Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis.

Area of Science:

• Molecular Biology
• Cell Biology
• Spectroscopy

Background:

• Pyronin Y is an environment-sensitive probe that labels double-stranded RNA in live cells.
• Current methods to identify specific RNA species labeled by Pyronin Y are limited due to challenges in isolating individual transcripts.
• Understanding which RNA species Pyronin Y binds is crucial for its application in cell biology.

Purpose of the Study:

• To develop and apply a spectroscopic technique to identify specific RNA species labeled by Pyronin Y in live cells.
• To overcome the limitations of isolating individual RNA transcripts for analysis.
• To demonstrate the capability of phasor analysis for distinguishing RNA subtypes.

Main Methods:

• Utilized the phasor approach to fluorescence lifetime analysis.
• Detected and analyzed RNA transcripts directly within their biological environment in live fibroblast cells.
• Correlated spectroscopic properties with cellular location to identify RNA species.

Main Results:

• Successfully identified at least six different RNA species labeled by Pyronin Y in live fibroblast cells.
• Demonstrated consistent lifetime differences among detected RNA species across different cells.
• Showcased the sensitivity of the phasor approach for distinguishing RNA subtypes.

Conclusions:

• The phasor approach to lifetime analysis is a sensitive spectroscopic technique for identifying Pyronin Y-labeled RNA subtypes in living cells.
• This study represents the first application of spectroscopy to differentiate Pyronin Y-bound RNA subtypes within their native cellular context.
• The findings open new avenues for studying RNA localization and function in real-time.