Data from fluorescence-based methods that measure in vivo hybridization efficacy of unique RNA regions can be used to infer regulatory activity and to identify novel RNA: RNA interactions. Here, we document the step-by-step analysis of fluorescence data collected using an in vivo regional RNA structural sensing system (iRS) for the purpose of identifying potential functional sites that are likely to be involved in regulatory interactions. We also detail a step-by-step protocol that couples this in vivo accessibility data with computational mRNA target predictions to inform the selection of potentially true targets from long lists of thermodynamic predictions.
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