Abstract
RNA-targeting small molecules, particularly RIBOnuclease TArgeting Chimeras (RIBOTACs), represent a powerful and promising therapeutic approach by selectively degrading RNAs through ribonuclease (RNase) recruitment. Despite their potential, the development of effective RNase recruitment tools is still in its early stages and remains a critical area of research. Ribonuclease L (RNase L) is a key ribonuclease targeted by RIBOTACs, yet the tools available for studying RNase L are limited. In this study, we introduce novel fluorescent ribonuclease binders that enhance the visualization and investigation of RNase L activity. Our findings provide new insights into RNase L dynamics and RNA degradation pathways, paving the way for more effective RNA-targeted degradation strategies. Furthermore, we explore the versatility of these conjugates for real-time tracking of RNase L localization, intracellular trafficking, and mechanistic studies. These fluorescent probes also enable high-throughput fluorescence-based assays to identify small molecules that bind and recruit RNase L, advancing RNA-targeted therapeutic approaches.