Differential Scanning Fluorimetry (DSF): A High-Throughput Screening Method for Monitoring RNA Stability

Victoria S. Ogunkunle, Nolan D. Blackford, Robert P. Silvers

Florida State University, Tallahassee, Florida.

Demand on a multiple assay toolbox for modern drug discovery for detection high-throughput screening of therapeutic ligands and their small molecule target can be achieved through an indispensable biophysical technique using a non-interactive dye in a thermal shift analysis, Differential scanning fluorimetry (DSF). Utilizing intercalating dye molecules which upon binding to the hydrophobic region of a structured RNA, the dynamics of the conformational stability can be determined via a temperature gradient.

Riboswitches are structured RNA that binds specific targets and induces gene regulation. Neomycin and PreQ1 riboswitches are well-characterized with the three-dimensional structure solved, and with different affinities for their respective ligands. Differential Scanning Fluorimetry (DSF) is a method used to quantify protein thermal stability with the use of intercalating dyes that bind preferentially to the hydrophobic region of the protein. We have employed the use of DSF to measure the thermal stability of different structured riboswitches with and without ligand bound.

Our data showed differences in the melting temperature in both bound and unbound states for preQ1 and Neomycin riboswitches. Neomycin riboswitch showed a wide range in the melting temperatures observed in the bound state using two of its binding ligands. The observed Tm of the riboswitch only is reported to be 51 ⁰C while there is a shift in the T_m values when bounded with paromomycin and neomycin drugs to 63.66 ⁰C and 76.24 ⁰C respectively. This shows that the neomycin riboswitch discriminates the presence of different functional groups on the ligands tested. Comparing the T_m values observed using DSF method and UV-Vis spectroscopy helps in validating our results.

DSF method was used to measure the thermal stability of structured mRNAs. We anticipate that this approach will be widely applicable to other functional RNAs as a high-throughput screening method for drug discovery, and deployment of DSF assay to determine binding affinities of small molecules over a wide range enhances drug discovery.