Tertiary RNA Folding-Targeted Drug Screening Strategy Using a Protein Nanopore
- 21 January 2021
- journal article
- research article
- Published by American Chemical Society (ACS) in Analytical Chemistry
- Vol. 93 (5), 2811-2819
- https://doi.org/10.1021/acs.analchem.0c03941
Abstract
Bacterial riboswitch RNAs are attractive targets for novel antibiotics against antibiotic-resistant superbacteria. Their binding to cognate metabolites is essential for the regulation of bacterial gene expression. Despite the importance of RNAs as therapeutic targets, the development of RNA-targeted, small-molecule drugs is limited by current biophysical methods. Here, we monitored the specific interaction between the adenine-sensing riboswitch aptamer domain (ARS) and adenine at the single-molecule level using α-hemolysin (αHL) nanopores. During adenine-induced tertiary folding, adenine-bound ARS intermediates exhibited characteristic nanopore events, including a two-level ionic current blockade and a ∼ 5.6-fold longer dwell time than that of free RNA. In a proof-of-concept experiment, tertiary RNA folding-targeted drug screening was performed using a protein nanopore, which resulted in the discovery of three new ARS-targeting hit compounds from a natural compound library. Taken together, these results reveal that αHL nanopores are a valuable platform for ultrasensitive, label-free, and single-molecule-based drug screening against therapeutic RNA targets.Keywords
Funding Information
- Korea Research Institute of Bioscience and Biotechnology
- National Research Foundation (NRF-2017R1A2B4006378, NRF-2017R1E1A1A01074403, NRF-2019M3A9C4076156, NRF-2019M3E5D4069903)
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