Monitoring Enzymatic RNA G-Quadruplex Unwinding Activities by Nuclease Sensitivity and Reverse Transcription Stop Assays.

Multiple different methods have been employed to investigate the unwinding of RNA G-quadruplexes by various helicase proteins. Each has their own pitfalls, namely, looking at non-native or chemically modified RNA sequences, biasing the unwinding process with competing trap nucleotides, and a lack of context sequence to the 5' and 3' of the RNA G-quadruplex structure. Herein we present two straightforward methods that allow for quadruplex unwinding to be monitored on native RNA sequences without the use of fluorescent modifications, specialized equipment, or trap nucleotides to be employed.