# Fluorescent Ligand Equilibrium Displacement: A High-Throughput Method for Identification of FMN Riboswitch-Binding Small Molecules

## TL;DR

This paper introduces a new high-throughput method called FLED to find small molecules that bind to the FMN riboswitch, offering a potential new approach for antibiotic development.

## Contribution

The novel FLED method enables label-free, in vitro screening of small molecules targeting the FMN riboswitch.

## Key findings

- FLED demonstrated robustness for high-throughput screening with a hit rate of 0.67% from an existing drug library.
- The method revealed a variety of effective concentration values for identified small molecules.
- FLED is adaptable and offers a strong alternative to existing riboswitch screening techniques.

## Abstract

Antibiotic resistance remains a pressing global concern, with most antibiotics targeting the bacterial ribosome or a limited range of proteins. One class of underexplored antibiotic targets is bacterial riboswitches, structured RNA elements that regulate key biosynthetic pathways by binding a specific ligand. We developed a methodology termed Fluorescent Ligand Equilibrium Displacement (FLED) to rapidly discover small molecules that bind the flavin mononucleotide (FMN) riboswitch. FLED leverages intrinsically fluorescent FMN and the quenching effect on RNA binding to create a label-free, in vitro method to identify compounds that can bind the apo population of riboswitch in a system at equilibrium. The response difference between known riboswitch ligands and controls demonstrates the robustness of the method for high-throughput screening. An existing drug discovery library that was screened using FLED resulted in a final hit rate of 0.67%. The concentration response of each hit was determined and revealed a variety of approximate effective concentration values. Our preliminary screening data support the use of FLED to identify small molecules for medicinal chemistry development as FMN riboswitch-targeted antibiotic compounds. This robust, label-free, and cell-free method offers a strong alternative to other riboswitch screening methods and can be adapted to a variety of laboratory setups.