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Abstract
By taking advantage of ssDNA self-folding into stable secondary structures, ssDNA can be applied as a scaffold for presenting peptide residues to mimic the interface of PPIs. We have developed a method for the T4 DNA ligase-catalyzed polymerization (LOOPER) of 5-phosphorylated pentanucleotides containing peptide fragments. This polymerization proceeds sequence-specifically to generate DNA-scaffolded peptides in excellent yields. It has been shown to tolerate peptides ranging from two to eight amino acids in length with a wide variety of functionalities. We validated the capabilities of this system in a mock selection for the enrichment of a His-tagged DNA-scaffolded peptide phenotype from a library, where almost 190-fold enrichment after one round of selection was observed. A high-throughput duplex DNA sequencing method was developed to facilitate the determination of fidelity for various codon sets and library sizes. With this process, we identified several codon sets that enable the efficient and sequence-specific incorporation of peptide fragments along a ssDNA template at fidelities up to 99%. These findings marked a significant advance in generating evolvable biomimetic polymers which should find ready application for the in vitro selection of molecular recognition.