-19-s2 against Bst DNA polymerase with that of other aptamers. 1 shows agarose gel electrophoresis photographs showing the inhibitory activity of Bst-19-s2 against various strand-displacing DNA polymerases. This figure shows the amino acid sequence homology of the DNA polymerase A domain of strand-displacing DNA polymerases. This figure shows a multiple alignment of the amino acid sequences of the DNA polymerase A domain of strand-displacing DNA polymerases.

The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. In this specification, numerical ranges expressed using "to" or "-" mean ranges that include the numerical values at both ends as upper and lower limits, unless otherwise specified. The upper and lower limits of each numerical range exemplified in this specification can be combined in any way.

In this disclosure, the term "oligonucleotide" refers to an oligomer or polymer composed of nucleotides as monomers, and can be used interchangeably with the terms "nucleic acid" and "polynucleotide." In an oligonucleotide, adjacent nucleotides are linked by covalent bonds such as phosphodiester bonds. Examples of oligonucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and chimeric nucleic acids containing both deoxyribonucleotides and ribonucleotides as building blocks.

In this disclosure, the term "nucleotide" includes deoxyribonucleotides found in natural DNA, i.e., dATP, dGTP, dCTP, and dTTP, and ribonucleotides found in natural RNA, i.e., ATP, GTP, CTP, and UTP, as well as analogs of these nucleotides. Examples of nucleotide analogs include dUTP (deoxyuridine triphosphate), dITP (deoxyinosine triphosphate), nucleotides in which the 2'-hydroxyl group of the ribose moiety has been replaced (e.g., nucleotides containing 2'-O-methyl-ribose, 2'-O-methoxyethyl-ribose, 2'-alkoxy-ribose, 2'-amino-ribose, and 2'-fluoro-ribose), nucleotides in which the base moiety has been modified (e.g., nucleotides containing 5-bromouracil, 5-methylcytosine, 5-iodouracil, 2-aminoadenine, and 6-methyladenine), and nucleotides in which the phosphate group has been replaced with a chemically modified phosphate group such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate.

The terms "region" and "portion" in reference to oligonucleotides are used interchangeably and both refer to one nucleotide or multiple contiguous nucleotides contained in an oligonucleotide.

In this specification, unless otherwise specified, nucleotide sequences are written from left to right in order from the 5' end to the 3' end. Furthermore, within nucleotide sequences, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents uridine.

In this disclosure, the terms "oligonucleotide aptamer" and "aptamer" refer to an oligonucleotide molecule capable of specifically binding to a strand-displacing DNA polymerase and are used interchangeably with the term "nucleic acid aptamer." An aptamer may be composed of one oligonucleotide molecule or two or more oligonucleotide molecules. Aptamers can form secondary structures, such as stem-loop structures, by hybridizing their single-stranded portions with complementary general-stranded portions within the same aptamer. The secondary structure of an aptamer can be determined using known structure prediction programs, such as mfold (M. Zuker, Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31(13), 3406-15.) or UNAFold (MARKHAM N. R. Markham, UNAFold: software for nucleic acid folding and hybridization, Methods in Molecular Biology. 2008:453:3-31.).

In this disclosure, the term "complementarity" refers to the ability of two nucleotides to pair and form Watson-Crick base pairs (A-T base pairs, G-C base pairs, A-U base pairs). This term can be used in reference to both individual nucleotides and nucleotide sequences. The percentage of complementarity between two nucleotide sequences is the percentage (%) of nucleotides that form Watson-Crick base pairs out of all nucleotides contained in the nucleotide sequence, with 100% complementarity meaning that the two nucleotide sequences are fully complementary. Two fully complementary nucleotide sequences are the same length and have no base pairs that do not form Watson-Crick base pairs, i.e., no mismatched base pairs. Less than 100% complementarity means that the two nucleotide sequences are partially complementary. In determining complementarity, chemically modified nucleotides are considered identical to unmodified nucleotides as long as they retain the ability to form Watson-Crick base pairs.

In this disclosure, the term "hybridize" refers to the binding of two oligonucleotides having complementary sequences through base pairing.

Nucleotide sequence identity refers to the percentage of identical nucleotides relative to the total overlapping nucleotides in an optimal alignment calculated using an algorithm known in the art (preferably one that can take into account the introduction of gaps into one or both of the sequences for optimal alignment). Identity can be calculated, for example, by aligning two nucleotide sequences using NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) with default settings.

In one aspect, the present disclosure provides an aptamer that has the ability to inhibit the activity of strand-displacing DNA polymerase (hereinafter also referred to as "activity inhibition ability"), and that includes oligonucleotide region 1 (hereinafter also referred to as "region 1") consisting of a sequence in which the sequence X1a is linked to the 3' end of a first sequence, TTTCCAG, or to the 3' end of a mutant sequence of the first sequence, and oligonucleotide region 2 (hereinafter also referred to as "region 2") consisting of a sequence in which the sequence X1b is linked to the 5' end of a second sequence, AGTGTGAACGCGCAGCC (SEQ ID NO: 1), or to the 5' end of a mutant sequence of the second sequence.

X1a and X1b are nucleotide sequences that can hybridize to each other to form a double-stranded structure. X1a and X1b are fully or partially complementary sequences, for example, having 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% complementarity.

As shown in the examples below, the aptamer of the present disclosure exhibits the ability to inhibit the activity of strand-displacing DNA polymerase when the double-stranded structure formed by X1a and X1b (hereinafter also referred to as "double-stranded structure X1") is four or more nucleotide pairs in length, but does not exhibit this ability when the double-stranded structure X1 is three or fewer nucleotide pairs in length. Without being bound by theory, it is believed that the secondary structure formed by the first sequence and the second sequence bound to one end of the double-stranded structure X1 exhibits the ability to bind to the strand-displacing DNA polymerase and inhibit its activity. Therefore, X1a and X1b may be any sequences as long as they are capable of forming a double-stranded structure containing four or more nucleotide pairs. The double-stranded structure X1 contains, for example, 5 or more, 6 or more, or 7 or more nucleotide pairs, or 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, or 7 or less nucleotide pairs. The double-stranded structure X1 contains, for example, 4 to 20, 4 to 15, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 4 to 8, or 4 to 7 nucleotide pairs. X1a and X1b may be the same length or different lengths as long as they are capable of forming a double-stranded structure. The end of the double-stranded structure X1 to which the first sequence and the second sequence are not bound may be a blunt end or a protruding end, and may be linked via sequence L1 to form a stem-loop structure.

