Poster abstracts

Poster number 11 submitted by Taylor Opolka

An Unexpected Branch in the Family Tree: Biochemical Investigation of Novel Branching Ligase Ribozymes

Taylor D. Opolka (Department of Chemistry and Biochemistry, University of Notre Dame), Natalie R. Kotlin (Department of Chemistry and Biochemistry, University of Notre Dame), Annyesha Biswas (Department of Chemistry and Biochemistry, University of Notre Dame), Saurja DasGupta (Department of Chemistry and Biochemistry, University of Notre Dame)

Abstract:
The RNA World Hypothesis posits that RNA played an essential role in life’s origins, acting as both genetic carrier and facilitator of enzymatic transformations (ribozymes). As such, the assembly of RNAs from shorter oligomers via the activity of RNA enzymes would have been pivotal to the success of the RNA World. In working toward an improved understanding of RNA-catalyzed RNA assembly and the evolutionary transition from RNA-based to extant biology, we utilized directed evolution to convert an existing ligase ribozyme that utilized RNA substrates activated at the 5′ end with prebiotically relevant phosphorimidazole groups, to a new ribozyme that catalyzes ligation using biologically relevant 5′ triphosphate-activated RNA substrates. In addition to the isolation of the desired ‘triphosphate ligase,’ this selection also led to a surprising discovery; a secondary class of low abundance sequences that catalyze branching RNA ligation between specific internal 2’ hydroxyls of the substrate and their own 5’ triphosphate groups. This is a distinct chemical pathway that has not been observed in any known enzyme in nature – protein or otherwise - and is reminiscent of the first step of RNA splicing, in which an internal 2’ hydroxyl group acts as a nucleophile toward the phosphate group of a neighboring internal nucleotide. The novelty of this catalytic activity warrants a thorough biochemical investigation of the two sequences that make up this ribozyme class – dubbed CS9 and CS10 – in which we aim to better understand their mechanisms and scope of activity, and the implications they may have for the study of the origin of life in the RNA World.

References:
[1] Osterberg, R.; Orgel, L.E.; Polyphosphate and trimetaphosphate formation under potentially prebiotic conditions. J. Mol. Evol. 1972, 1 (3), 241-8. doi:10.1007/BF01660243. [2] Martin, L.L.; Unrau, P.J.; Muller, U.F. RNA synthesis by in vitro select-ed ribozymes for recreating an RNA world. Life. 2015, 5 (1), 247-268. doi:10.3390/life5010247  [3] Walton, T.; DasGupta, S.; Duzdevich, D.; Oh, S. S.; Szostak, J. W.  In vitro selection of ribozyme ligases that use prebiotically plausible 2-aminoimidazole-Activated substrates. Proc. Natl. Acad. Sci. U.S.A. 2020, 117 (11), 5741–5748. doi:10.1073/pnas.1914367117. [4] DasGupta, S.; Weiss, Z.; Nisler, C.; Szostak. J.W. Evolution of the substrate specificity of an RNA ligase ribozyme from phosphorimidazole to triphosphate activation. Proc. Natl. Acad. Sci. U.S.A. 2024, 121 (38), e2407325121. doi:10.1073/pnas.2407325121.

Keywords: Ribozymes, Origin of Life, Ligase