Multiplexed sequential imaging in living cells with orthogonal fluorogenic RNA aptamer/dye pairs

Area of Science:

• Cell Biology
• Molecular Imaging
• Biotechnology

Background:

• Multiplexed fluorescence imaging is crucial for understanding complex cellular processes.
• Current methods struggle with detecting more than two-to-three targets simultaneously in living cells.

Purpose of the Study:

• To introduce and validate a novel multiplexed imaging strategy, seqFRIES, for enhanced live-cell target detection.
• To overcome the technical challenges associated with high-multiplexing fluorescence imaging.

Main Methods:

• Developed sequential Fluorogenic RNA Imaging-Enabled Sensor (seqFRIES) using genetically encoded orthogonal fluorogenic RNA aptamers.
• Employed sequential rounds of imaging and rapid stripping of cell membrane permeable dye molecules.
• Identified and optimized four orthogonal fluorogenic RNA aptamer/dye pairs for live-cell imaging.

Main Results:

• Demonstrated highly orthogonal and multiplexed imaging in living bacterial and mammalian cells using seqFRIES.
• Achieved a four-color semi-quantitative imaging process completed in approximately 20 minutes.
• Successfully performed simultaneous detection of critical signaling molecules and mRNA targets within individual living cells.

Conclusions:

• seqFRIES is a validated, rapid, and effective strategy for multiplexed live-cell imaging.
• The developed orthogonal fluorogenic RNA/dye pairs enable dynamic and multi-target detection.
• This approach is expected to advance live-cell imaging and cell biology research.