In one embodiment, double-stranded structure X1 contains more A-T base pairs than G-C base pairs. Double-stranded structure X1 may contain zero G-C base pairs, or one or more G-C base pairs, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1.

In one embodiment, the double-stranded structure X1 comprises five or more, six or more, or seven or more nucleotide pairs, provided that the nucleotide at the 5'-terminal position of X1a and the nucleotide at the 3'-terminal position of X1b do not form a G-C base pair.

Double-stranded structure X1 may contain one or more, for example, 1 to 3, 1 to 2, or 1 mismatched base pair. Double-stranded structure X1 may contain one or more, for example, 1 to 3, 1 to 2, or 1 bulge structure. Double-stranded structure X1 may contain one or more, for example, 1 to 3, 1 to 2, or 1 internal loop structure.

A mutant sequence of a first sequence is a sequence in which one or more nucleotides, for example, 1 to 3, 1 to 2, or 1, have been deleted, substituted, or added from the first sequence, and has the same sequence as the first sequence apart from the deleted, substituted, or added nucleotides. A mutant sequence of a second sequence is a sequence in which one or more nucleotides, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, have been deleted, substituted, or added from the second sequence, and has the same sequence as the second sequence apart from the deleted, substituted, or added nucleotides.

There are no limitations on the type of mutation (deletion, substitution, addition) in the mutant sequence, or the type of substituted nucleotide in the case of a substitution mutation, as long as the ability to inhibit the activity of strand-displacing DNA polymerase is maintained. Nucleotide substitutions can be, for example, T for C or G, C for T, or A for C. In one embodiment, the mutant sequence of the first sequence is a sequence in which one nucleotide has been deleted, substituted, or added in the first sequence, where the nucleotide substitution is T for C or G, or C for T. In one embodiment, the mutant sequence of the second sequence is a sequence in which one nucleotide has been deleted, substituted, or added in the second sequence, where the nucleotide substitution is T for C or G, C for T, or A for C.

Examples of mutant sequences of the first sequence are CTTCCAG, TTCCAG, TTTTCAG, and TTTCTAG. Examples of mutant sequences of the first sequence are CTTCCAG, TTCCAG, TTTTCAG, TTTCTAG, and TTTCCA. Another example of a mutant sequence of the first sequence is a sequence in which one nucleotide is deleted, substituted, or added in any of the sequences CTTCCAG, TTCCAG, TTTTCAG, or TTTCTAG. Another example of a mutant sequence of the first sequence is a sequence in which one nucleotide is deleted, substituted, or added in any of the sequences CTTCCAG, TTCCAG, TTTTCAG, TTTCTAG, or TTTCCA. Examples of mutant sequences of the second sequence are AGTGTGAACGCGCAGCT (SEQ ID NO: 2), AGTTGGAACCGCGCAGC (SEQ ID NO: 3), AGTGTGAACGCGCAGTC (SEQ ID NO: 4), AGTGTGAACGCGTAGCC (SEQ ID NO: 5), AGTGTGAATGCGCAGCC (SEQ ID NO: 6), GTGTGAACGCGCAGCC (SEQ ID NO: 7), AGCGTGAACGCGCAGCC (SEQ ID NO: 8), and AGTGGGAACGCGCAGCC (SEQ ID NO: 9). Another example of a mutant sequence of the second sequence is a sequence in which one or two nucleotides are deleted, substituted, or added in any of AGTGTGAACGCGCAGCT (SEQ ID NO: 2), AGTTGTGAACGCGCAGC (SEQ ID NO: 3), AGTGTGAACGCGCAGTC (SEQ ID NO: 4), AGTGTGAACGCGTAGCC (SEQ ID NO: 5), AGTGTGAATGCGCAGCC (SEQ ID NO: 6), GTGTGAACGCGCAGCC (SEQ ID NO: 7), AGCGTGAACGCGCAGCC (SEQ ID NO: 8), and AGTGGGAACGCGCAGCC (SEQ ID NO: 9).

Aptamers in which the first sequence is replaced with a mutated sequence of the first sequence, aptamers in which the second sequence is replaced with a mutated sequence of the second sequence, and aptamers in which the first sequence and the second sequence are replaced with their respective mutated sequences can all inhibit the activity of strand-displacing DNA polymerase.

In one embodiment, the nucleotide at the 3'-end of region 1, i.e., the nucleotide at the 3'-end of X1a, and the nucleotide at the 5'-end of region 2, i.e., the nucleotide at the 5'-end of X1b, form the 3'-end and 5'-end of the same or different oligonucleotide molecules. In this embodiment, the aptamer of the present disclosure is composed of one oligonucleotide molecule containing region 1 at its 3'-end and another oligonucleotide molecule containing region 2 at its 5'-end, or is composed of one oligonucleotide molecule containing region 1 at its 3'-end and region 2 at its 5'-end. The nucleotide at the 3'-end of X1a and the nucleotide at the 5'-end of X1b may each independently be further supplemented with one or more nucleotides, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide.

In one embodiment, the nucleotide at the 3'-end of region 1, i.e., the nucleotide at the 3'-end of X1a, and the nucleotide at the 5'-end of region 2, i.e., the nucleotide at the 5'-end of X1b, are linked via sequence L1. In this embodiment, the aptamer of the present disclosure is composed of a single oligonucleotide molecule comprising region 1, L1, and region 2.

L1 may be any nucleotide sequence, and its length is not limited, as long as adjacent X1a and X1b can form a double-stranded structure. The length of L1 is, for example, 3 or more nucleotides, and may be 3 to 20, 3 to 15, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, or 3 nucleotides. L1 can form a stem-loop structure together with X1a and X1b. L1 may be, for example, GAA, CCGG, GAAA, or a complementary sequence thereof.

The aptamer of the present disclosure may include sequence X2a in the 5'-terminal region of region 1 and sequence X2b in the 3'-terminal region of region 2. X2a and X2b are nucleotide sequences that can form a double-stranded structure by hybridizing with each other. X2a and X2b are fully or partially complementary sequences, and have, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100% complementarity.

As shown in the examples below, the aptamer of the present disclosure can inhibit the activity of strand-displacing DNA polymerase whether or not it contains a double-stranded structure formed by X2a and X2b (hereinafter also referred to as "double-stranded structure X2"). Therefore, X2a and X2b may be of any length and any sequence, as long as they are capable of forming a double-stranded structure. Double-stranded structure X2 contains, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 15, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide pair. X2a and X2b may be the same length or different lengths, as long as they are capable of forming a double-stranded structure. The ends of double-stranded structure X2 distal to region 1 and region 2 may be blunt or overhanging, and may be linked via sequence L2 to form a stem-loop structure.

