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Imaging and Tracking RNA in Live Mammalian Cells via Fluorogenic Photoaffinity Labeling.

Alexandria L Quillin1, Diane B Karloff1,2, Tewoderos M Ayele2

  • 1Department of Chemistry, Washington University, St. Louis, Missouri 63130, United States.

ACS Chemical Biology
|February 15, 2025
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Summary
This summary is machine-generated.

This study introduces a novel photoaffinity labeling method for robust cellular RNA tracking. Covalent attachment via UV light improves imaging sensitivity and enables precise visualization of RNA dynamics, overcoming limitations of noncovalent methods.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Cell Biology

Background:

  • Aptamer-based RNA labeling offers a genetically encodable alternative to protein-based methods.
  • Noncovalent aptamer-fluorogen interactions limit RNA imaging robustness and tracking fidelity due to dye washout and dissociation.
  • Existing methods struggle with dye transfer between RNA molecules, compromising dynamic studies.

Purpose of the Study:

  • To develop a covalent RNA labeling strategy to overcome limitations of noncovalent aptamer-based systems.
  • To enable robust, spatiotemporally controlled RNA imaging and tracking within live cells.
  • To enhance sensitivity and temporal resolution for studying RNA dynamics.

Main Methods:

  • Functionalization of aptamer ligands with a photoactivatable diazirine group for UV-induced covalent RNA attachment.
  • Incorporation of a photoaffinity linker into malachite green and fusion of its aptamer to target RNAs (snRNA and mRNA).
  • Live-cell imaging experiments to assess labeling efficiency, sensitivity, and RNA dynamics.

Main Results:

  • Demonstrated improved sensitivity for live-cell RNA imaging upon UV irradiation.
  • Successfully visualized RNA dynamics over time scales of minutes with covalent labeling.
  • Showcased the robustness of covalent linkage, preventing dye transfer and enabling accurate RNA tracking.

Conclusions:

  • Photoaffinity labeling provides a robust method for covalent RNA labeling, enhancing imaging capabilities.
  • This approach enables precise, time-resolved studies of RNA localization and dynamics.
  • The method holds promise for diverse investigations into cellular RNA behavior and interactions.