Protocol for evaluating compound uptake and RNase L co-localization in live cells using fluorescence-based binding, competition assay, and confocal microscopy.

Using ribonuclease targeting chimera (RIBOTAC) technology, fluorescent probes enable real-time visualization of RNase L localization and interaction dynamics. Here, we present a protocol to assess probe uptake, binding specificity, and RNase L co-localization in live cells using a fluorescent-based binding and competition assay combined with confocal microscopy. We provide step-by-step instructions for live-cell imaging and quantitative fluorescence analysis, enabling researchers to monitor RNA degradation pathways and evaluate the effects of RNA-targeting small molecules with high spatial resolution. For complete details on the use and execution of this protocol, please refer to Khaskia et al.(1).