Ribozymes with broad tRNA aminoacylation activity
AssigneeThe Research Foundation of State University of New York
InventorsHiraoki Suga, Hiroshi Murakami, Hirohide Saito
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Abstract
The present invention provides catalytic RNA molecules having cis or trans aminoacylation activity. The catalytic RNA molecules having cis aminoacylation activity comprise a catalytic domain and an aminoacylation domain. The catalytic RNA molecules having trans aminoacylation activity only have the catalytic domain. A method is provided for constructing and screening of these molecules. These molecules are suitable for aminoacylating with specific amino acids.
Claims36
A polynucleotide comprising a sequence encoding a ribozyme, said ribozyme being capable of aminoacylating a tRNA with a non-cognate amino acid.
A method of aminoacylating a tRNA with a non-cognate amino acid comprising the steps of:
- a) providing a ribozyme capable of aminoacylating tRNA with a non-cognate amino acid;
- b) providing a tRNA;
- c) providing a non-cognate amino acid for the tRNA;
- and
- d) combining a), b) and c) to effect aminoacylation of the tRNA with the non-cognate amino acid.
A method of making a polypeptide having a desired non-cognate amino acid at a selected position comprising the steps of:
- a) providing a ribozyme capable of aminoacylating a tRNA with a non-cognate amino acid;
- b) aminoacylating a tRNA with a non-cognate amino acid by the ribozyme of a);
- c) providing an mRNA having a codon corresponding to the anticodon of the tRNA of b);
- and,
- d) combining b), c) and a translation mixture to obtain a polypeptide having the desired non-cognate amino acid at the selected position.
A kit for aminoacylating tRNAs with non-cognate amino acids comprising:
- a) a ribozyme capable of aminoacylating tRNAs with non-cognate amino acids;
- b) one or more non-cognate amino acids.
- c) tRNAs.
A method of making a polypeptide having a first amino acid substituted in place of a second amino acid comprising the steps of:
- a) providing a tRNA having an anticodon corresponding to the second amino acid and being aminoacylated with the first amino acid;
- b) combining
- 1) an mRNA encoding the polypeptide, wherein the mRNA comprises a codon for the second amino acid;
- 2) the tRNA from a);
- 3) an in vitro translation mixture;
- whereby the translation product of the combination from b) is a polypeptide comprising the first amino acid in place of the second amino acid.
Dependencies are inferred from the claim text.