Method for producing protein-nucleic acid conjugate, and method for detecting target substance

1. A method for producing a protein-nucleic acid conjugate, the method comprising:

a 3′-terminal addition step of adding at least one nucleoside derivative to a 3′-terminal of a nucleic acid using a 3′-terminal addition enzyme which adds a nucleotide to a 3′-terminal of a nucleic acid, the nucleoside triphosphate derivative comprising a glutamine (Gln) residue, and

a protein binding step of binding a protein to the glutamine (Gln) residue of the nucleoside triphosphate derivative, using a transglutaminase (TGase), the protein comprising a lysine (Lys) residue and a labeling moiety,

wherein the nucleoside triphosphate derivative is represented by formula (1) shown below:

wherein each of X and Y independently represents an alkylene group having a carbon number of 1 to 48 or an alkenylene group having a carbon number of 2 to 48 which may be substituted with an ethenylene group, —(C 2 H 4 O) n — group or —(C 3 H 6 O) n — group, where n═2, 4, 8, 12 or 24,

wherein Z represents an alkyl group having a carbon number of 1 to 48, an alkoxy group having a carbon number of 1 to 48, an aryl group having a carbon number of 6 to 48, an aryloxy group having a carbon number of 6 to 48, an arylalkyl group having a carbon number of 7 to 48, or an arylalkyloxy group having a carbon number of 7 to 48, which may be substituted with a dinitrophenyl group or an L-3,4-dihydroxyphenyl group,

wherein at least one of Y and Z may be independently substituted with an amino acid other than Lys,

wherein B represents a hydrogen atom or a hydroxyl group,

wherein C represents a hydrogen atom or a hydroxyl group,

wherein m represents 0 or 1, and

wherein the 3′-terminal addition enzyme introduces the at least one nucleoside triphosphate derivate in a non-template dependent manner.

2. The method for producing a protein-nucleic acid conjugate according to claim 1 , wherein

the 3′-terminal addition enzyme is at least one of terminal deoxynucleotidyl transferase (TdT), Family A-type DNA polymerase, and poly(A) RNA polymerase.

3. The method for producing a protein-nucleic acid conjugate according to claim 1 , wherein

X represents an ethenylene group, Y represents a methylene group, and Z represents a benzyloxy group.

4. A method for detecting a target substance, the method comprising:

binding, via nucleic acid moieties

a protein-nucleic acid conjugate, which is obtained by a method for producing a protein-nucleic acid conjugate comprising:

a 3′-terminal addition step of adding at least one nucleoside triphosphate derivative to a 3′-terminal of a nucleic acid using a 3′-terminal addition enzyme which adds a nucleotide to a 3′-terminal of a nucleic acid, the nucleoside triphosphate derivative comprising a glutamine (Gln) residue, and

a protein binding step of binding a protein to the glutamine (Gln) residue of the nucleoside triphosphate derivative, using a transglutaminase (TGase) the protein comprising a lysine (Lys) residue and a labeling moiety, and

a target substance which exists within a target material, and

detecting the bound protein-nucleic acid conjugate by the labeling moiety,

wherein the nucleoside triphosphate derivative is represented by formula (1) shown below:

wherein each of X and Y independently represents an alkylene group having a carbon number of 1 to 48 or an alkenylene group having a carbon number of 2 to 48 which may be substituted with an ethenylene group, —(C 2 H 4 O) n — group or —(C 3 H 6 O)n- group where n═2, 4, 8, 12 or 24,

wherein Z represents an alkyl group having a carbon number of 1 to 48, an alkoxy group having a carbon number of 1 to 48, an aryl group having a carbon number of 6 to 48, an aryloxy group having a carbon number of 6 to 48, an arylalkyl group having a carbon number of 7 to 48, or an arylalkyloxy group having a carbon number of 7 to 48, which may be substituted with a dinitrophenyl group or an L-3,4-dihydroxyphenyl group,

wherein at least one of Y and Z may be independently substituted with an amino acid other than Lys,

wherein B represents a hydrogen atom or a hydroxyl group,

wherein C represents a hydrogen atom or a hydroxyl group, and

wherein m represents 0 or 1, and

wherein the 3′-terminal additional introduces the at least one nucleoside triphoshphate derivate in a non dependent manner.

5. The method for producing a protein-nucleic acid conjugate according to claim 1 , wherein the 3′-terminal addition enzyme is terminal deoxynucleotidyl transferase (TdT).

6. The method for producing a protein-nucleic acid conjugate according to claim 1 , wherein the 3′-terminal addition enzyme is Family A-type DNA polymerase.

7. The method for producing a protein-nucleic acid conjugate according to claim 1 , wherein the 3′-terminal addition enzyme is poly(A) RNA polymerase.

8. The method for producing a protein-nucleic acid conjugate according to claim 4 , wherein the 3′-terminal addition enzyme is at least one of terminal deoxynucleotidyl transferase (TdT), Family A-type DNA polymerase, and poly(A) RNA polymerase.

9. The method for producing a protein-nucleic acid conjugate according to claim 4 , wherein X represents an ethenylene group, Y represents a methylene group, and Z represents a benzyloxy group.

10. The method for producing a protein-nucleic acid conjugate according to claim 4 , wherein the 3′-terminal addition enzyme is terminal deoxynucleotidyl transferase (TdT).

11. The method for producing a protein-nucleic acid conjugate according to claim 4 , wherein the 3′-terminal addition enzyme is Family A-type DNA polymerase.

12. The method for producing a protein-nucleic acid conjugate according to claim 4 , wherein the 3′-terminal addition enzyme is poly(A) RNA polymerase.