Parallel determination of competitive binding and target specificity of a binding compound library by high-throughput dna sequencing

A method of simultaneously determining competitive binding and target specificity of binding compounds of a library relative to a predetermined reference compound, the method comprising the steps of:

• (a) to reacting under binding conditions in a plurality of reactions a predetermined reference compound and a library of binding compounds with a ligand, wherein the predetermined reference compound is present in each reaction at a different concentration and wherein each binding compound is a protein encoded by a linked nucleic acid;
• (b) separating in each reaction binding compounds forming complexes with the ligand from binding compounds free of ligand;
• (c) sequencing for each reaction nucleic acids of a sample of binding compounds forming complexes with the ligand to determine frequencies of the nucleic acids;
• (d) sequencing for each reaction nucleic acids of a sample of expressed binding compounds free of ligand or a sample of expressed binding compounds from the library to determine frequencies of the nucleic acids;
• (e) determining affinities of the binding compounds of the samples from each reaction, wherein the affinities are determined by comparing the frequency of nucleotide sequences identified with a binding compound forming complexes with the ligand and the frequency of the same nucleotide sequences identified with the binding compound free of the ligand or with the frequency of the same nucleotide sequences that encode the same binding compound in the library;
• and
• (f) identifying a binding compound as competitively binding with the predetermined reference compound to the ligand whenever the affinities of the binding compound arc a monotonically decreasing function of predetermined reference compound concentration, or identifying a binding compound as non-competitive with the predetermined reference compound in binding to the ligand whenever the affinities of the binding compound are a substantially unchanging function of predetermined reference compound concentration.