High-Fidelity RNA Copying via 2′,3′-Cyclic Phosphate Ligation
Adriana Calaça Serrão, Sreekar Wunnava, Avinash Vicholous Dass, Lennard Ufer, Philipp Schwintek, Christof B. Mast, Dieter Braun
Abstract
High Resolution Image Download MS PowerPoint Slide Templated ligation offers an efficient approach to replicate long strands in an RNA world. The 2′,3′-cyclic phosphate (>P) is a prebiotically available activation that also forms during RNA hydrolysis. Using gel electrophoresis and high-performance liquid chromatography, we found that the templated ligation of RNA with >P proceeds in simple low-salt aqueous solutions with 1 mM MgCl 2 under alkaline pH ranging from 9 to 11 and temperatures from −20 to 25 °C. No additional catalysts were required. In contrast to previous reports, we found an increase in the number of canonical linkages to 50%. The reaction proceeds in a sequence-specific manner, with an experimentally determined ligation fidelity of 82% at the 3′ end and 91% at the 5′ end of the ligation site. With splinted oligomers, five ligations created a 96-mer strand, demonstrating a pathway for the ribozyme assembly. Due to the low salt requirements, the ligation conditions will be compatible with strand separation. Templated ligation mediated by 2′,3′-cyclic phosphate in alkaline conditions therefore offers a performant replication and elongation reaction for RNA on early Earth.