Discovery and engineering of polymerases and ligases for the synthesis of modified nucleic acids

Oligonucleotides have emerged as a rapidly expanding modality in therapeutics, diagnostics, and synthetic biology, increasing manufacturing demand. However, solid phase oligonucleotide synthesis, the dominant production technology, is hampered by cumulative yield losses as sequence length increases, poor scalability for stereochemically defined backbones, and high solvent and reagent consumption. In response, enzymatic routes that extend or assemble oligonucleotides in water, at ambient temperature, and with high chemo- and regioselectivity have progressed from explorative technologies to process-relevant platforms. While recent reviews have mapped this landscape from chemoenzymatic manufacturing perspectives, here we focus on recent approaches that harness engineered polymerases and ligases for the synthesis of oligonucleotides bearing non-natural building blocks. We examine how computational tools accelerate the discovery and engineering of nucleic acid modifying enzymes and highlight recent development examples. In addition, we provide a web application that simplifies discovery of scaffolds across biocatalyst families involved in oligonucleotide elongation and assembly (https://buller-lab.github.io/DNA-RNA_Polymerases_Ligases/).