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A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states.
Rivera, Maira; Ayon, Omma S; Diaconescu-Grabari, Suzana; Pottel, Joshua; Moitessier, Nicolas; Mittermaier, Anthony; McKeague, Maureen.
Afiliação
  • Rivera M; Department of Chemistry, Faculty of Science, McGill University, Montreal, QC H3A 0B8, Canada.
  • Ayon OS; Department of Chemistry, Faculty of Science, McGill University, Montreal, QC H3A 0B8, Canada.
  • Diaconescu-Grabari S; Department of Chemistry, Faculty of Science, McGill University, Montreal, QC H3A 0B8, Canada.
  • Pottel J; Molecular Forecaster Inc. 910-2075 Robert Bourassa, Montreal, QC H3A 2L1, Canada.
  • Moitessier N; Department of Chemistry, Faculty of Science, McGill University, Montreal, QC H3A 0B8, Canada.
  • Mittermaier A; Molecular Forecaster Inc. 910-2075 Robert Bourassa, Montreal, QC H3A 2L1, Canada.
  • McKeague M; Department of Chemistry, Faculty of Science, McGill University, Montreal, QC H3A 0B8, Canada.
Nucleic Acids Res ; 52(6): 3164-3179, 2024 Apr 12.
Article em En | MEDLINE | ID: mdl-38375901
ABSTRACT
The capacity of riboswitches to undergo conformational changes in response to binding their native ligands is closely tied to their functional roles and is an attractive target for antimicrobial drug design. Here, we established a probe-based fluorescence anisotropy assay to monitor riboswitch conformational switching with high sensitivity and throughput. Using the Bacillus subtillis yitJ S-Box (SAM-I), Fusobacterium nucleatum impX RFN element of (FMN) and class-I cyclic-di-GMP from Vibrio cholerae riboswitches as model systems, we developed short fluorescent DNA probes that specifically recognize either ligand-free or -bound riboswitch conformational states. We showed that increasing concentrations of native ligands cause measurable and reproducible changes in fluorescence anisotropy that correlate with riboswitch conformational changes observed by native gel analysis. Furthermore, we applied our assay to several ligand analogues and confirmed that it can discriminate between ligands that bind, triggering the native conformational change, from those that bind without causing the conformational change. This new platform opens the possibility of high-throughput screening compound libraries to identify potential new antibiotics that specifically target functional conformational changes in riboswitches.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ensaios de Triagem em Larga Escala / Riboswitch Idioma: En Ano de publicação: 2024 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ensaios de Triagem em Larga Escala / Riboswitch Idioma: En Ano de publicação: 2024 Tipo de documento: Article