Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM)

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Visualizing RNA dynamics in live cells is crucial for understanding cellular processes.
• Current methods often use fluorescently tagged RNA aptamers, but face limitations in multiplexing and cellular contrast.
• Engineering efforts focus on enhancing RNA visualization techniques for simultaneous multi-RNA tracking.

Purpose of the Study:

• To develop a novel platform for live-cell RNA visualization with enhanced capabilities.
• To improve cellular contrast and reduce tag size compared to existing methods.
• To enable multiplexed visualization of multiple RNAs within the same cell.

Main Methods:

• Development of the Riboglow-FLIM platform utilizing fluorescence lifetime imaging microscopy.
• Engineering of RNA tags derived from bacterial riboswitch sequences.
• Demonstration of simultaneous visualization of two distinct RNAs using orthogonal lifetime-based tags.

Main Results:

• Riboglow-FLIM offers superior cell contrast compared to intensity-based detection methods.
• The platform utilizes smaller RNA tags, minimizing cellular perturbation.
• Successful simultaneous visualization of two RNAs was achieved using orthogonal riboswitch variants.

Conclusions:

• Riboglow-FLIM provides a versatile and effective platform for live-cell RNA visualization.
• The use of riboswitch variants enables robust multiplexing for simultaneous multi-RNA tracking.
• This technology advances the study of RNA dynamics and function in complex cellular environments.