Evolution and Evaluation of Engineered DNA Ligases for Improved Blunt-End Ligation

Abstract

DNA ligases are fundamental enzymes in molecular biology and biotechnology where they perform essential reactions, e.g. to create recombinant DNA and for adaptor attachment in next-generation sequencing. T4 DNA ligase is the most widely used commercial ligase owing to its ability to catalyse ligation of blunt-ended DNA termini. However, even for T4 DNA ligase, blunt-end ligation is an inefficient activity compared to cohesive-end ligation, or its evolved activity of sealing single-strand nicks in double-stranded DNA. Previous research from Dr Wayne Patrick showed that fusion of T4 DNA ligase to a DNA-binding domain increases the enzyme’s affinity for DNA substrates, resulting in improved ligation efficiency. It was further shown that changes to the linker region between the ligase and DNA-binding domain resulted in altered ligation activity. To assist in optimising this relationship, we designed a competitive ligase selection protocol to enrich for engineered ligase variants with greater blunt-end ligation activity. This selection involves expressing a DNA ligase from its plasmid construct, and ligating a linear form of its plasmid, sealing a double-strand DNA break in the chloramphenicol resistance gene, permitting bacterial growth. Previous researcher Dr Katherine Robins created two linker libraries of 33 and 37 variants, from lead candidate ligase-cTF and (the less active form of p50-ligase variant) ligase-p50, respectively. Five rounds of selection were applied to each library. One linker variant, denoted ligase-CA3 showed the greatest improvement, comprising 42% of the final selected ligase-cTF population. In contrast, a lead linker variant from the ligase-p50 library was not obtained. In this study one additional round of selection was applied to the ligase-p50 library to test whether a lead variant would emerge. However, the linker variants selected at the end of Round 6 did not suggest a clear lead candidate, so one of the top variants (ligase-PPA17) was selected to represent this population in a fluorescence-based ligation assay that I optimised. Following identification of optimal reaction buffers to improve protein stability and DNA ligation, six engineered variants were compared for blunt-, cohesive-end, and nick sealing ligation activities. All five engineered variants exhibited improved blunt-end ligation activity over T4 DNA ligase. Ligase-PPA17 (1.9-fold improvement over T4 DNA ligase) was best performing for blunt-end ligation. This study found no evidence that ligase-CA3 was significantly improved over its predecessor, ligase-cTF in blunt-end ligation, however it was the best performing variant at cohesive-end ligation. Overall, we have evolved DNA ligase variants with improved blunt-end ligation activity over T4 DNA ligase which may be more advantageous in molecular biology and biotechnology for a variety of applications.