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

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

  • STAR Protoc. 2025 Jun 14;6(3):103904. doi: 10.1016/j.xpro.2025.103904.
Elias Khaskia 1,  Raphael I Benhamou 2
Affiliations

Affiliations

  • 1 The Institute for Drug Research of the School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. Electronic address: elias.khaskia@mail.huji.ac.il.
  • 2 The Institute for Drug Research of the School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem 91120, Israel. Electronic address: raphael.benhamou@mail.huji.ac.il.
PMID: 40517387 DOI: 10.1016/j.xpro.2025.103904
Abstract

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.

Keywords

Cell Biology; Microscopy; Molecular Biology; Molecular/Chemical Probes.

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