Naturally occurring peptides often possess macrocyclic and N-methylated backbone. These features grant them structural rigidity, high affinity to targets, proteolytic resistance, and occasionally membrane permeability. Because such peptides are produced by either nonribosomal peptide synthetases or enzymatic posttranslational modifications, it is yet a formidable challenge in degenerating sequence or length and preparing libraries for screening bioactive molecules. Here, we report a new means of synthesizing a de novo library of "natural product-like" macrocyclic N-methyl-peptides using translation machinery under the reprogrammed genetic code, which is coupled with an in vitro display technique, referred to as RaPID (random nonstandard peptides integrated discovery) system. This system allows for rapid selection of strong binders against an arbitrarily chosen therapeutic target. Here, we have demonstrated the selection of anti-E6AP macrocyclic N-methyl-peptides, one of which strongly inhibits polyubiqutination of proteins such as p53.

We report a methodology for the ribosomal synthesis of backbone-cyclized peptides involving genetic code reprogramming to introduce one or more nonproteinogenic amino acids. Expression of linear peptides bearing a cysteine-proline dipeptide sequence followed by glycolic acid results in self-rearrangement to a C-terminal diketopiperadine-thioester, which non-enzymatically generates a cyclized peptide. We demonstrate the ribosomal synthesis of several naturally occurring backbone-cyclized peptides and a library based on a bicyclic scaffold, and we identify bioactive sequences by screening and deconvolution.

Natural peptide products often contain N-methylated backbones, and such a modification plays a crucial role in making natural peptides peptidase resistant and membrane permeable. Here, we demonstrate the ribosomal synthesis of N-methyl-peptides by means of genetic code reprogramming. Two key technologies, a ribozyme-based de novo tRNA acylation (flexizyme) system and an E. coli reconstituted cell-free translation (PURE) system, were used in order to reassign arbitrarily chosen codons to N(alpha)-methylated amino acids ((Me)aa). Using this combination, we determined the general structural requirement of "accessible"(Me)aa and demonstrated their multiple incorporations into the nascent peptide chain according to the assignments made on mRNA, giving linear and cyclic N-methyl-peptides in high purities. This platform technology offers a convenient tool for the construction of N-methyl-peptide libraries, potentially leading to the discovery of therapeutic peptides.

Here, we describe a novel method that enables high-speed in vitro selection of functional peptides, peptidomimetics, and proteins via a simple procedure. We first developed a new cell-free translation system, the TRAP system (transcription-translation coupled with association of puromycin linker), which automatically produces a polypeptide library through a series of sequential reactions: transcription, association of puromycin-DNA linker, translation, and conjugation between the nascent polypeptide and puromycin-DNA linker. We then applied the TRAP system for the selection of macrocyclic peptides against human serum albumin. Six rounds of selection using TRAP display were performed in approximately 14 h, yielding macrocyclic peptides with nanomolar affinity to their target protein. Because TRAP display enables high-speed selection of functional polypeptides, it will facilitate the generation of various polypeptides that are useful for biological and therapeutic applications.

Here we report a new methodology for mRNA-programmed synthesis of peptoids and peptoid-peptide hybrids by means of translation machinery under the reprogrammed genetic code. We initially screened N-substituted glycines (rGly) for their single incorporation into a nascent peptide chain and found translation machinery accepts a variety of rGly for elongation. Moreover, we have shown consecutive elongations of rGly and mRNA-directed synthesis of cyclic peptoid-peptide hybrids. This methodology offers a powerful tool for mRNA-programmed library synthesis of peptoids and peptoid-peptide hybrids with linear and cyclic scaffolds, potentially leading to the discovery of drug candidates with proteolytic stability and membrane permeability.