Protocol for detecting in vitro riboswitch conformational switching using a fluorescence anisotropy single-stranded...

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Riboswitches are RNA molecules that regulate gene expression in response to small molecule metabolites.
• Understanding riboswitch conformational dynamics is crucial for deciphering their regulatory mechanisms and for drug discovery.
• Existing methods for studying riboswitch conformational changes can be limited in sensitivity or throughput.

Purpose of the Study:

• To develop a high-sensitivity fluorescence anisotropy-based protocol for monitoring ligand-induced riboswitch conformational switching in vitro.
• To enable the screening of novel riboswitch ligands based on their ability to induce specific conformational changes.

Main Methods:

• Design of single-stranded fluorescent DNA probes that selectively bind to distinct riboswitch conformational states.
• Measurement of DNA probe binding affinity and riboswitch conformational switching using a 96-well plate reader.
• Detailed procedures for data analysis of fluorescence anisotropy measurements.

Main Results:

• The fluorescence anisotropy single-stranded targeting (FASST) protocol provides a sensitive method to detect riboswitch conformational changes.
• The protocol allows for the quantitative assessment of DNA probe binding affinity to specific riboswitch states.
• Demonstrated the capability to monitor ligand-induced alterations in riboswitch conformation.

Conclusions:

• The FASST protocol offers a robust and sensitive approach for studying riboswitch conformational dynamics.
• This method has significant potential for high-throughput screening of novel riboswitch-targeting ligands.
• Facilitates the discovery of small molecules that modulate riboswitch function for therapeutic or research applications.