Generation and screening of an oligonucleotide-encoded synthetic peptide library.

Abstract

We have prepared a library of -106 different peptide sequences on small, spherical (10-pm 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 fluorescenceactivated 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 hig afminty. 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-afflmity ligands for biological receptors.

The Primary Antibody Repertoire Represents a Linked Network of Degenerate Antigen Specificities

“…Interestingly, studies using combinatorial peptide libraries have revealed that affinity-matured Abs also display the ability to bind to more than one peptide epitope (32)(33)(34)(35)(36)(37)(38)(39)(40). In these cases, consensus motifs within the epitope sequence, however, could be identified, suggesting that such crossreactivity derived from molecular similarity between these sequences (mimotopes) (32)(33)(34)(35)(36)(37)(38)(39)(40). In contrast to this, our present results are more similar to those obtained for some autoreactive Abs where the consensus motif was either more degenerate or absent (41).…”

Evolution of cyclic peptide protease inhibitors

“…Considerable effort has focused on reprogramming this natural biosynthetic machinery to generate diverse libraries of structurally complex peptides that can be subsequently selected for a desired biological activity. Although various synthetic and in vitro methods have also been developed to generate large peptide libraries from natural and unnatural amino acids and enrich them using affinity-based methods (e.g., phage and ribosomal display, DNA directed synthesis, and encoded synthetic peptide libraries) (8)(9)(10)(11)(12)(13), to date the synthesis and bacterial selection of cyclic peptides containing unnatural amino acid (UAA) building blocks has remained elusive.…”

A paradigm for drug discovery employing encoded combinatorial libraries.

“…Many strategies for the generation of chemical diversity have been proposed (4)(5)(6)(7). With a single exception (4), these approaches have been confined to the synthesis of flexible oligomeric ligands of relatively high molecular weight (e.g., peptides and oligonucleotides) which tend to be poor therapeutic agents, in part because of their lack of availability and stability in vivo.…”