A universal orthogonal imaging platform for living-cell RNA detection using fluorogenic RNA aptamers

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• MicroRNAs (miRNAs) are key post-transcriptional gene regulators with implications for disease and therapeutics.
• Existing miRNA detection methods often lack sensitivity or multiplexing capabilities for real-time cellular analysis.

Purpose of the Study:

• To develop and validate a novel class of miRNA-induced light-up RNA sensors (miLS) for sensitive and orthogonal imaging of intracellular miRNAs.
• To demonstrate the capability of miLS for simultaneous detection and visualization of multiple miRNA targets in living cells.

Main Methods:

• Design of miLS based on toehold-mediated strand displacement and fluorogenic RNA aptamers (Pepper and Squash).
• Incorporation of a sensor switch for enhanced flexibility and convertibility.
• Characterization of sensor performance, including detection limits and signal-to-noise ratios for specific miRNA targets (miR-21 and miR-122).
• Establishment of an orthogonal dual-color imaging platform for simultaneous miRNA visualization in living cells.

Main Results:

• miLS demonstrated sensitive detection of miR-21 (0.48 nM) and miR-122 (0.2 nM) with high signal-to-noise ratios (up to 44x).
• The dual-color system utilizing Pepper (red) and Squash (green) enabled orthogonal and simultaneous imaging of different intracellular miRNAs.
• Successful visualization of sequence-specific miRNA activity in living cells.

Conclusions:

• The developed miLS platform offers a flexible, sensitive, and orthogonal approach for miRNA imaging in living systems.
• This technology provides a powerful tool for studying intricate RNA functions and disease mechanisms at the cellular level.
• The orthogonal imaging capability opens new avenues for multiplexed analysis of RNA dynamics in real-time.