Poster abstracts
Poster number 52 submitted by Joseph Heili
Activation of caged functional RNAs by oxidative desulfurization of 2-thiouridine
Joseph Heili (BMBB & MCDBG, University of Minnesota), Wakana Sato (BMBB & MCDBG, University of Minnesota), Nathan Gaut (BMBB & MCDBG, University of Minnesota), Tanner Hoog (BMBB & MCDBG, University of Minnesota), Kate Adamala (BMBB & MCDBG, University of Minnesota), Aaron Engelhart (BMBB & MCDBG, University of Minnesota)
Abstract:
The discovery, about 35 years ago, that RNA can act as both a heritable information source and as a functional molecule has led many to consider the possibility there was an RNA world during the origins of life. Researchers in this field have demonstrated high-fidelity, rapid templated nonenzymatic polymerization of RNA rich in GC sequences using activated nucleotide derivatives. In contrast, AU-rich sequences exhibit diminished fidelity and speed. This is due in part to G-U wobble pairing (which is ca. isoenergetic with A-U base pairing) as well as the lower stability of the A-U base pair relative to the G-C base pair.
Recently, it was shown that the diminished wobble pairing, and stronger base pairing with A, exhibited by 2-thiouridine (2sU) allows it to substitute for uridine in nonenzymatic RNA polymerization, enabling faster, higher-fidelity base pairing. While this solves the problem of wobble pairing in nonezymatic copying, it brings about a second. In functional RNA, G-U wobble pairs are common secondary structure elements and metal ion binding motifs. Furthermore, the stronger base pairing of 2sU-containing duplexes resulting from such copying reactions inhibit strand separation, which is required to produce a folded, single-stranded functional RNA.
We sought to take advantage of a recent finding showing that 2sU can be oxidatively desulfurized to U, reasoning that we could exploit this phenomenon to create switchable functional RNAs by employing RNAs with critical G-U wobble pairs (or O2 interactions). In preliminary work with a fluorescent aptamer and ribozyme nuclease containing critical interactions with the O2 of U, we have observed that RNAs transcribed with 2sU exhibit diminished function, which can be rescued by the oxidative desulfurization reaction. We also have investigated the kinetics of the oxidative desulfurization reaction, finding the optimal reaction conditions to protect RNA while activating the caged function. Capitalizing on this phenomenon, we are developing switchable functional RNAs for both prebiotic chemistry and synthetic biology applications using engineered functional RNAs.
Keywords: Functional RNA Aptamer, Ribozyme, 2-thiouridine