Activatable Fluorescence-Encoded Nanoprobes Enable Simple Multiplexed RNA Imaging in Live Cells

Area of Science:

• Molecular Biology
• Biotechnology
• Cellular Imaging

Background:

• Current single-molecule RNA fluorescence imaging methods struggle with multiplexing due to fluorophore spectral overlap.
• Existing multiplex imaging strategies often require complex designs and cell permeability enhancements, hindering rapid live-cell applications.
• There is a need for simplified, efficient methods for simultaneous detection of multiple RNA targets in live cells.

Purpose of the Study:

• To develop a novel nanoprobe strategy for convenient and rapid multiplexed RNA imaging in single live cells.
• To overcome the limitations of spectral overlap and complex sample preparation in current RNA imaging techniques.
• To enable simultaneous qualitative and quantitative analysis of multiple RNA targets within live cells.

Main Methods:

• Development of an activatable fluorescence-encoded nanoprobe (AFENP) strategy.
• Utilizing fluorescence-encoded functional modules for qualitative analysis and activated nucleic acid assemblies for quantitative testing.
• Employing two distinguishable fluorophores (fluorescein and rhodamine B) with seven distinct intensity levels for multiplexing.

Main Results:

• The self-assembled AFENP enables simplified and rapid simultaneous in situ detection and imaging of seven types of targets in live single cells.
• Fluorescent signals are activated only in the presence of the target, eliminating the need for washing procedures.
• The strategy avoids additional pretreatment to increase cell permeability, streamlining the imaging process.

Conclusions:

• The activatable fluorescence-encoded nanoprobe (AFENP) strategy provides a practical platform for simple and multiplexed RNA imaging in live single cells.
• This approach overcomes key limitations of existing methods, offering enhanced convenience and speed.
• The AFENP technology holds promise for widespread adoption in multiple gene expression analysis and live-cell imaging.