Totally synthetic affinity reagents

Claim 1

A method for identifying a protein, polypeptide and/or peptide which binds to a ligand of choice, comprising: screening a library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins containing (a) a binding domain encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB) n J Z 3' with a second nucleotide sequence of the formula 3'Z'O U (NNV).sub.m Y5' where X and Y are restriction enzyme recognition sites, such that X≠Y; N is A, C, G or T; B is G, T or C; V is G, A or C; n is an integer, such that 10≦n≦100; m is an integer, such that 10≦m≦100; Z and Z' are each a sequence of 6, 9 or 12 nucleotides, such that Z and Z' are complementary to each other; and J is A, C, G, T or nothing; O is A, C, G, T or nothing; and U is G, A, C or nothing; provided, however, if any one of J, O or U is nothing then J, O and U are all nothing, and converting the annealed oligonucleotides to a double stranded oligonucleotide, and (b) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain, by contacting the plurality of heterofunctional fusion proteins with said ligand of choice under conditions conducive to ligand binding and isolating any fusion prosthesis which bind said ligand.

Claim 2

A method for identifying a protein, polypeptide and/or peptide which binds to a ligand of choice, comprising: screening a library of recombinant vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins containing (a) a binding domain encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB) n J Z 3' with a second nucleotide sequence of the formula 3'Z'O U (NNV).sub.m Y5' where X and Y are restriction enzyme recognition sites, such that X≠Y; N is A, C, G or T; B is G, T or C; V is G, A or C; n is an integer, such that 10≦n≦100; m is an integer, such that 10≦m≦100; Z and Z' are each a sequence of 6, 9 or 12 nucleotides, such that Z and Z' are complementary to each other; and J is A, C, G, T or nothing; O is A, C, G, T or nothing; and U is G, A, C or nothing; provided, however, if any one of J, O or U is nothing then J, O and U are all nothing, and converting the annealed oligonucleotides to a double stranded oligonucleotide, and (b) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain, by contacting the plurality of heterofunctional fusion proteins with said ligand of choice under conditions conducive to ligand binding and isolating any fusion prosthesis which bind said ligand.

Claim 14

A method for identifying a protein and/or peptide which binds to a ligand of choice, comprising: (a) generating a library of vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins containing (i) a binding domain encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB) n J Z 3' with a second nucleotide sequence of the formula 3'Z'O U (NNV)m Y 5' where X and Y are restriction enzyme recognition sites, such that X≠Y; N is A, C, G or T; B is G, T or C; V is G, A or C; n is an integer, such that 10≦n≦100; m is an integer, such that 10≦m≦100; Z and Z' are each a sequence of 6, 9 or 12 nucleotides, such that Z and Z' are complementary to each other; and J is A, C, G, T or nothing; O is A, C, G, T or nothing; and U is G, A, C or nothing; provided, however, if any one of J, O or U is nothing then J, O and U are all nothing, and converting the annealed oligonucleotides to a double stranded oligonucleotide, (ii) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain; and (b) screening the library of vectors by contacting the plurality of heterofunctional fusion proteins with said ligand of choice under conditions conducive to ligand binding and isolating any heterofunctional fusion protein which binds said ligand.

Claim 15

A method for identifying a protein and/or peptide which binds to a ligand of choice, comprising: (a) generating a library of vectors in which each vector encodes one of a plurality of heterofunctional fusion proteins containing (i) a binding domain encoded by a double stranded oligonucleotide assembled by annealing a first nucleotide sequence of the formula 5' X (NNB) n J Z 3' with a second nucleotide sequence of the formula 3'Z'O U (NNV)m Y 5' where X and Y are restriction enzyme recognition sites, such that X≠Y; N is A, C, G or T; B is G, T or C; V is G, A or C; n is an integer, such that 10≦n≦100; m is an integer, such that 10≦m≦100; Z and Z' are each a sequence of 6, 9 or 12 nucleotides, such that Z and Z' are complementary to each other; and J is A, C, G, T or nothing; O is A, C, G, T or nothing; and U is G, A, C or nothing; provided, however, if any one of J, O or U is nothing then J, O and U are all nothing, and converting the annealed oligonucleotides to a double stranded oligonucleotide, (ii) an effector domain encoded by an oligonucleotide sequence encoding a protein or peptide that enhances expression or detection of the binding domain; and (b) screening the library of vectors by contacting the plurality of heterofunctional fusion proteins with said ligand of choice under conditions conducive to ligand binding and isolating any heterofunctional fusion protein which binds said ligand.