In some embodiments, the double-stranded structure X2 contains 1 to 8, 2 to 8, or 3 to 8 nucleotide pairs.

Double-stranded structure X2 may contain one or more, for example, 1 to 3, 1 to 2, or 1 mismatch. Double-stranded structure X2 may contain one or more, for example, 1 to 3, 1 to 2, or 1 bulge structure. Double-stranded structure X2 may contain one or more, for example, 1 to 3, 1 to 2, or 1 internal loop structure.

In one embodiment, the nucleotide at the 5'-terminal position of X2a and the nucleotide at the 3'-terminal position of X2b form the 5'-terminal and 3'-terminal ends of the same or different oligonucleotide molecules. In this embodiment, the aptamer of the present disclosure is composed of one oligonucleotide molecule comprising X2a at its 5'-terminal end and region 1 in the 3'-terminal region of X2a, and another oligonucleotide molecule comprising X2b at its 3'-terminal end and region 2 in the 5'-terminal region of X2b, or is composed of one oligonucleotide molecule comprising X2a at its 5'-terminal end, region 1 in the 3'-terminal region of X2a, region 2 in the 3'-terminal region of region 1, and X2b at its 3'-terminal end. The nucleotide at the 5'-terminal position of X2a and the nucleotide at the 3'-terminal position of X2b may each independently have one or more nucleotides added, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide added.

In one embodiment, the nucleotide at the 5'-terminal position of X2a and the nucleotide at the 3'-terminal position of X2b are linked via sequence L2. In this embodiment, the aptamer of the present disclosure is composed of a single oligonucleotide molecule comprising Region 2-X2b-L2-X2a-Region 1.

L2 may be any nucleotide sequence, and its length is not limited, as long as adjacent X2a and X2b can form a double-stranded structure. The length of L2 is, for example, 3 or more nucleotides, and may be 3 to 20, 3 to 15, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, or 3 nucleotides. L2 can form a stem-loop structure together with X2a and X2b. L2 may be, for example, GAA, CCGG, GAAA, or a complementary sequence thereof.

The oligonucleotide molecules constituting the aptamers of the present disclosure may be composed of deoxyribonucleotides, ribonucleotides, or analogs of these nucleotides, for example, deoxyribonucleotides. Furthermore, the oligonucleotide molecules may have a 3'-cap and/or a 5'-cap.

The oligonucleotide molecules that make up the aptamers of the present disclosure may be linear or cyclic. Here, a linear oligonucleotide molecule refers to an oligonucleotide in which the phosphate group at the 5' end and the hydroxyl group at the 3' end are not bound to other nucleotides. Furthermore, a cyclic oligonucleotide molecule refers to an oligonucleotide that does not have a free end.

In certain embodiments, the aptamer of the present disclosure is any of the following a) to g):
a) A combination of one linear oligonucleotide molecule consisting of TTTCCAG-X1a and one linear oligonucleotide molecule consisting of X1b-AGTGTGAACGCGCAGCC
b) one oligonucleotide molecule consisting of TTTCCAG-X1a-L1-X1b-AGTGTGAACGCGCAGCC
c) a combination of one linear oligonucleotide molecule consisting of X2a-TTTCCAG-X1a and one linear oligonucleotide molecule consisting of X1b-AGTGTGAACGCGCAGCC-X2b
d) one linear oligonucleotide molecule consisting of X1b-AGTGTGAACGCGCAGCC-X2b-L2-X2a-TTTCCAG-X1a
e) one circular oligonucleotide molecule consisting of X1b-AGTGTGAACGCGCAGCC-X2b-L2-X2a-TTTCCAG-X1a, in which the nucleotide at the 3'-terminal position of X1a and the nucleotide at the 5'-terminal position of X1b are linked to each other;
f) one linear oligonucleotide molecule consisting of X2a-TTTCCAG-X1a-L1-X1b-AGTGTGAACGCGCAGCC-X2b
g) one circular oligonucleotide molecule consisting of X2a-TTTCCAG-X1a-L1-X1b-AGTGTGAACGCGCAGCC-X2b, in which the nucleotide at the 5'-terminal position of X2a and the nucleotide at the 3'-terminal position of X2b are linked to each other;

As described above, the oligonucleotide molecules included in a) to g) above may have one or more nucleotides, for example, 1 to 3, 1 to 2, or 1, deleted, substituted, or added in the TTTCCAG portion of the first sequence, and may have one or more nucleotides, for example, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, deleted, substituted, or added in the AGTGTGAACGCGCAGCC portion of the second sequence. Furthermore, when X1a, X1b, X2a, or X2b are free ends, they may each independently have one or more nucleotides, for example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, added. In addition, in the cyclic oligonucleotide molecule of e), the nucleotide at the 3'-terminal position of X1a and the nucleotide at the 5'-terminal position of X1b may be linked via sequence L1, and in the cyclic oligonucleotide molecule of g), the nucleotide at the 5'-terminal position of X2a and the nucleotide at the 3'-terminal position of X2b may be linked via sequence L2.

The total nucleotide length of the oligonucleotide molecules constituting the aptamer of the present disclosure is, for example, 25 or more nucleotides, 30 or more nucleotides, 35 or more nucleotides, 40 or more nucleotides, 45 or more nucleotides, 50 or more nucleotides, 55 or more nucleotides, or 60 or more nucleotides, or, for example, 200 or less nucleotides, 150 or less nucleotides, 120 or less nucleotides, 110 or less nucleotides, 100 or less nucleotides, 90 or less, 800 or less nucleotides, 75 or less nucleotides, 70 or less, 65 or less, or 60 or less nucleotides. Here, the total nucleotide length refers to the nucleotide length of the oligonucleotide molecule when the aptamer is composed of a single oligonucleotide molecule, and refers to the sum of the nucleotide lengths of all of the oligonucleotide molecules when the aptamer is composed of multiple oligonucleotide molecules.

X1a, X1b, X2a, X2b, L1, and L2 may be designed to prevent unintended hybridization within or between the oligonucleotide molecules that make up the aptamer. For example, X1a and X1b may be designed not to hybridize with sequences other than X1b and X1a, respectively; X2a and X2b may be designed not to hybridize with sequences other than X2b and X2a, respectively; and L1 and L2 may be designed not to hybridize with other sequences.

