Visualizing Diverse RNA Functions in Living Cells With Spinach^TM Family of Fluorogenic Aptamers

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• RNA localization, processing, and turnover are critical for cellular functions and disease development.
• Live-cell RNA imaging is essential for understanding RNA dynamics and mechanisms.
• Current imaging methods have limitations, including delivery issues, large payloads, and background noise.

Purpose of the Study:

• To present a standardized protocol for live-cell RNA imaging using the Spinach family of fluorogenic RNA aptamers.
• To enable robust and reproducible tracking of RNA localization and dynamics in various cellular applications.
• To provide a simpler, more efficient alternative to existing RNA imaging techniques.

Main Methods:

• Design and cloning of Spinach-tagged RNA constructs.
• Selection and handling of cognate small-molecule fluorophores.
• Expression in mammalian cells, dye loading, and fluorescence microscopy for image acquisition and analysis.
• Utilizing controls for aptamer expression and signal specificity, with guidance for multiplexing and troubleshooting.

Main Results:

• Demonstrated successful live-cell RNA imaging using Spinach aptamers across different RNA types and cellular compartments (nucleus and cytoplasm).
• Achieved high-contrast imaging with compact, genetically encodable Spinach tags, simplifying the imaging process.
• Showcased applications in tracking mRNA therapeutics, monitoring transcription/decay, and observing RNA aggregate formation.

Conclusions:

• Spinach aptamer technology provides a powerful, versatile, and user-friendly tool for live-cell RNA imaging.
• The standardized protocol enhances robustness and reproducibility for diverse RNA-focused applications.
• This method facilitates deeper insights into RNA biology, cellular mechanisms, and disease processes.