Biodiscovery of Novel RNA Ligases with Biotechnological Potential - New Insights into the RNA Ligase 3 Family

Abstract

Polynucleotide ligases constitute a class of enzymes which catalyse the formation of phosphodiester bonds between proximal 5’-PO4 and 3’-OH groups in DNA, RNA and DNA/RNA-hybrid substrates. The reaction proceeds through three steps, where steps 1 and 2 prepare the substrate for ligation in step 3. The focus of this thesis is on ssRNA ligases of the RNA ligase (Rnl) 3 family. RNA ligases can repair breaks in oligonucleotides and create entirely new RNA molecules. They have biotechnological potential in applications ranging from diagnostics to preparation of sequencing libraries from RNA samples. One member of the Rnl3 family has already been commercialized for the latter, as well as for producing adenylated substrate for preparative purposes.

This thesis applies a biodiscovery pipeline to investigate the potential of 22 putative RNA ligases as new enzyme leads. Potential candidates were selected based on their expression in Escherichia coli, activity in the first two steps of the reaction, success of upscaling, and novelty. Selected candidates were purified using fast protein liquid chromatography (FPLC) and activity was further characterized. Activity was assessed after the first two reaction steps, after step 3 in isolation, and throughout the complete ligation reaction. For the latter two, new protocols were developed as part of this thesis. These did not demand additional equipment and were less laborious than the existing protocols on which they were based.

Four novel, thermotolerant candidates were found to adenylate ssDNA but surprisingly none exhibited ligation activity in the conditions tested here. Substrate adenylation could not be improved by increasing adenosine triphosphate (ATP) and enzyme concentrations. Indications of that adenylation by one candidate (X07) was increased at higher temperatures (80°C) were found but require further studies. Another hit (X06) consistently showed adenylation activity up to 90%, however due to poor reproducibility this needs further investigation. Knowledge gained from this thesis should be used to improve the purification protocol and thereby sample quality. Dimerization is thought to be important for ligation, and we believe means to study dimerization should be a priority in future work.