In certain embodiments, the aptamer of the present disclosure may be designed so that the sequence of the oligonucleotide molecule that constitutes the aptamer contains a nucleotide that is selectively recognized by a specific enzyme, for example, at either position in the sequence of region 1 or region 2. Here, the specific enzyme is an enzyme that has the ability to recognize the nucleotide in question and cleave an oligonucleotide containing it, but does not recognize other nucleotides, such as deoxyribonucleotides found in natural DNA. Examples of such nucleotides and enzymes include dUTP and uracil DNA glycosylase, and dITP and endonuclease V. An aptamer containing such a nucleotide inhibits the activity of a strand-displacing DNA polymerase when coexisting with the aptamer, and is degraded by reacting with an enzyme that selectively recognizes the nucleotide, allowing the strand-displacing DNA polymerase to regain its activity.

Examples of aptamers of the present disclosure are shown in Tables 1A, 1B and 2 below.

The present disclosure also provides an aptamer whose sequence is identical to the above-described aptamer, but which lacks at least one internucleotide covalent bond. This aptamer may lack any internucleotide bond, for example, a bond between nucleotides in either the sequence of region 1 or region 2, as long as it is capable of forming the secondary structure of the above-described aptamer upon binding to a strand-displacing DNA polymerase, i.e., a secondary structure formed by the first sequence and the second sequence bound to one end of the double-stranded structure X1.

The linear oligonucleotide molecules that make up the aptamers of the present disclosure can be prepared by known chemical synthesis methods. Furthermore, the cyclic oligonucleotide molecules that make up the aptamers of the present disclosure can be prepared by ligating the 5' and 3' ends of the corresponding linear oligonucleotide molecules using a chemical such as cyanogen bromide or an enzyme such as T4 ligase. The aptamers of the present disclosure can be prepared by denaturing the constituent oligonucleotide molecules by heating, followed by slowly cooling them to ambient temperature to allow annealing.

The aptamer disclosed herein has the ability to inhibit the activity of strand displacement DNA polymerase and can inhibit the activity of strand displacement DNA polymerase at ambient temperatures, for example, 0 to 30°C. The ability to inhibit the activity of strand displacement DNA polymerase refers to the ability to at least partially reduce the activity of strand displacement DNA polymerase, for example, by 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 100%. A 100% reduction in strand displacement DNA polymerase activity means complete inhibition of strand displacement DNA polymerase activity. The percentage of activity inhibition can be calculated as the percentage decrease in enzyme activity in the presence of the aptamer compared to the absence of the aptamer by creating a calibration curve showing the relationship between the amount of reaction product synthesized within a certain time period under certain reaction conditions and enzyme activity, and calculating the corresponding enzyme activity from the amount of reaction product synthesized in the presence and absence of the aptamer under the same reaction conditions and time period.

Strand displacement DNA polymerases are enzymes that have DNA polymerase activity and strand displacement activity and can synthesize DNA strands while dissociating hydrogen bonds in template DNA. Examples of strand displacement DNA polymerases include Bst DNA polymerases, such as Bst DNA polymerase Large fragment, Bst 2.0 DNA polymerase (New England Biolabs), and Bst 3.0 DNA polymerase (New England Biolabs), Bsu DNA polymerase, Csa DNA polymerase, DNA polymerase I Large (Klenow) Fragment, 96-7 DNA polymerase, and Bca DNA polymerases, such as BcaBEST (registered trademark) DNA polymerase. Examples of such polymerases include BcaBEST (registered trademark) DNA polymerase and BcaBEST (registered trademark) DNA polymerase ver. 2.0 (both 5'→3' exonuclease-deficient DNA polymerases derived from Bacillus caldotenax, TAKARA Bio), Aac DNA polymerase (DNA polymerase derived from Alicyclobacillus acidocaldarius), Vent (Exo-) DNA polymerase (New England Biolabs), and Deep Vent (Exo-) DNA polymerase (New England Biolabs).

Bst DNA polymerase is a protein consisting of 878 amino acids derived from Geobacillus stearothermophilus (formerly known as Bacillus stearothermophilus). Its amino acid sequence is registered in GenBank under accession number AAB52611.1. The large fragment of Bst DNA polymerase is a protein lacking the N-terminal 292 amino acids of Bst DNA polymerase, i.e., a protein consisting of the C-terminal 586 amino acids of the amino acid sequence registered in GenBank under accession number AAB52611.1. The large fragment of Bst DNA polymerase has 5'→3' DNA polymerase activity but does not have 5'→3' exonuclease activity, making it ideal for use in DNA amplification.

The ability of the aptamer disclosed herein to inhibit the activity of strand displacement DNA polymerase is reversible; when the temperature is raised from ambient temperature, the ability to inhibit activity is partially or completely lost, but when the temperature is returned to ambient temperature, the ability to inhibit activity is restored. Without being bound by theory, it is believed that the aptamer disclosed herein forms a secondary structure that is capable of binding to strand displacement DNA polymerase at ambient temperature, but when the temperature is raised from ambient temperature, the secondary structure changes, losing its ability to bind to strand displacement DNA polymerase and weakening its ability to inhibit activity, and when the temperature is lowered again, the secondary structure is formed again, allowing it to bind to strand displacement DNA polymerase and inhibit its activity.

The aptamer disclosed herein exhibits the ability to inhibit the activity of strand displacement DNA polymerase at temperatures lower than the temperature at which DNA amplification by strand displacement DNA polymerase is performed (hereinafter also referred to as the "reaction temperature"), for example, within the range of 0-30°C, 0-35°C, or 0-40°C, while it loses its ability to inhibit the activity of strand displacement DNA polymerase at reaction temperatures within the range of, for example, 45-70°C, 50-70°C, 55-70°C, 60-70°C, 45-65°C, 50-65°C, 55-65°C, or 60-65°C. The reaction temperature may vary depending on the strand displacement DNA polymerase. The reaction temperature for Bst DNA polymerase is preferably 55-70°C, more preferably 60-65°C.

