Microfluidic screening of electrophoretic mobility shifts elucidates riboswitch binding function

Area of Science:

• Molecular Biology
• Biophysics
• Biochemistry

Background:

• Riboswitches are RNA sensors that regulate gene expression by altering conformation upon binding small molecule metabolites.
• Existing methods for studying riboswitch-ligand interactions are often time-consuming and lack high-throughput capabilities.
• A need exists for a rapid, quantitative tool to screen riboswitches and their ligand binding.

Purpose of the Study:

• To introduce and validate a microfluidic mobility shift assay for high-throughput, quantitative analysis of riboswitch-ligand binding.
• To demonstrate the assay's ability to rapidly screen and validate candidate riboswitches.
• To establish a scalable platform for characterizing riboswitch conformational changes and binding kinetics.

Main Methods:

• Development of a microfluidic mobility shift assay for precise quantitation of riboswitch-ligand binding.
• Screening and validation of five candidate SAM-I riboswitches from thermophilic and cryophilic bacteria.
• Quantitation of equilibrium dissociation constants (K(d)) using the microfluidic assay and comparison with in-line probing results.

Main Results:

• The microfluidic assay screens riboswitches in 0.3% of the time required for traditional benchtop assays (3.2 min vs. 1020 min).
• The assay provides enhanced resolution, quantitation, and repeatability for detecting small mobility shifts and conformational changes.
• Equilibrium dissociation constants (K(d)) were accurately quantified for both known and candidate SAM-I riboswitches.

Conclusions:

• The microfluidic mobility shift assay is a rapid, sensitive, and scalable tool for studying riboswitch-ligand interactions.
• This technology accelerates the discovery and selection of novel riboswitches.
• The assay facilitates the development of new antibiotics targeting bacterial riboswitches.