Poster abstracts
Poster number 25 submitted by Richard A. Lease
A structured antisense RNA motif can filter out RNA:RNA hybridization mismatches
Samuel D. Stimple, Hopen Yang (Dept. of Chemical and Biomolecular Engineering), Ashwin Lahiry (Dept. of Microbiology), David Wood (Dept. of Chemical and Biomolecular Engineering; Dept. of Microbiology), Richard A. Lease (Dept. of Chemical and Biomolecular Engineering; Dept. of Chemistry and Biochemistry; The Ohio State University, Columbus OH 43210)
Abstract:
Programmed RNA self-assembly can be used to target regulation of gene expression for metabolic engineering or medical applications. When designing an RNA to be implemented in a cell or organism, it will be increasingly important to avoid mismatched RNA base pairing to prevent off-target interactions and undesired side effects. Bacteria produce antisense regulatory small RNAs (sRNAs) that act by forming specific sRNA:mRNA base pairing interactions to post-transcriptionally regulate mRNA fates. Here we describe fingerloops, structured synthetic antisense motifs that mimic native regulatory RNA structures and strongly reduce off-target RNA pairing interactions. Fingerloop antisense sequences are encoded in a hairpin stem-loop within one strand of the RNA helix and into the single-stranded loop. We demonstrate that synthetic fingerloop helices derived from E. coli DsrA sRNA antisense structural motifs encode a target-discrimination function in vivo that is superior to single-stranded antisense regions commonly found in sRNAs. These antisense motifs can be retargeted to bind non-native target mRNA leader sequences. The optimized fingerloop appears to sharply decrease pairing between the sRNA and a mismatched target mRNA with single-base discrimination, as assessed by changes in target reporter gene translation. We hypothesize that RNA loop sequences constrain a helix-nucleation step to filter out mismatches in sRNA:mRNA base pairing. The fingerloop structures may enhance the specificity of E. coli DsrA for two of its mRNA targets (hns, rpoS) that are global transcriptional stress-response regulators. Fingerloops may also represent the simplest and smallest RNA nanotechnological devices, and constitute a new RNA structural motif.
References:
Lahiry, A., Stimple, S.D., Wood, D.W., and Lease, R.A. (2017) Retargeting a dual-acting sRNA for multiple mRNA transcript regulation. ACS Synth. Biol. 6:648-658. DOI: 10.1021/acssynbio.6b00261
Stimple, S.D., Lahiry, A., Taris, J.E., Wood, D.W. and Lease, R.A. (2018) A Modular Genetic System for High-Throughput Profiling and Engineering of Multi-Target Small RNAs In Arluison, V. and Valverde, C. (Eds.), Bacterial Regulatory RNA: Methods and Protocols. Meth. Mol. Biol. 1737:373-391 (ISBN 978-1-4939-7633-1; DOI: 10.1007/978-1-4939-7634-8_21).
Keywords: RNA structure, antisense RNA regulation, RNA-RNA interactions