Genetically Encoded Ratiometric RNA-Based Sensors for Quantitative Imaging of Small Molecules in Living Cells

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Accurate intracellular concentration determination is vital for cell biology research.
• Fluorogenic RNA sensors offer potential for live-cell target detection.
• Quantifying cellular targets with genetically encoded sensors remains challenging.

Purpose of the Study:

• To develop a modular sensor system for quantifying intracellular target concentrations.
• To address the limitations of current genetically encoded sensors in cellular quantification.
• To enable ratiometric measurement of cell-to-cell variations in target levels.

Main Methods:

• Engineered a modular sensor system using orthogonal fluorogenic RNA aptamers (DNB and Broccoli).
• Applied the DNB-to-Broccoli fluorescence ratio for quantitative analysis.
• Utilized live-cell imaging techniques for sensor validation.

Main Results:

• Developed a ratiometric sensor system capable of quantifying intracellular target concentrations.
• Demonstrated the ability to measure cell-to-cell variations in target levels.
• Validated the broad applicability of the sensor system for various compounds.

Conclusions:

• The DNB-to-Broccoli ratiometric sensor system provides a robust method for quantifying intracellular molecules.
• This system overcomes previous challenges in applying RNA sensors for cellular concentration measurements.
• The developed sensors are broadly applicable for live-cell imaging and quantification in diverse biological and chemical contexts.