Abstract
The evolution of serum stable RNA aptamers is of great interest for the development of aptamer-based therapeutics. A novel mutant polymerase, KOD DGLNK, can create fully 2’-OMe-modified RNA, which has excellent serum stability. However, reverse transcription of 2’-OMe-RNA presents challenges. To circumvent this, we propose incorporating KOD DGLNK within Hairpin SELMA, a directed evolution platform that avoids reverse transcription. Our aim is to evolve fully 2’-OMe-RNA aptamers towards thrombin and evaluate the efficacy of KOD DGLNK within Hairpin SELMA. In this study, we successfully used SELMA incorporating KOD DGLNK to enrich a random library towards a sequence with known binding affinity. We then conducted 4 rounds of selection towards thrombin; we observed preliminary enrichment in thrombin binders and a convergence in the library to higher melting features. Although additional rounds of selection are necessary to obtain low nanomolar binders of thrombin, our preliminary results demonstrate the potential of KOD DGLNK within Hairpin SELMA as a powerful tool for the evolution of fully modified aptamers.