Fluorescent Dyes for Differential Scanning Fluorimetry of RNA

Differential scanning fluorimetry (DSF) to determine the thermal stability of RNA has only recently been adapted from proteins using the nucleic acid dye RiboGreen. Here, we investigate the suitability of seven commercially available nucleic acid dyes for use in DSF on RNA—acridine orange, ethidium bromide, RiboGreen, OliGreen, SYBR Green II, SYBR Gold, and TOTO‐1 iodide—using a double‐stranded RNA model and the 27‐nt neomycin‐sensing riboswitch aptamer. Concentration‐dependent DSF curves revealed pronounced dye‐specific effects on both initial fluorescence and apparent melting temperatures. RiboGreen and OliGreen yielded concentration‐independent melting temperatures within experimental error, whereas SYBR derivatives, acridine orange, and ethidium bromide stabilized RNA progressively with increasing dye concentration, shifting the melting temperature by up to +9 °C. Taken together, the data identify RiboGreen and OliGreen as probes for use in DSF on RNA that minimize dye‐induced artifacts while preserving robust signal‐to‐noise, and they provide a framework for tailoring DSF conditions to specific RNA targets and ligand‐binding screens. Other tested dyes displaying concentration‐dependent melting temperatures are suitable as well but require appropriate care when utilizing them in DSF on RNA.