Patent ID: 8715984
Filing Date: 2014-05-06
Classification: C12N,C12P,C12Q,G01N

Abstract:
1. A method of screening a modified tRNA for improving aminoacylation reaction of a tRNA by a noncanonical amino acid, comprising: (a) preparing a modified suppressor tRNA comprising an unnatural base at the first or the second position of the anticodon of a suppressor tRNA, wherein said unnatural base comprises a 2-oxo-1H-pyridine-3-yl (y) group which may have a substituted group at position 5, a 2-oxo-1H-imidazole-3-yl (z) group, a 2-formyl-1H-pyrrole- 1-yl (Pa) group which may have a substituted group at position 4 or a 2-nitro-1H-pyrrole-1-yl (Pn) group which may have a substituted group at position 4; (b) measuring an aminoacylation activity of the modified tRNA with standard and noncanonical amino acids, and a mutant aminoacyl-tRNA svnthetase that is: a mutant aminoacyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 in which the glutamic acid at position 418 is replaced with aspartic acid, asparagine or glutamine; a mutant aminoacyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 in which the glutamic acid at position 420 is replaced with aspartic acid, asparagine, glutamine, lysine or arginine; a mutant aminoacvl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 in which the glutamic acid at position 418 is replaced with aspartic acid, asparagine or glutamine, and the glutamic acid at position 420 is replaced with aspartic acid, asparagine or glutamine: or a mutant aminoacvl-tRNA synthetase having the amino acid sequence of SEQ ID NO:2 in which the glutamic acid at position 418 is replaced with asparagine, the glutamic acid at position 420 is replaced with asparagine and the threonine at position 423 is replaced with valine; and (c) selecting the modified tRNA, in which an incorporating amount of the noncanonical amino acid(s) is increased or an incorporating rate of the noncanonical amino acid is improved compared to that of the standard amino acid(s), as a modified tRNA having improved aminoacylation activity with a noncanonical amino acid.