Parallel Discovery Strategies Provide a Basis for Riboswitch Ligand Design

Author:

Tran, Brandon

Pichling, Patricio

Tenney, Logan

Connelly, Colleen M

Moon, Michelle H

Ferré-D'Amaré, Adrian R

Schneekloth,Jay

Jones, Christopher P
Author Address

Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA., Chemical Biology Laboratory, National Cancer Institute, Frederick, MD, USA., Chemical Biology Laboratory, National Cancer Institute, Frederick, MD, USA. Electronic address: jay.schneekloth@nih.gov., Biochemistry and Biophysics Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA. Electronic address: christopher.jones2@nih.gov.,

Abstract:

Riboswitches are mRNA domains that make gene-regulatory decisions upon binding their cognate ligands. Bacterial riboswitches that specifically recognize 5-aminoimidazole-4-carboxamide riboside 5'-monophosphate (ZMP) and 5'-triphosphate (ZTP) regulate genes involved in folate and purine metabolism. Now, we have developed synthetic ligands targeting ZTP riboswitches by replacing the sugar-phosphate moiety of ZMP with various functional groups, including simple heterocycles. Despite losing hydrogen bonds from ZMP, these analogs bind ZTP riboswitches with similar affinities as the natural ligand, and activate transcription more strongly than ZMP in vitro. The most active ligand stimulates gene expression ~3 times more than ZMP in a live Escherichia coli reporter. Co-crystal structures of the Fusobacterium ulcerans ZTP riboswitch bound to synthetic ligands suggest stacking of their pyridine moieties on a conserved RNA nucleobase primarily determines their higher activity. Altogether, these findings guide future design of improved riboswitch activators and yield insights into how RNA-targeted ligand discovery may proceed. Published by Elsevier Ltd.

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