Development of tRNA Scaffold for Facilitated Recombinant RNA Purification

Abstract

This study explored novel methods for RNA purification using recombinant RNA engineering. Existing RNA purification methods often involve denaturing processes that are time-consuming and compromise RNA stability and folding. In this study, two innovative approaches were developed for RNA purification. First, mRNA capping was applied to recombinant RNA to function as an affinity tag for RNA. Second, split and circularly permuted tRNA scaffolds were designed to carry a target RNA stably. The integration of these two strategies enables the development of a new non-denaturing RNA purification method. The Vaccinia virus capping enzyme and recombinant RNA were successfully co-expressed in Escherichia coli. Binding assays with the cap-binding protein eIF4E did not confirm the capping. Next, recombinant tRNA scaffolds were designed using the E. coli tRNA operons (LeuP-LeuV and AlaW-AlaX) fused to the FMN riboswitch as a target RNA. While the expressions of the tRNALeu P-V scaffold-FMN riboswitch fusions were confirmed, evaluating factors such as identifying the exact target band on a gel and high expression levels was challenging. In contrast, the tRNAAla W-X scaffold-FMN riboswitch fusions exhibited higher expression levels. Notably, the FMN riboswitch-tRNAAlaW-X construct showed more promise. Although further optimization is necessary, this study suggests new pathways for RNA purification technologies.