Live-cell imaging of RNA dynamics using bright and stable fluorescent RNAs

Area of Science:

• Molecular Biology
• Cell Biology
• Biotechnology

Background:

• Cellular RNA dynamics are complex, involving coordinated expression, splicing, localization, translation, and degradation.
• Understanding RNA biological functions requires methods for high spatiotemporal resolution monitoring.
• Fluorescent RNAs (FRs) offer a promising approach for live-cell RNA imaging.

Purpose of the Study:

• To provide accessible strategies for visualizing diverse RNA species in live bacteria and mammalian cells using FRs.
• To detail protocols for multiplexed RNA imaging and super-resolution live imaging.
• To enable real-time monitoring of RNA localization and dynamics.

Main Methods:

• Development and application of high-performance FRs (Pepper, Clivia, Okra).
• Tagging RNA of interest with aptamer sequences for FR binding.
• Utilizing orthogonal FRs for multiplexed imaging.
• Implementing super-resolution live imaging techniques.

Main Results:

• Demonstrated easy, efficient, and generalizable strategies for FR-based RNA visualization.
• Achieved high cellular brightness, photostability, and low ion dependence with developed FRs.
• Successfully imaged diverse RNA species in both bacterial and mammalian cells.
• Enabled multiplexed and super-resolution live imaging of RNA.

Conclusions:

• FRs provide powerful tools for real-time, high-resolution RNA imaging in live cells.
• The developed protocols are broadly applicable for studying RNA localization and dynamics.
• This approach facilitates a deeper understanding of RNA biological functions.