Artificial base pair capable of forming specific base pair

A method of hybridizing a nucleic acid containing an unnatural base comprising:

• hybridizing a first nucleic acid strand containing a nucleotide having an unnatural base represented by Formula I:
• wherein,
• A1 represents N or CH;
• A2 represents CH;
• R1 represents hydrogen or an amino group;
• and
• R2 represents a substituent selected from the group consisting of a 2-thienyl group, a 2-thiazolyl group, a 2-imidazolyl group, a 2,2′-bithien-5-yl group, a 2-(2-thiazolyl)thien-5-yl group, a 5-(2-thienyl)thiazol-2-yl group, and a 2,2′,5′,2″-terthien-5-yl group or a derivative thereof as the base with a second nucleic acid strand which contains or which does not contain a nucleotide having an unnatural base represented by Formula I or a derivative thereof as the base,
• wherein the unnatural base represented by Formula I or the derivative thereof in the first nucleic acid strand forms a base pair with any natural base or the unnatural base represented by Formula I or the derivative thereof in the second nucleic acid strand;
• wherein the unnatural base derivative thereof is represented by Formula I in which the R2 is further modified by addition of C1-C3 alkyl group, a C1-C3 alkenyl group, or a C1-C3 alkynyl group;
• and
• wherein the unnatural base derivative thereof is represented by Formula I further comprising a protecting group selected from a group consisting of:
• a phenoxyacetyl group, an isobutyryl group, and a dimethyl formamidyl group.

A method of identifying a single-base mutation, comprising the steps of:

• (a) preparing a probe complementary to a sequence containing a single-base mutation site to be identified in a target nucleic acid, wherein the probe includes a nucleotide having an unnatural base represented by Formula I:
• wherein,
• A1 represents N or CH;
• A2 represents CH;
• R1 represents hydrogen or an amino group;
• and
• R2 represents a substituent selected from the group consisting of a 2-thienyl group, a 2-thiazolyl group, a 2-imidazolyl group, a 2,2′-bithien-5-yl group, a 2-(2-thiazolyl)thien-5-yl group, a 5-(2-thienyl)thiazol-2-yl group, and a 2,2′,5′,2″-terthien-5-yl group or a derivative thereof as the base at a position adjacent to the nucleotide complementary to the single-base mutation site to be identified;
• wherein the unnatural base derivative thereof is represented by Formula I in which the R2 is further modified by addition of C1-C3 alkyl group, a C1-C3 alkenyl group, or a C1-C3 alkynyl group;
• and
• wherein the unnatural base derivative thereof is represented by Formula I further comprising a protecting group selected from a group consisting of:
• a phenoxyacetyl group, an isobutyryl group, and a dimethyl formamidyl group;
• (b) hybridizing a target nucleic acid strand with the probe of (a);
• and
• (c) identifying that a target nucleic acid hybridized with the probe has a base complementary to the probe at the single-base mutation site to be identified.

A method of identifying a single-base mutation, comprising the steps of:

• (a) preparing a primer complementary to a sequence containing a single-base mutation site to be identified in a target nucleic acid, wherein the primer includes a nucleotide having an unnatural base represented by Formula I:
• wherein,
• A1 represents N or CH;
• A2 represents CH;
• R1 represents hydrogen or an amino group;
• and
• R2 represents a substituent selected from the group consisting of a 2-thienyl group, a 2-thiazolyl group, a 2-imidazolyl group, a 2,2′-bithien-5-yl group, a 2-(2-thiazolyl)thien-5-yl group, a 5-(2-thienyl)thiazol-2-yl group, and a 2,2′,5′,2″-terthien-5-yl group or a derivative thereof as the base at a position adjacent to the nucleotide complementary to the single-base mutation site to be identified;
• wherein the unnatural base derivative thereof is represented by Formula I in which the R2 is further modified by addition of C1-C3 alkyl group, a C1-C3 alkenyl group, or a C1-C3 alkynyl group;
• and
• wherein the unnatural base derivative thereof is represented by Formula I further comprising a protecting group selected from a group consisting of:
• a phenoxyacetyl group, an isobutyryl group, and a dimethyl formamidyl group;
• (b) performing PCR employing a target nucleic acid as the template and using the primer of (a), a nucleotide substrate having an unnatural base represented by Formula I or a derivative thereof as the base, a natural nucleotide substrate, and a DNA polymerase;
• and
• (c) identifying that a target nucleic acid amplified by the PCR has a base complementary to the primer at a single-base mutation site to be identified.

