Translation of DNA into Evolvable Sequence-Defined Synthetic Polymers

Laboratory directed evolution have enabled the discovery of numerous functional natural and synthetic macromolecules with tailor-made functions. However, approaches that use enzymes to effect the crucial translation from an information carrier molecule such as DNA or RNA to synthetic polymers are limited to producing close analogs of nucleic acids, either due to a strict requirement to hybridize with a nucleic acid template or as a consequence of the limited substrate scope of polymerase enzymes.;In Chapter Two, we developed a DNA-templated translation system that enables the enzyme-free translation of DNA templates into sequence-defined synthetic polymers with no necessary structural relationship with nucleic acids. We demonstrate the efficiency, sequence-specificity, and generality of this translation system by polymerizing building blocks including polyethylene glycol (PEG), beta-amino acids, and alpha-(D)-amino acids in a DNA-programmed manner. Sequence-defined synthetic polymers with molecular weights of up to 26 kDa containing 16 consecutively coupled building blocks can be translated from DNA templates using this strategy.;In Chapter Three, we further incorporated the translation strategy developed in Chapter Two into a synthetic polymer in vitro selection system that integrates a carefully designed genetic code to enable a DNA template library with diversity of 7x1010 members, 16 macrocyclic substrates with structurally and functionally diverse beta-peptide building blocks, optimized DNA-templated translation conditions, and molecular biology methods to prepare DNA templates surviving in vitro selection and PCR amplification for subsequent rounds of translation and selection. We anticipate these developments have the potential to enable the laboratory evolution of a wide range of synthetic polymers and to reveal the evolutionary potential of macromolecules beyond the reach of previous translation systems.;In Chapter Four, we developed a strategy to use T4 DNA ligase to mediate the ligation of short oligonucleotides with various different non-natural side-chain modifications. We described the incorporation of eight different functional groups encoded by eight trinucleotide codons. The translation system exhibits high sequence specificity and efficiency and could generate a polymer of 50 consecutive building blocks. Finally, we demonstrate an in vitro selection system of highly functionalized nucleic acid polymers based on the ligase-mediated translation strategy.