Journal
CHEMICAL SCIENCE
Volume 2, Issue 1, Pages 157-165Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/c0sc00406e
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Funding
- UK Biotechnology and Biological Sciences Research Council
- Biotechnology and Biological Sciences Research Council [BB/D005817/1] Funding Source: researchfish
- BBSRC [BB/D005817/1] Funding Source: UKRI
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Riboswitches are regions of mRNA that bind selectively certain metabolites, thereby regulating gene expression. We have developed a method, which uses a combination of biophysical techniques (equilibrium dialysis, waterLOGSY and T-2 relaxation-edited NMR spectroscopy and isothermal titration calorimetry) that allows screening and identification of novel ligands for riboswitches. By this method a library of 1300 fragments was screened on the E. coli thiamine pyrophosphate (TPP) riboswitch thiM, resulting in the identification of 17 hits. The compounds showed K-D values from 22 to 670 mu M, with four compounds having KD values <100 mu M. The fragments are structurally diverse and their ligand efficiency indicates good prospects for structure-guided elaboration. In addition, a riboswitch functional assay based on in vitro transcription translation (IVTT) of the reporter gene luciferase was developed. This assay system is an alternative to in vivo assays and constitutes a valuable tool to determine the effect of small molecules on riboswitch-regulated gene expression.
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