The aptamer disclosed herein can inhibit the activity of strand-displacing DNA polymerase at temperatures lower than the reaction temperature, thereby enabling DNA amplification reactions that suppress the production of nonspecific amplification products at temperatures lower than the reaction temperature. The aptamer disclosed herein can be suitably used in DNA amplification reactions that use strand-displacing DNA polymerase, particularly in isothermal DNA amplification reactions. Examples of such DNA isothermal amplification reactions include LAMP (Loop-Mediated Isothermal Amplification), RCA (Rolling Circle Amplification), NEAR (Nicking Enzyme Amplification Reaction), SDA (Strand Displacement Amplification), SMAP (SMartAmplification Process), NASBA (Nucleic Acid Sequence-Based Amplification), ICAN (registered trademark) (Isothermal and Chimeric primer-initiated Amplification of Nucleic Acids), TRC (transcription-reverse transcription concerted), and TMA (transcription-mediated amplification).

In one aspect, the present disclosure provides a method for amplifying a nucleic acid, the method comprising the steps of: preparing a reaction mixture containing a target nucleic acid, at least one oligonucleotide primer, a strand-displacing DNA polymerase, and the above-described aptamer; increasing the temperature of the reaction mixture; and extending the primer with the strand-displacing DNA polymerase.

The reaction mixture is an aqueous solution containing a target nucleic acid that serves as a template for nucleic acid amplification, at least one oligonucleotide primer that contains a sequence complementary to a portion of the sequence of the target nucleic acid, a strand-displacing DNA polymerase, and an effective amount of the above-mentioned aptamer for inhibiting the activity of the strand-displacing DNA polymerase. The reaction mixture can be prepared at a temperature lower than the reaction temperature, preferably at a temperature sufficiently lower than the reaction temperature (e.g., 0-4°C), and at this time, the activity of the strand-displacing DNA polymerase is inhibited by the aptamer.

When preparing the reaction mixture, there are no restrictions on the order in which the target nucleic acid, oligonucleotide primer, strand-displacing DNA polymerase, and the above-mentioned aptamer are mixed. The reaction mixture may be prepared by pre-mixing the aptamer and strand-displacing DNA polymerase and then adding the other components, or it may be prepared by adding and mixing all the components at once.

The reaction mixture also contains reagents necessary for the primer extension reaction using a strand-displacing DNA polymerase, such as dNTPs, metal ions (e.g., magnesium ions, manganese ions), buffers, etc. When the target nucleic acid is RNA, the reaction mixture further contains a reverse transcriptase. The reaction mixture may also contain a dye (e.g., calcein, hydroxynaphthol blue) necessary for detecting the amplification product.

In the reaction mixture, the aptamer is present in an amount effective to inhibit the activity of the strand-displacing DNA polymerase. In one embodiment, the strand-displacing DNA polymerase is Bst DNA polymerase, and the effective amount is 50 nmol or more per unit of polymerase, e.g., 50-500 nmol, 50-400 nmol, 50-300 nmol, 50-200 nmol, 50-150 nmol, or 50-100 nmol. In one embodiment, the effective amount is 60 nmol or more per unit of Bst DNA polymerase Large Fragment, e.g., 60-500 nmol, 60-400 nmol, 60-300 nmol, 60-200 nmol, 60-150 nmol, or 60-100 nmol. Here, one unit of polymerase is defined as the amount of enzyme required to incorporate 10 nmol of dNTP into acid-insoluble material at 65°C for 30 minutes.

Increasing the temperature of the reaction mixture weakens the aptamer's inhibitory ability, and the strand-displacing DNA polymerase becomes active. The temperature of the reaction mixture is increased from a temperature lower than the reaction temperature to the reaction temperature, for example, from a temperature within the range of 0-30°C, 0-35°C, or 0-40°C, to a temperature within the range of 45-70°C, 50-70°C, 55-70°C, 60-70°C, 45-65°C, 50-65°C, 55-65°C, or 60-65°C. At the reaction temperature, the primers are extended by the strand-displacing DNA polymerase, and the target nucleic acid is amplified.

In one embodiment, the strand-displacing DNA polymerase is Bst DNA polymerase, and the reaction temperature is preferably 55 to 70°C, more preferably 60 to 65°C. In this case, the aptamer preferably has a double-stranded structure X1 that contains 5 or more, 6 or more, or 7 or more nucleotide pairs, with the proviso that the nucleotide at the 5'-terminal position of X1a and the nucleotide at the 3'-terminal position of X1b do not form a G-C base pair. Furthermore, the double-stranded structure X2 preferably contains 1 to 8, 2 to 8, or 3 to 8 nucleotide pairs.

In one aspect, the present disclosure provides a composition comprising a strand-displacing DNA polymerase and an effective amount of the above-described aptamer. The composition may be in a liquid state, such as an aqueous solution, or in a dry state. The composition can be used in the above-described method for amplifying nucleic acids.

In one aspect, the present disclosure provides a kit for nucleic acid amplification comprising at least one oligonucleotide primer, a strand-displacing DNA polymerase, and an effective amount of the above-described aptamer. In the kit, the strand-displacing DNA polymerase and the aptamer may be in the form of the above-described composition. The kit may further comprise reagents necessary for the primer extension reaction using the strand-displacing DNA polymerase, a dye necessary for detecting the amplification product, a reverse transcriptase, equipment used in the reaction, instructions for use, etc. The kit can be used in the above-described method for amplifying nucleic acids.

The present invention will be explained in more detail by the following examples, but the present invention is not limited to these examples.

[Example 1]
Single-stranded oligonucleotides (DNA) consisting of the sequences shown in SEQ ID NOS: 10 to 74 were synthesized by Eurofins Corporation. Tables 3 and 4 show the complete nucleotide sequences of each oligonucleotide, as well as sequences capable of forming intramolecular duplexes (for Bst-19-s2-f and Bst-19-s2-r, sequences capable of hybridizing to each other to form duplexes) single- and double-underlined. These oligonucleotides are Bst-19-s2 and variants based on Bst-19-s2. The secondary structure of Bst-19-s2 (Gibbs free energy change dG = -5.82) calculated using UNAFold (MARKHAM N. R. Markham, UNAFold: software for nucleic acid folding and hybriziation, Methods in Molecular Biology. 2008:453:3-31.) is shown in Figure 1. The alterations in the variants are shown in Tables 1A, 1B, and 2.

