Systematic detection of m⁶A-modified transcripts at single-molecule and single-cell resolution.

Area of Science:

• Molecular Biology
• Epigenetics
• Cell Biology

Background:

• Epigenetic modifications regulate mRNA stability and translation.
• Understanding RNA modifications at the single-cell level is crucial for cell biology.

Purpose of the Study:

• To develop a microscopy-based platform for quantifying RNA modifications in single cells.
• To correlate RNA modification patterns with single-cell phenotypes and gene expression.

Main Methods:

• Direct capture of mRNA from cell lysates onto oligo-dT-coated coverslips.
• Label-free, amplification-free visual detection and sequencing of individual m⁶A-modified transcripts.
• Integration of a nanoscale device for single-cell isolation and multi-modal analysis (m⁶A detection, gene expression, cell surface markers).
• Coupling single-molecule m⁶A detection with fluorescence in situ hybridization (FISH).

Main Results:

• Demonstrated a microscopy platform for single-cell RNA modification quantification.
• Observed reduced cellular m⁶A levels during MUTZ3 leukemia cell differentiation (CD34+ to CD14+).
• Successfully related individual gene mRNA and m⁶A levels to specific single-cell phenotypes using FISH.

Conclusions:

• The developed platform enables direct, amplification-free quantification of RNA modifications in single cells.
• This technology allows for the investigation of RNA modification dynamics in relation to cellular states and differentiation.
• The single-cell multi-modal assay suite is valuable for studying RNA modifications in rare cell populations.