Tertiary RNA Folding-Targeted Drug Screening Strategy Using a Protein Nanopore.


Lee DH(1)(2), Oh S(1)(3), Lim K(1), Lee B(4), Yi GS(4), Kim YR(5), Kim KB(6), Lee CK(2), Chi SW(1)(3), Lee MK(1)(3).
Author information:
(1)Disease Target Structure Research Center, KRIBB, Daejeon 34141, Republic of Korea.
(2)College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea.
(3)Department of Proteome Structural Biology, KRIBB School of Bioscience, University of Science and Technology, Daejeon 34113, Republic of Korea.
(4)Department of Bio and Brain Engineering, KAIST, Daejeon 34141, Republic of Korea.
(5)Graduate School of Biotechnology & Department of Food Science and Biotechnology, College of Life Sciences, Kyung Hee University, Yongin 17104, Republic of Korea.
(6)Department of Materials Science and Engineering, Seoul National University, Seoul 08826, Republic of Korea.


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.