For each of the oligonucleotides listed in Tables 3 and 4, a 1 μM PBS solution was prepared, and annealing was carried out using a thermal cycler under the following conditions.
97℃ 2min→85℃ 1min(0.3℃/sec)→75℃ 1min(0.3℃/sec)→65℃ 1min(0.3℃/sec)→55℃ 1min(0.3℃/sec)→45℃ 1min(0.3℃/sec)→35℃ 1min(0.3℃/sec)→25℃ 1min(0.3℃/sec) →4℃ 1min→25℃ forever

Next, the inhibitory activity against Bst DNA polymerase was evaluated by the RCA method as follows. A reaction mixture was prepared containing 100 nM of Universal Primer (SEQ ID NO: 80), 4 ng of M13 ssDNA (TAKARA Bio), 1.5 mM each of dNTPs, 1 unit of Bst DNA Polymerase (Nippon Gene Co., Ltd.), and 100 nM of annealed oligonucleotides in 10 μl of 1x Bst Reaction Buffer (a 10-fold dilution of 10x Bst Reaction Buffer (Nippon Gene Co., Ltd.)), and the reaction was carried out for 30 minutes at 30°C, 35°C, 40°C, or 60°C. For Bst-19-s2-f and Bst-19-s2-r, reaction mixtures containing either alone and both were prepared, and the reaction was carried out.

The reaction mixture was analyzed by agarose gel electrophoresis using a 0.8% agarose gel. 1 kbp Ladder PLUS (Nihon Genetics) was used as a marker. Strand displacement DNA polymerases continue DNA synthesis while dissociating double-stranded DNA even after the template has become double-stranded DNA, producing amplification products with higher molecular weights than the template. The presence of a band at a different position from M13 ssDNA allows evaluation of activity against Bst DNA polymerase and its inhibition by the aptamer.

The results are shown in Figures 2 to 7. In the figures, "Bst polymerase -" or "Bst pol. -" indicates the lane corresponding to the negative control reaction mixture containing no Bst DNA polymerase, in which only a band corresponding to the template M13 ssDNA was detected. Also, in the figures, "Aptamer-" indicates the lane corresponding to the positive control reaction mixture containing no oligonucleotide, in which a smear band corresponding to the amplification product produced by Bst DNA polymerase was detected. The inhibition of Bst DNA polymerase activity by each oligonucleotide was evaluated by comparison with these negative and positive controls.

Bst-19-s2 inhibited Bst DNA polymerase activity at 30°C and 40°C, but this inhibition disappeared at 60°C (Figs. 2, 4, and 6). Bst-19-s2-f and Bst-19-s2-r, which are partial sequences derived by truncating the CCGG portion of Bst-19-s2 (corresponding to sequence L1), did not inhibit Bst DNA polymerase activity alone at either temperature. However, when the two sequences were mixed and annealed, Bst-19-s2-f and -r inhibited Bst DNA polymerase activity at 30°C, but no inhibition was observed at 60°C (Fig. 5). Furthermore, Bst-19-s2-m26, which was derived by linking the 5' and 3' ends of Bst-19-s2 with the common tetraloop sequence GAAA and truncating the CCGG portion, exhibited inhibition similar to that observed with Bst-19-s2 (Figs. 6 and 7). This indicates that for the inhibition of Bst DNA polymerase activity, it is important that the aptamer has a secondary structure formed by the first and second sequences bound to one end of the double-stranded structure X1, that a loop structure is not essential in the double-stranded structure X1, and that the aptamer may be composed of one or two oligonucleotide molecules. Furthermore, Bst-19-s2-m29, in which the CCGG portion of Bst-19-s2 (corresponding to sequence L1) was replaced with the general tetraloop sequence GAAA, exhibited inhibition similar to that of Bst-19-s2 (Figure 5), indicating that the inhibitory ability of the aptamer against Bst DNA polymerase activity does not depend on the sequence of the loop portion, even when a loop structure is present in the double-stranded structure X1.

Oligonucleotides in which the length or sequence of sequence X1a or X1b forming the double-stranded structure X1 in Bst-19-s2-m26 has been changed (Bst-19-s2-m26-1, Bst-19-s2-m26-2, Bst-19-s2-m26-3, Bst-19-s2-m26-4, Bst-19-s2-m26-5, Bst-19-s2-m26-6, Bst-19-s2-m26-7, Bst-19-s2-m26-8, Bst-19 The following oligonucleotides (Bst-19-s2-m26-r, Bst-19-s2-m26-9, Bst-19-s2-m26-10, Bst-19-s2-m26-11 and Bst-19-s2-m26-12) inhibited the activity of Bst DNA polymerase at 30°C but not at 60°C when X1a and X1b were four nucleotides or longer in length, whereas they did not inhibit activity even at 30°C when X1a and X1b were three nucleotides or shorter in length (Figures 6 and 7). In addition, oligonucleotides in which the length or sequence of the sequence X1a or X1b forming the double-stranded structure X1 in Bst-19-s2 has been changed (Bst-19-s4-m1, Bst-19-s5-m1, Bst-19-s6-m1, Bst-19-s7-m1, Bst-19-s2-m1, Bst-19-s2-m7, Bst-19-s2-m8, Bst-19-s2-m9, Bst-19-s2-m10, Bst-19-s2-m11, Bst-19-s2-m12, Bst-19-s2-m13, Bst-19-s2-m14, Bst-19-s2-m15, Bst-19-s2-m16, Bst-19-s2-m17, Bst-19-s2-m18, Bst-19-s2-m19 ...9, Bst-19-s2-m19, Bst-19- All of the oligonucleotides (Bst-19-s2-m15, Bst-19-s2-m16, Bst-19-s2-m17, Bst-19-s2-m18, Bst-19-s2-m19, Bst-19-s2-m20, Bst-19-s2-m21, Bst-19-s2-m22, Bst-19-s2-m23, Bst-19-s2-m30 and Bst-19-s2-m37) inhibited the activity of Bst DNA polymerase at 30°C or 35°C, and none of the oligonucleotides except Bst-19-s2-m37 inhibited the activity of Bst DNA polymerase at 60°C (Figures 3 to 5). Taken together with these results, it was shown that to inhibit the activity of Bst DNA polymerase, X1a and X1b contained in the aptamer need only be four nucleotides in length, and are independent of the sequence of X1a and X1b. Furthermore, to eliminate the activity inhibition by the aptamer at 60°C, when the double-stranded structure X1 contains seven or more nucleotide pairs, it is preferable to design the nucleotide at the 5'-terminal position of X1a and the nucleotide at the 3'-terminal position of X1b so that they do not form a G-C base pair.

