Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression

Shaifaly Parmar, Desta Doro Bume, Colleen M. Connelly, Robert E. Boer, Peri R. Prestwood, Zhen Wang, Henning Labuhn, Krishshanthi Sinnadurai, Adeline Feri, Jimmy Ouellet, Philip Homan, Tomoyuki Numata and John S. Schneekloth ()
Additional contact information
Shaifaly Parmar: National Cancer Institute
Desta Doro Bume: National Cancer Institute
Colleen M. Connelly: National Cancer Institute
Robert E. Boer: National Cancer Institute
Peri R. Prestwood: National Cancer Institute
Zhen Wang: Depixus SAS
Henning Labuhn: Depixus SAS
Krishshanthi Sinnadurai: Depixus SAS
Adeline Feri: Depixus SAS
Jimmy Ouellet: Depixus SAS
Philip Homan: National Institutes of Health
Tomoyuki Numata: Kyushu University
John S. Schneekloth: National Cancer Institute

Nature Communications, 2024, vol. 15, issue 1, 1-13

Abstract: Abstract Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

Date: 2024
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DOI: 10.1038/s41467-024-52235-3

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