Method and kit for generating high affinity binding agents
View Patent ↗A combined ribosome-display and phage-display method and kit for carrying out the method are provided. The method includes screening a ribosome-display library of binding agents to identify binding agents that interact with one or more target molecules of interest, converting the RNA encoding the binding agents to a phage-display format by amplification and primer extension, and the screening the phage-display library to enrich for binding agents that interact with one or more target molecules of interest.
1. A method for generating high affinity monobodies to a target molecule comprising:
(a) annealing a first population of oligonucleotides and a second population of oligonucleotides, each encoding a library of binding sites, to a single-stranded ribosome-display vector comprising nucleic acids encoding a monobody scaffold;
(b) primer extending the first population of oligonucleotides and second population of oligonucleotides to generate heteroduplexes, wherein one strand of the heteroduplexes encodes the monobody scaffold comprising the library of binding sites;
(c) amplifying both strands of the heteroduplexes;
(d) subjecting the amplified products of step (c) to in vitro transcription and in vitro translation to produce ribosome-mRNA-monobody scaffold complexes;
(e) contacting the ribosome-mRNA-monobody scaffold complexes with one or more tagged target molecules to bind the ribosome-mRNA-monobody scaffold complexes to the tagged target molecules;
(f) removing unbound ribosome-mRNA-monobody scaffold complexes;
(g) dissociating the ribosome-mRNA-monobody scaffold complexes bound to the tagged target molecules;
(h) reverse transcribing the mRNA molecules of (g) to generate cDNA;
(i) amplifying the cDNA molecules of (h) with primers that anneal to nucleic acids encoding the monobody scaffold to generate amplified nucleic acids encoding the monobody scaffold and binding sites;
(j) annealing the amplified nucleic acids of (i) to a phage-display vector comprising nucleic acids encoding the monobody scaffold;
(k) primer extending the annealed nucleic acids of (j) to generate heteroduplexes, wherein one strand of the heteroduplexes encodes the monobody scaffold and binding sites;
(l) expressing the monobody scaffold and binding sites encoded by the phage-display vector; and
(m) screening the expressed products of (l) with one or more of the tagged target molecules thereby generating high affinity monobodies to the one or more target molecules.
2. The method of claim 1 , in which the first population of oligonucleotides comprise a BC loop with the nucleotide sequence of SEQ ID NO:15.
3. The method of claim 1 , in which the second population of oligonucleotides comprise a FG loop with the nucleotide sequence of SEQ ID NO:16.
4. The method of claim 1 , in which the monobody comprises the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.