All of the oligonucleotides (Bst-19-s2-m27, Bst-19-s2-m28, Bst-19-s2-m31, Bst-19-s2-m32, Bst-19-s2-m33, Bst-19-s2-m34, Bst-19-s2-m35, Bst-19-s2-m36, Bst-19-s2-m37, Bst-19-s2-m38, and Bst-19-s2-m39) containing a single nucleotide substitution in the first or second sequence of Bst-19-s2 inhibited the activity of Bst DNA polymerase at 30°C, whereas none of the oligonucleotides except Bst-19-s2-m37 inhibited the activity of Bst DNA polymerase at 60°C (Figure 5). This demonstrates that even if mutations are present in the first or second sequence, the aptamer still has the ability to inhibit the activity of Bst DNA polymerase.

Oligonucleotides in Bst-19-s2 in which the length or sequence of sequence X2a or X2b forming the double-stranded structure X2 has been changed (Bst-19-s4, Bst-19-s5, Bst-19-s6, Bst-19-s7, Bst-19-s4-m1, Bst-19-s5-m1, Bst-19-s6-m1, Bst-19-s7-m1, Bst-19, Bst-19-s1, Bst-19 All of the oligonucleotides (Bst-19-s1, Bst-19-s2, Bst-19-s3, Bst-19-s8, Bst-19-s9, Bst-19-s10, Bst-19-s11, and Bst-19-s12) inhibited the activity of Bst DNA polymerase at 30°C or 40°C, and none of the oligonucleotides other than Bst-19 and Bst-19-s1 inhibited the activity of Bst DNA polymerase at 60°C (Figures 2 and 6). Taken together with other results, this indicates that an aptamer does not need to have a double-stranded structure X2 to inhibit the activity of Bst DNA polymerase, but that in order to eliminate the activity inhibition by the aptamer at 60°C, it is preferable to design the double-stranded structure X2 to contain up to eight nucleotide pairs.

[Example 2]
Reaction mixtures were prepared in the same manner as in Example 1, except that the Bst-19-s2 concentration in the reaction mixture was varied between 10 and 100 nM. The reaction mixtures were incubated at 30°C for 30 minutes. Figure 8 shows the agarose gel electrophoresis results of the reaction mixtures. Bst-19-s2 was confirmed to inhibit the activity of Bst DNA polymerase at concentrations of 50 nM or higher. This concentration corresponds to 50 nmol or more per unit of Bst DNA polymerase.

[Example 3]
To compare their inhibitory activities against Bst DNA polymerase, reaction mixtures were prepared as in Example 1 using Bst-19-s2, five aptamers Bst-X-1 to Bst-X-5 (SEQ ID NOS: 75-79) described in Chinese Patent Application Publication No. 114990126, and the aptamer included with Bst 2.0 WarmStart® DNA Polymerase (NEW ENGLAND Biolabs). Reactions were performed at 30°C, 40°C, or 60°C for 30 minutes. Agarose gel electrophoresis of the reaction mixtures is shown in Figure 9. Unlike Bst-19-s2, none of Bst-X-1 to Bst-X-5 or WarmStart sufficiently inhibited Bst DNA polymerase even at 30°C, and this tendency was more pronounced at 40°C.

[Example 4]
The activity inhibition by the aptamer Bst-19-s2 was evaluated using various strand-displacing DNA polymerases. A reaction mixture (10 μl) containing 100 nM of Universal Primer (SEQ ID NO: 80), 4 ng of M13 ssDNA (TAKARA Bio), 1.5 mM each of dNTPs, 1 unit of strand-displacing DNA polymerase, and 200 nM of annealed Bst-19-s2 was prepared and incubated at 30°C for 20 minutes. The strand-displacing DNA polymerases and buffers used are listed below.
Bst DNA polymerase (New England Biolabs)
1x ThermoPol Reaction Buffer (New England Biolabs) + 8 mM MgSO ( _final concentration)
Bst 2.0 DNA polymerase (New England Biolabs)
1x Isothermal Amplification Buffer (New England Biolabs) + 8 mM _MgSO4 (final concentration)
Bst 3.0 DNA polymerase (New England Biolabs)
1x Isothermal Amplification Buffer (New England Biolabs) + 8 mM _MgSO4 (final concentration)
Bsu DNA polymerase 1x NEBuffer 2 (New England Biolabs)
Csa DNA polymerase 1x Csa Reaction Buffer (Nippon Gene Co., Ltd.)
Klenow Fragment
1x NEBuffer 2 (New England Biolabs)
96-7 DNA polymerase (Nippon Gene Co., Ltd.)
1x 96-7 Reaction Buffer (Nippon Gene Co., Ltd.)
BcaBEST (registered trademark) DNA polymerase ver.2.0 (TAKARA Bio)
1x BcaBEST Buffer (TAKARA Bio)
This buffer contained 200 μM of each dNTP, and the amount of dNTPs added was adjusted so that the final concentration of each in the reaction mixture was 1.5 mM.

The results of agarose gel electrophoresis of the reaction mixture are shown in Figure 10. Bst-19-s2 exhibited weak inhibitory activity against Klenow fragment and strong inhibitory activity against other strand-displacing DNA polymerases.

The amino acid residues in the active site of Bst DNA polymerase and its related strand-exchange DNA polymerases are highly conserved and are known to form a domain structure known as the DNA polymerase A domain (registered in the protein domain database SMART under accession number SM00482). Within this domain, amino acid residues involved in substrate binding or catalysis (D705, E710, Y766, R841, N845, Q849, R668, D882, E883) have been identified (Igor Oscorbin et al., Computational and Structural Biotechnoly Journal, 2023 Sep 12:21:4519-4535. doi: 10.1016/j.csbj.2023.09.008.). The aptamers disclosed herein, including Bst-19-s2, are thought to interact with this active domain and exhibit inhibitory activity.

