A T4 DNA ligase-catalysed oligonucleotide polymerisation process has been recently developed to enable the incorporation of multiple functional groups throughout a nucleic acid polymer. T4 DNA ligase requires ATP as a cofactor to catalyse phosphodiester bond formation during the polymerisation process. Herein, we describe the structure–activity relationship of ATP within the context of T4 DNA ligase-catalyzed oligonucleotide polymerisation. Using high-throughput sequencing, we study not only the influence of ATP modification on polymerisation efficiency, but also on the fidelity and sequence bias of the polymerisation process.

We describe the development and analysis of the T4 DNA ligase-catalyzed DNA templated polymerization of pentanucleotides modified with peptide fragments toward the generation of ssDNA-scaffolded peptides. A high-throughput duplex DNA sequencing method was developed to facilitate the determination of fidelity for various codon sets and library sizes used during the polymerization process. 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% and with low sequence bias. These findings mark a significant advance in generating evolvable biomimetic polymers and should find ready application to the in vitro selection of molecular recognition.

Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function.Keywords: aptamer; combinatorial assembly; modified nucleic acids; molecular evolution; sequence-controlled; synthetic copolymer libraries

We have developed a method for the T4 DNA ligase-catalyzed DNA-templated polymerization of 5′-phosphorylated pentanucleotides containing peptide fragments. The polymerization proceeds sequence-specifically to generate DNA-scaffolded peptides in excellent yields. The method has been shown to tolerate peptides ranging from two to eight amino acids in length with a wide variety of functionality. 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, which exhibited a 190-fold enrichment after one round of selection. This strategy demonstrates a promising new approach to allowing the generation and in vitro selection of high-affinity reagents based upon single-stranded DNA scaffolding of peptide fragments.

In vitro selection of nucleic acid polymers can readily deliver highly specific receptors and catalysts for a variety of applications; however, it is suspected that the functional group deficit of nucleic acids has limited their potential with respect to proteinogenic polymers. This has stimulated research toward expanding their chemical diversity to bridge the functional gap between nucleic acids and proteins to develop a superior biopolymer. In this study, we investigate the effect of codon library size and composition on the sequence specificity of T4 DNA ligase in the DNA-templated polymerization of both unmodified and modified oligonucleotides. Using high-throughput DNA sequencing of duplex pairs, we have uncovered a 256-membered codon set that yields sequence-defined modified ssDNA polymers in high yield and with high fidelity.Keywords: aptamers; DNA sequencing; DNA-templated; SELEX; T4 DNA ligase

A T4 DNA ligase-catalysed oligonucleotide polymerisation process has been recently developed to enable the incorporation of multiple functional groups throughout a nucleic acid polymer. T4 DNA ligase requires ATP as a cofactor to catalyse phosphodiester bond formation during the polymerisation process. Herein, we describe the structure–activity relationship of ATP within the context of T4 DNA ligase-catalyzed oligonucleotide polymerisation. Using high-throughput sequencing, we study not only the influence of ATP modification on polymerisation efficiency, but also on the fidelity and sequence bias of the polymerisation process.