Visualizing Newly Synthesized RNA by Bioorthogonal Labeling-Primed DNA Amplification

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Monitoring RNA synthesis is crucial for understanding cellular functions and disease mechanisms.
• Visualizing newly synthesized RNA in single cells presents significant technical challenges.

Purpose of the Study:

• To develop a novel method for visualizing newly synthesized RNA in single cells.
• To investigate the subcellular distribution and spatial organization of newly synthesized RNA.

Main Methods:

• Developed a bioorthogonal labeling strategy using a novel N6-allyladenosine nucleoside to label newly synthesized RNA.
• Utilized tetrazine-functionalized primers for bioorthogonal reaction with allyl-modified RNA.
• Employed rolling circle amplification (RCA) for signal amplification, generating long DNA strands for fluorescence probe capture.

Main Results:

• Successfully visualized newly synthesized RNA in single cells using the developed method.
• Demonstrated that newly synthesized RNAs exhibit cell type-specific spatial organization.
• Observed significant cell-to-cell heterogeneity in the distribution of newly synthesized RNA.

Conclusions:

• The developed bioorthogonal labeling-primed DNA amplification strategy is effective for visualizing newly synthesized RNA in single cells.
• Newly synthesized RNA distribution is spatially organized in a cell type-specific manner.
• Significant cell-to-cell variability exists in newly synthesized RNA localization, highlighting its dynamic nature.