Silent Encoding of Chemical Post-Translational Modifications in Phage-Displayed Libraries

In vitro selection of chemically modified peptide libraries presented on phage, while a powerful technology, is limited to one chemical post-translational modification (cPTM) per library. We use unique combinations of redundant codons to encode cPTMs with "silent barcodes" to trace multiple modifications within a mixed modified library. As a proof of concept, we produced phage-displayed peptide libraries Ser-[X]4-Gly-Gly-Gly, with Gly and Ser encoded using unique combinations of codons (TCA-[X]4-GGAGGAGGA, AGT-[X]4-GGTGGTGGT, etc., where [X]4 denotes a random NNK library). After separate chemical modification and pooling, mixed-modified libraries can be panned and deep-sequenced to identify the enriched peptide sequence and the accompanying cPTM simultaneously. We panned libraries bearing combinations of modifications (sulfonamide, biotin, mannose) against matched targets (carbonic anhydrase, streptavidin, concanavalin A) to identify desired ligands. Synthesis and validation of sequences identified by deep sequencing revealed that specific cPTMs are significantly enriched in panning against the specific targets. Panning on carbonic anhydrase yielded a potent ligand, sulfonamide-WIVP, with Kd = 6.7 ± 2.1 nM, a 20-fold improvement compared with the control ligand sulfonamide-GGGG. Silent encoding of multiple cPTMs can be readily incorporated into other in vitro display technologies such as bacteriophage T7 or mRNA display.