The amino acid sequence of the active domain of the strand-displacing DNA polymerase whose inhibitory activity was confirmed in this example was analyzed using the UniProt Align tool. The information on the amino acid sequence used for the analysis is shown below.
Amino acid sequence of the DNA polymerase A domain of Bst DNA polymerase (GenBank: AAB52611.1) (SEQ ID NO: 81)
Amino acid sequence of the DNA polymerase A domain of Bca DNA polymerase (GenBank: BAA02361.1) (SEQ ID NO: 82)
Amino acid sequence (SEQ ID NO: 83) of the DNA polymerase A domain of Bsu DNA polymerase (NCBI Reference Sequence: NP_390787.1)
Amino acid sequence (SEQ ID NO: 84) of the DNA polymerase A domain of 96-7 DNA polymerase (described as SEQ ID NO: 3 in WO2011/055737)
Amino acid sequence (SEQ ID NO: 85) of the DNA polymerase A domain of Csa DNA polymerase (NCBI Reference Sequence: WP_188816633.1)
Amino acid sequence (SEQ ID NO: 86) of the DNA polymerase A domain of Klenow fragment (NCBI Reference Sequence: NP_418300.1)

The amino acid sequence homology of the DNA polymerase A domain is shown in Figure 11. The DNA polymerase A domain of all strand-displacing DNA polymerases had a homology of 50% or more to the DNA polymerase A domain of Bst DNA polymerase. In particular, the DNA polymerase A domains of Bca DNA polymerase, Bsu DNA polymerase, 96-7 DNA polymerase, and Csa DNA polymerase, for which Bst-19-s2 showed strong inhibitory activity, had a homology of 70% or more to the DNA polymerase A domain of Bst DNA polymerase. This suggests that the aptamer of the present disclosure, including Bst-19-s2, exhibits inhibitory activity against strand-displacing DNA polymerases having a DNA polymerase A domain with an amino acid sequence homology of 50% or more, preferably 70% or more, to the DNA polymerase A domain of Bst DNA polymerase.

Furthermore, a multiple alignment of the amino acid sequences of the DNA polymerase A domain is shown in Figure 12. It was suggested that the aptamers disclosed herein, including Bst-19-s2, exhibit inhibitory activity against strand-displacing DNA polymerases having DNA polymerase A domains that contain amino acid residues conserved in the DNA polymerase A domains of all strand-displacing DNA polymerases, preferably amino acid residues conserved in the DNA polymerase A domains of the five types of DNA polymerase A domains: Bca DNA polymerase, Bsu DNA polymerase, 96-7 DNA polymerase, and Csa DNA polymerase.

Claims (18)

an oligonucleotide region 1 consisting of a sequence in which the sequence X1a is linked to the 3' end of the first sequence TTTCCAG or to the 3' end of a mutant sequence of the first sequence, and an oligonucleotide region 2 consisting of a sequence in which the sequence X1b is linked to the 5' end of the second sequence AGTGTGAACGCGCAGCC (SEQ ID NO: 1) or to the 5' end of a mutant sequence of the second sequence;
X1a and X1b are each independently a sequence of 4 nucleotides or more in length, and form a double-stranded structure by hybridizing with each other;
The mutant sequence of the first sequence is a sequence in which one or two nucleotides are deleted, substituted, or added in the first sequence,
The mutant sequence of the second sequence is a sequence in which 1 to 3 nucleotides are deleted, substituted, or added in the second sequence,
The 3' end of X1a and the 5' end of X1b may each have zero or one or more additional nucleotides and form the 3' and 5' ends of the same or different oligonucleotide molecules, or
The nucleotide at the 3'-terminal position of X1a and the nucleotide at the 5'-terminal position of X1b are linked via the sequence L1.
An aptamer capable of inhibiting the activity of strand-displacing DNA polymerase. The aptamer of claim 1, wherein the mutant sequence of the first sequence is CTTCCAG, TTCCAG, TTTTCAG, TTTCTAG, TTTCCA, or any of these sequences in which one nucleotide has been deleted or substituted. The aptamer of claim 1, wherein the mutant sequence of the second sequence is AGTGTGAACGCGCAGCT (SEQ ID NO: 2), AGTTGGAACCGCGCAGC (SEQ ID NO: 3), AGTGTGAACGCGCAGTC (SEQ ID NO: 4), AGTGTGAACGCGTAGCC (SEQ ID NO: 5), AGTGTGAATGCGCAGCC (SEQ ID NO: 6), GTGTGAACGCGCAGCC (SEQ ID NO: 7), AGCGTGAACGCGCAGCC (SEQ ID NO: 8), AGTGGGAACGCGCAGCC (SEQ ID NO: 9), or a sequence in which one or two nucleotides are deleted or substituted in any of these sequences. The aptamer of claim 1, wherein the double-stranded structure formed by X1a and X1b contains 4 to 50 nucleotide pairs. The aptamer of claim 1, wherein the double-stranded structure formed by X1a and X1b contains a mismatch. The aptamer of claim 1, wherein the double-stranded structure formed by X1a and X1b includes a bulge structure. The aptamer of claim 1, wherein the double-stranded structure formed by X1a and X1b contains 1 to 3 G-C base pairs. The aptamer of claim 1, wherein the double-stranded structure formed by X1a and X1b contains seven or more nucleotide pairs, with the proviso that the nucleotide at the 5'-terminal position of X1a and the nucleotide at the 3'-terminal position of X1b do not form a G-C base pair. The oligonucleotide region 1 contains the sequence X2a in the 5'-terminal region, and the oligonucleotide region 2 contains the sequence X2b in the 3'-terminal region,
X2a and X2b hybridize to each other to form a double-stranded structure,
The 5' end of X2a and the 3' end of X2b may each have zero or one or more additional nucleotides and form the 5' and 3' ends of the same or different oligonucleotide molecules, or
The aptamer of claim 1, wherein the nucleotide at the 5'-terminal position of X2a and the nucleotide at the 3'-terminal position of X2b are linked via sequence L2. The aptamer of claim 9, wherein sequence X2a is linked to the 5' end of oligonucleotide region 1 and sequence X2b is linked to the 3' end of oligonucleotide region 2. The aptamer of claim 9, wherein the double-stranded structure formed by X2a and X2b contains a mismatch. The aptamer of claim 9, wherein the double-stranded structure formed by X2a and X2b includes a bulge structure. The aptamer of claim 9, wherein the double-stranded structure formed by X2a and X2b contains 1 to 8 nucleotide pairs. Preparing a reaction mixture comprising a target nucleic acid, at least one oligonucleotide primer, a strand-displacing DNA polymerase, and the aptamer of any one of claims 1 to 13;
A method for amplifying a nucleic acid comprising: increasing the temperature of a reaction mixture; and extending a primer with a strand-displacing DNA polymerase. The method of claim 14, wherein the aptamer is present in the reaction mixture in an amount of 50 nmol or more per 1 unit of strand-displacing DNA polymerase. The method of claim 14, wherein the temperature of the reaction mixture is increased from a temperature in the range of 0 to 30°C to a temperature in the range of 45 to 70°C. A composition comprising a strand-displacing DNA polymerase and the aptamer described in any one of claims 1 to 13. A kit for nucleic acid amplification, comprising at least one oligonucleotide primer, a strand-displacing DNA polymerase, and the aptamer according to any one of claims 1 to 13.