Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1999-04-14
2002-08-27
Gitomer, Ralph (Department: 1618)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069700
Reexamination Certificate
active
06440695
ABSTRACT:
BACKGROUND OF THE INVENTION
Large combinatorial libraries of biopolymers are starting points for isolating new enzymes, binding motifs and other useful molecules. For example, current technologies can generate populations of nucleic acids with complexities on the order of 10
15
molecules and then isolate and identify a single molecule with a desired activity. Random polypeptide populations have greater chemical diversity than do polynucleotides, making them an attractive alternative to nucleic acids. Current systems are limited in their ability to easily generate large complex libraries of polypeptides that are in a form that allows the isolation and identification of rare molecules with a desired activity.
SUMMARY OF THE INVENTION
The present invention relates to tRNA analogues; polypeptide-tRNA-analogue-mRNA fusions; diverse libraries of encoded polypeptides; and a method of producing the diverse libraries. tRNA analogues of the present invention comprise a tRNA (such as a yeast tRNA); an amino acid moiety that acts as an acceptor substrate, but not as a donor substrate, for ribosome-directed peptidyl transfer; and a reactive or activatible moiety near or within the anticodon stemp loop of the tRNA that can mediate the stable coupling of the tRNA analogue to mRNA. An amino acid moiety is stably linked to the tRNA if the linkage between the two or the chemical environment allows the amino acid moiety to act as an acceptor substrate but not as a donor substrate for ribosome-directed peptidyl transfer. In a specific embodiment, the tRNA analogue is a 3′-amino-3′-deoxyadenosine-substituted tRNA in which the 3′-terminal A of yeast tRNA
phe
is replaced or substituted by 3′-amino-3′-deoxyadenosine and then the substituted tRNA is charged with phenylalaine. This tRNA analogue is termed PHE-N-tRNA. In another specific embodiment, the tRNA analogue is a puromycin-substituted tRNA in which the 3′-terminal A of yeast tRNA
phe
is replaced or substituted by puromycin, with the result that the amino acid moiety is the methoxytyrosine moiety of puromycin. In both embodiments, the reactive or activatible moiety near or within the anticodon stem-loop is the modified Y base of yeast tRNA
phe
.
Also the subject of this invention are polypeptide-tRNA analogue-mRNA fusions, in which: the polypeptide can be a peptide or polypeptide of any size; the tRNA analogue is as described herein; and the mRNA is mRNA which encodes the polypeptide component of the fusion. In a fusion, the tRNA analogue component is: (a) located between the polypeptide and the mRNA which encodes the polypeptide; (b) linked to the polypeptide by a stable bond between the terminal amino acid residue of the polypeptide and the amino acid moiety of the tRNA analogue; and (c) linked to the mRNA by crosslinks between a reactive or activatible moiety of the tRNA analogue and the mRNA. In one embodiment, the polypeptide-tRNA analogue-mRNA fusion includes a PHE-N-tRNA (and, thus, the amino acid moiety which acts as an acceptor substrate but cannot act as a donor substrate is phenylalanine) and the tRNA is yeast tRNA
phe
. In another embodiment, the polypeptide-tRNA analogue-mRNA fusion includes a puromycin-substituted tRNA (and, thus, the amino acid moiety which acts as an acceptor substrate but cannot act as a donor substrate is the methoxytyrosine moiety of puromycin) and the tRNA is yeast tRNA
phe
.
Diverse libraries or collections of encoded polypeptides are also the subject of this invention. A diverse library or collection comprises the encoded polypeptides, which are the polypeptide-tRNA analogue-mRNA fusions described herein. Methods of producing such libraries are also the subject of this invention.
The invention further relates to a method of screening a diverse encoded polypeptide library to identify target members (library members with desired biological or biochemical properties or activities, such as binding to a particular ligand or enzymatic activity). In one embodiment of screening a diverse encoded polypeptide library to identify a target member or members, the diverse library is initially enriched in molecules with desired properties. This is done, for example, to identify a binding partner or ligand of interest using known enrichment methods, such as affinity enrichment using an immobilized ligand or binding partner or to identify a library member with enzymatic activity by assessing affinity of a library member to a product of a reaction in which the enzyme has modified itself or a substrate to which the library member is attached. Library members identified in this way are target members. In a further step in the method, a library which has been enriched in target members (an enriched fusion or encoded diverse polypeptide library) is amplified and subjected to additional enrichment. For example, the enriched library is reverse transcribed, thereby producing cDNAs of the mRNA components; the cDNAs are, optionally, amplified. The initially produced cDNAs or the resulting PCR products are subjected to in vitro transcription, thereby producing an amplified pool of mRNAs that encode members of the enriched fusion library. The amplified pool of mRNAs is subjected to in vitro translation in the presence of the tRNA analogue of the present invention, producing an amplified versionof the enriched encoded polypeptide library. Library members (fusions) amplified in this manner are, optionally, subjected to further enrichment and amplification, as necessary, until target members are enriched to a level where they are present in sufficient numbers to be detected. They are detected using any known method, such as binding to a ligand of interest or catalyzing a reaction of interest. mRNAs of target members are cloned and then individual fusions made from cloned mRNAs are screened for the desired properties (e.g., by ligand binding or catalyzing a reaction of interest). Library members identified in this way are target members, which are also a subject of this invention.
Target members are polypeptide-tRNA analogue-mRNA fusions. The translation products of the enriched mRNA (the polypeptide component of a target member) which display properties of interest and modified or engineered derivatives of the translation products which display properties of interest are target polypeptide fragments. These fragments are also a subject of this invention. As used herein, the term target polypeptide fragments includes fragments released or separated from target members in which they occur and fragments produced or synthesized by another method (e.g., chemical synthesis, mRNA translation in the absence of the tRNA analogue or a recombinant DNA method in which DNA encoding a desired target polypeptide fragment is expressed). Target polypeptide fragments include, but are not limited to, protein catalysts, single-chain monoclonal antibodies, binding pair members (ligand or binding partner), receptors or their ligands, and enzymes and their substrates.
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Bartel David P.
Merryman Charles Everett
Gitomer Ralph
Hamilton Brook Smith & Reynolds P.C.
Whitehead Institute for Biomedical Research
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