# A sensitive and scalable fluorescence anisotropy single stranded RNA targeting approach for monitoring riboswitch conformational states

## TL;DR

This paper introduces a new fluorescence-based method to monitor riboswitch conformational changes, which could aid in developing antibiotics.

## Contribution

A novel fluorescence anisotropy assay is developed for sensitive and scalable monitoring of riboswitch conformational states.

## Key findings

- Fluorescence anisotropy changes correlate with riboswitch conformational changes observed by native gel analysis.
- The assay can distinguish ligands that induce conformational changes from those that do not.
- The method is suitable for high-throughput screening of potential antibiotic compounds.

## 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.

Graphical Abstract