Protein and peptide libraries
AssigneeThe General Hospital Corporation
InventorsDouglas A. Treco, Alonso Ricardo
Life of this patent
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Abstract
Provided herein are, inter alia, methods for linking an mRNA molecule to a polypeptide (e.g., a peptide or a protein) by linking the mRNA molecule to a linking amino acid in the polypeptide, or by linking the mRNA molecule to a linking tRNA to which the polypeptide is attached, via reactions not catalyzed by the ribosome, and methods for making polypeptide libraries. Also provided are mRNA-protein complexes and mRNA-tRNA-protein complexes, libraries containing these complexes, and methods of using these complexes.
Claims24
(canceled)
A method for linking an mRNA molecule to a polypeptide, the method comprising:
- (a) providing an mRNA molecule comprising a reactive nucleoside that is reactive with a crosslinker, wherein the crosslinker is an alkylated nucleoside;
- (b) providing a translation system comprising a linking aminoacyl-tRNA comprising an amino acid residue linked to a linking tRNA by a covalent bond, wherein the linking tRNA comprises an anticodon comprising a crosslinker that is an alkylated nucleoside;
- (c) translating the mRNA molecule in the translation system to produce a polypeptide into which the amino acid residue, still linked to the tRNA, is incorporated;
- and
- (d) during or after step (c), crosslinking the reactive nucleoside of the mRNA molecule to the crosslinker of the anticodon of the linking tRNA, thereby linking the mRNA molecule to the polypeptide through the linking tRNA.
(canceled)
-20. (canceled)
A method for linking an mRNA molecule to a polypeptide, the method comprising:
- (a) providing an mRNA molecule comprising a nucleophilic nucleoside partner;
- (b) providing a translation system comprising a linking aminoacyl-tRNA comprising an amino acid residue linked to a linking tRNA by a covalent bond, wherein the linking tRNA comprises an anticodon comprising a crosslinker, wherein the crosslinker is an electrophilic nucleoside that reacts with a nucleophilic nucleoside partner via hybridization-triggered alkylation;
- (c) translating the mRNA molecule in the translation system to produce a polypeptide into which the amino acid residue, still linked to the linking tRNA, is incorporated;
- and
- (d) during or after step (c), crosslinking the crosslinker of the linking tRNA to the nucleophilic nucleoside partner of the mRNA molecule, thereby linking the mRNA molecule to the polypeptide through the linking tRNA.
A method for linking an mRNA molecule to a polypeptide, the method comprising:
- (a) providing an mRNA molecule comprising a first member of a complementary crosslinker pair;
- (b) providing a translation system comprising a linking aminoacyl-tRNA comprising an amino acid residue linked to a linking tRNA by a covalent bond, wherein the linking tRNA comprises an anticodon comprising a second member of the complementary crosslinker pair, wherein the complementary crosslinker pair is selected from the following pairs:
- (i) 2-thiouridine and an adenosine containing an olefin substitution at the 2-position of the purine ring, (ii) 4-thiouridine and 2-amino-6 vinylpurine, and (iii) 4-thiouridine and an adenosine containing a vinyl substituent at the 8-position of the ring;
- (c) translating the mRNA molecule in the translation system to produce a polypeptide into which the amino acid residue, still linked to the linking tRNA, is incorporated;
- and
- (d) during or after step (c), crosslinking the first member and the second member of the complementary crosslinker pair, thereby linking the mRNA molecule to the polypeptide through the linking tRNA.
-54. (canceled)
Dependencies are inferred from the claim text.