Modification of D- and T-arms of tRNA enhancing D-amino acid and β-amino acid uptake
The purpose of the present invention is to provide, for translation in a cell-free translation system, a novel translation system capable of synthesizing a peptide having therein consecutive non-proteinogenic amino acids. The present invention provides a tRNA containing the base sequence represented by SEQ ID NO: 1 and encoding a non-proteinogenic amino acid.
1. A tRNA comprising the base sequence of SEQ ID NO: 1 and encoding a non-proteinogenic amino acid
(SEQ ID NO: 1)
N 1 N 2 GCN 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 GCN 12 N 13
wherein N 1 to N 13 each represents an arbitrary base, N 3 to N 11 form a D-loop, and N 1 N 2 GC forms a base pair with N 13 N 12 CG, and
said tRNA is an elongator tRNA whereby the base at its 5′ end forms a base pair with corresponding base on the 3′ side of its acceptor stem.
2. The tRNA according to claim 1 , further comprising the base sequence of SEQ ID NO: 2
(SEQ ID NO: 2)
AGGGG(N 14 ) m CCCCU
wherein N 14 represents an arbitrary base, m stands for an integer of 1 or more, (N 14 ) m form a T-loop, and AGGGG forms a base pair with UCCCC.
3. The tRNA according to claim 2 , wherein the non-proteinogenic amino acid is a D-amino acid, a β-amino acid, or an α,α-disubstituted amino acid.
4. A translation system for the synthesis of a peptide having two or more consecutive non-proteinogenic amino acids, comprising the tRNA of claim 2 .
5. A method of constructing a peptide library, comprising a step of translating in a cell-free translation system by using the tRNA of claim 2 .
6. A method of constructing a library of a complex between a peptide and an mRNA encoding the peptide, comprising a step of translating in a cell-free translation system by using the tRNA of claim 2 .
7. The tRNA according to claim 1 , wherein the non-proteinogenic amino acid is a D-amino acid, a β-amino acid, or an α,α-disubstituted amino acid.
8. A translation system for the synthesis of a peptide having two or more consecutive non-proteinogenic amino acids, comprising the tRNA of claim 7 .
9. A method of constructing a peptide library, comprising a step of translating in a cell-free translation system by using the tRNA of claim 7 .
10. A method of constructing a library of a complex between a peptide and an mRNA encoding the peptide, comprising a step of translating in a cell-free translation system by using the tRNA of claim 7 .
11. A translation system for the synthesis of a peptide having two or more consecutive non-proteinogenic amino acids, comprising the tRNA of claim 1 .
12. A method of constructing a peptide library, comprising a step of translating in a cell-free translation system by using the tRNA of claim 1 .
13. A method of constructing a library of a complex between a peptide and an mRNA encoding the peptide, comprising a step of translating in a cell-free translation system by using the tRNA of claim 1 .
14. A tRNA comprising the base sequence of SEQ ID NO: 1 and encoding a non-proteinogenic amino acid
N 1 N 2 GCN 3 N 4 N 5 N 6 N 7 N 8 N 9 N 10 N 11 GCN 12 N 13 (SEQ ID NO: 1)
wherein N 1 to N 13 each represents an arbitrary base, N 3 to N 11 form a D-loop, and N 1 N 2 GC forms a base pair with N 13 N 12 CG, and further comprising the base sequence of SEQ ID NO: 2
(SEQ ID NO: 2)
AGGGG(N 14 ) m CCCCU
wherein N 14 represents an arbitrary base, m stands for an integer of 1 or more, (N 14 ) m form a T-loop, and AGGGG forms a base pair with UCCCC, and
said tRNA is an elongator tRNA whereby the base at its 5′ end forms a base pair with corresponding base on the 3′ side of its acceptor stem.
15. The tRNA according to claim 14 , wherein the integer is 2 or more.