Generation and Screening of an Oligonucleotide-Encoded Synthetic Peptide Library

• Published in: Proceedings of the National Academy of Sciences - PNAS 1993-11, Vol.90 (22), p.10700-10704
• Main Authors: Needels, Michael C., Jones, David G., Tate, Emily H., Heinkel, Gregory L., Kochersperger, Lynn M., Dower, William J., Barrett, Ronald W., Gallop, Mark A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Antibodies; Antibodies, Monoclonal - metabolism; Base Sequence; Biochemistry; Biological and medical sciences; Combinatorial chemistry; Diverse techniques; DNA; Epitopes; Flow Cytometry; Fundamental and applied biological sciences. Psychology; In Vitro Techniques; Libraries; Ligands; Molecular and cellular biology; Molecular Sequence Data; Nucleotides; Oligodeoxyribonucleotides - chemistry; Oligonucleotides; Peptides - chemical synthesis; Peptides - chemistry; Polymerase chain reaction; Protein Binding; Receptors
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.22.10700

We have prepared a library of ≈106different peptide sequences on small, spherical (10-μ m diameter) beads by the combinatorial chemical coupling of both L- and D-amino acid building blocks. To each bead is covalently attached many copies of a single peptide sequence and, additionally, copies of a unique single-stranded oligonucleotide that codes for that peptide sequence. The oligonucleotide tags are synthesized through a parallel combinatorial procedure that effectively records the process by which the encoded peptide sequence is assembled. The collection of beads was screened for binding to a fluorescently labeled anti-peptide antibody using a fluorescence-activated cell sorting instrument. Those beads to which the antibody bound tightly were isolated by fluorescence-activated sorting, and the oligonucleotide identifiers attached to individual sorted beads were amplified by the PCR. Sequences of the amplified DNAs were determined to reveal the identity of peptide sequences that bound to the antibody with high affinity. By combining the capacity for information storage in an oligonucleotide code with the tremendous level of amplification possible through the PCR, we have devised a means for specifying the identity of each member of a vast library of molecules synthesized from both natural and unnatural chemical building blocks. In addition, we have shown that the use of flow cytometry instrumentation permits facile isolation of individual beads that bear high-affinity ligands for biological receptors.