Talk abstracts

Talk on Friday 01:45-02:00pm submitted by McCauley Meyer

Nucleotide-level resolution of RNA folding interactions within peptide-based complex coacervates

McCauley Meyer (Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802), Saehyun Choi, Fatma Pir Cakmak, Philip C. Bevilacqua, Christine D. Keating (Department of Chemistry, Pennsylvania State University, University Park, PA 16802)

Abstract:
The RNA World Hypothesis states that RNA or an RNA-like polymer may have acted as both the initial genetic material and the catalyst for the reactions of life. In the 1980s, the first ribozymes were discovered, demonstrating that RNA could act as a catalyst. Since then, it has become apparent that RNA folding is integral to function in a way similar to protein enzyme folding. Because of this, it’s important to try to understand RNA folding under prebiotically relevant conditions.
On the early Earth, a problem that would have been faced by the first enzymes was the scarcity of organic material. To overcome this issue, organic material would need to be localized and concentrated on either a mineral surface or in some type of compartment, like a protocell. An ideal protocell candidate should partition molecules required for catalysis such as Mg2+, nucleotides, RNAs, amino acids, and peptides. A model protocell able to do this is one made of complex coacervates.
Herein, I describe turbidity measurements, FRET, local pH, and RNA folding studies within complex coacervate droplets made out of (Lys)n-(Asp)n, and (Lys)10-ATP1. tRNAphe from S. cerevisiae, was used for these RNA folding studies. tRNAphe was subjected to in-line probing (ILP) under the following conditions (0.5 mM Mg2+, 15 mM KCl, and 10 mM Tris, pH 8.3) initially to determine its native fold. Then, tRNAphe was placed inside of (Lys)n-(Asp)n and (Lys)10-ATP coacervates where we found that under all of these coacervate conditions, the tRNA had lost its tertiary contacts and the acceptor stem was unfolded. Upon changing Mg2+ conditions and charge-ratio of polyanions to polycations, more native folding of tRNAphe was observed. Future studies will focus on evaluating if under the same conditions ribozymes fold and function well. These experiments provide one of the first detailed views of RNA folding in protocells and pave the way for combining Next-Generation sequencing to study RNA folding within protocells.

References:
1.Pir Cakmak, F.; Choi, S.†; Meyer, M. O.†; Bevilacqua, P. C.; and Keating, C. D. Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments. Submitted and uploaded to BioRxiv: https://www.biorxiv.org/content/10.1101/2020.02.23.961920v1

Keywords: RNA folding, complex coacervates, liquid-liquid phase separation