A method of capturing a nucleic acid using a probe containing an unnatural base, comprising the steps of:

• (a) preparing a probe containing a nucleotide having an unnatural base represented by Formula I:
• wherein,
• A1 represents N or CH;
• A2 represents CH;
• R1 represents hydrogen or an amino group;
• and
• R2 represents a substituent selected from the group consisting of a 2-thienyl group, a 2-thiazolyl group, a 2-imidazolyl group, a 2,2′-bithien-5-yl group, a 2-(2-thiazolyl)thien-5-yl group, a 5-(2-thienyl)thiazol-2-yl group, and a 2,2′,5′,2″-terthien-5-yl group or a derivative thereof as the base, and immobilizing the probe to a solid phase,
• wherein the unnatural base derivative thereof is represented by Formula I in which the R2 is further modified by addition of C1-C3 alkyl group, a C1-C3 alkenyl group, or a C1-C3 alkynyl group;
• and
• wherein the unnatural base derivative thereof is represented by Formula I further comprising a protecting group selected from a group consisting of:
• a phenoxyacetyl group, an isobutyryl group, and a dimethyl formamidyl group;
• (b) adding a tag composed of a sequence complementary to the probe of (a) to a nucleic acid to be captured, wherein the tag includes a nucleotide having an unnatural base represented by Formula I or a derivative thereof as the base at a position complementary to the nucleotide having the unnatural base represented by Formula I or the derivative thereof as the base in the probe;
• and
• (c) capturing a nucleic acid by hybridizing the probe immobilized to the solid phase in (a) with the nucleic acid provided with the tag in (b).

A method of synthesizing a peptide by an in vitro translation system, comprising the steps of:

• (a) preparing a transfer RNA containing a nucleotide having an unnatural base represented by Formula I:
• wherein,
• A1 represents N or CH;
• A2 represents CH;
• R1 represents hydrogen or an amino group;
• and
• R2 represents a substituent selected from the group consisting of a 2-thienyl group, a 2-thiazolyl group, a 2-imidazolyl group, a 2,2′-bithien-5-yl group, a 2-(2-thiazolyl)thien-5-yl group, a 5-(2-thienyl)thiazol-2-yl group, and a 2,2′,5′,2″-terthien-5-yl group or a derivative thereof as the base at an anticodon position,
• wherein the unnatural base derivative thereof is represented by Formula I in which the R2 is further modified by addition of C1-C3 alkyl group, a C1-C3 alkenyl group, or a C1-C3 alkynyl group;
• and
• wherein the unnatural base derivative thereof is represented by Formula I further comprising a protecting group selected from a group consisting of:
• a phenoxyacetyl group, an isobutyryl group, and a dimethyl formamidyl group;
• (b) preparing a messenger RNA containing a nucleotide having an unnatural base represented by Formula I or a derivative thereof as the base in a coding sequence, where the nucleotide having an unnatural base represented by Formula I or a derivative thereof as the base in the messenger RNA is arranged as a codon corresponding to the anticodon position of the transfer RNA containing the unnatural base so that the nucleotides each having an unnatural base represented by Formula I or a derivative thereof as the base form a base pair in hybridization between codon and anticodon;
• and
• (c) synthesizing a peptide by in vitro translation using the transfer RNA of (a), the messenger RNA of (b), and ribosome.