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Protocol for evaluating compound uptake and RNase L co-localization in live cells using fluorescence-based binding,
Elias Khaskia1, Raphael I Benhamou1
1The Institute for Drug Research of the School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel.
STAR Protocols
|June 15, 2025
Summary
This study introduces fluorescent probes for real-time visualization of RNase L using ribonuclease targeting chimera (RIBOTAC) technology. The protocol allows researchers to track RNA degradation pathways and assess small molecule effects in live cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Ribonuclease L (RNase L) plays a crucial role in RNA degradation pathways.
- Real-time visualization of enzyme localization and dynamics is essential for understanding cellular processes.
- Ribonuclease targeting chimera (RIBOTAC) technology offers a novel approach for targeted RNA manipulation.
Purpose of the Study:
- To present a protocol for assessing fluorescent probe uptake, binding specificity, and RNase L co-localization in live cells.
- To enable real-time monitoring of RNA degradation pathways.
- To facilitate the evaluation of RNA-targeting small molecules.
Main Methods:
- Development and application of fluorescent probes for RNase L visualization.
- Utilization of a fluorescent-based binding and competition assay.
- Confocal microscopy for live-cell imaging and quantitative fluorescence analysis.
Main Results:
- Successful real-time visualization of RNase L localization and interaction dynamics.
- Demonstration of probe uptake, binding specificity, and co-localization with RNase L.
- Quantitative fluorescence analysis provides insights into RNA degradation pathways.
Conclusions:
- The developed protocol enables high-resolution monitoring of RNA degradation.
- This method is valuable for studying the effects of RNA-targeting small molecules.
- Fluorescent probes combined with RIBOTAC technology offer a powerful tool for live-cell investigations of RNase L activity.
