Method for producing diverse libraries of encoded polypeptides
AssigneeWhitehead Institute for Biomedical Research
InventorsCharles Everett Merryman, David P. Bartel
Life of this patent
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Abstract
tRNA analogues which comprise a tRNA, such as tRNAphe; an amino acid moiety which acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer and, thus, is stably linked to the acceptor stem of the tRNA; and a reactive or activatible moiety near or within the anticodon stem loop of the tRNA that can medidate the covalent coupling of the tRNA analogue to mRNA. Also described are polypeptide-tRNA analogue-mRNA fusions; libraries of encoded polypeptides; methods of producing and screening the libraries; and target members and their uses.
Claims11
A polypeptide-tRNA analogue-mRNA fusion, comprising:
- (a) a polypeptide;
- (b) a tRNA analogue comprising:
- (i) an amino acid moiety linked to the tRNA wherein the link is modified structurally so that the amino acid moiety acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer, wherein the amino acid moiety is at the 3′ end of the tRNA;
- and
- (ii) ac activatible moiety near or within the anticodon stem-loop that can become reactive and thereby mediate the specific, stable coupling of the tRNA analogue to mRNA;
- and
- (c) mRNA which encodes the polypeptide of (a),
- wherein the tRNA analogue is:
- located between the polypeptide and the mRNA;
- linked to the carboxy terminus of the polypeptide by a covalent bond between the terminal amino acid residue of the polypeptide and the amino acid moiety and, linked to the mRNA by covalent bonds between the reactive moiety of the tRNA analogue and the mRNA.
A polypeptide-tRNA analogue-mRNA fusion, comprising:
- (a) a polypeptide;
- (b) a tRNA analogue comprising:
- (i) an amino acid moiety linked to the tRNA wherein the link is modified structurally so that the amino acid moiety acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer, wherein the amino acid moiety is at the 3′ end of the tRNA;
- and
- (ii) an activatible moiety near or within the anticodon stem-loop that can become reactive and thereby mediate the specific, stable coupling of the tRNA analogue to mRNA;
- and
- (c) mRNA which encodes the polypeptide of (a),
- wherein the tRNA analogue is:
- located between the polypeptide and the mRNA;
- linked to the polypeptide by a covalent bond between the terminal amino acid residue of the carboxy terminus of the polypeptide and the amino acid moiety;
- and linked to the mRNA by the action of UV irradiation that produces a covalent bond between a modified base near or within the tRNA stem loop and the mRNA.
A polypeptide-tRNA analogue-mRNA fusion, comprising:
- (a) a polypeptide;
- (b) a tRNA analogue comprising:
- (i) an amino acid moiety linked to the tRNA wherein the link is modified structurally so that the amino acid moiety acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer, wherein the amino acid moiety is at the 3′ end of the tRNA;
- and
- (ii) an activatible Y base within the anticodon stem-loop that can become reactive and thereby mediate the specific, stable coupling of the tRNA analogue to mRNA;
- and
- mRNA which encodes the polypeptide of (a),
- wherein the tRNA analogue is:
- located between the polypeptide and the mRNA;
- linked to the carboxy terminus of the polypeptide by a covalent bond between the terminal amino acid residue of the polypeptide and the amino acid moiety and, linked to the mRNA by covalent bonds between the Y-base of the tRNA analogue and the mRNA.
Dependencies are inferred from the claim text.