Non-destructive transcriptomics via vesicular export

Area of Science:

• Molecular Biology
• Genomics
• Cell Biology

Background:

• Current transcriptomics methods require cell fixation or lysis, preventing longitudinal studies of RNA expression in living cells.
• Analyzing dynamic changes in cellular states over time is crucial for understanding biological processes.

Purpose of the Study:

• To develop a non-destructive method for longitudinal RNA expression analysis in living cells.
• To enable multi-time-point monitoring of cellular transcriptomes and responses to perturbations.

Main Methods:

• Developed non-destructive transcriptomics by vesicular export (NTVE) using stabilized RNA reporter barcodes packaged into virus-like particles (VLPs).
• Utilized an engineered poly(A)-binding protein adapter for endogenous transcript export.
• Employed bioorthogonal affinity handles for multichannel tracking of co-cultured cells.
• Demonstrated NTVE's ability to capture transcriptome changes in response to perturbations within the same cells over time.

Main Results:

• NTVE exports endogenous transcripts with high concordance to conventional RNA-seq.
• The platform successfully captures dynamic transcriptome changes in response to genetic and chemical perturbations.
• NTVE was used for daily non-destructive transcriptomic profiling during hiPSC differentiation, monitoring lineage-specific marker dynamics.

Conclusions:

• NTVE provides a novel platform for non-destructive, longitudinal transcriptomic analysis in living cells.
• This method facilitates the study of dynamic cellular processes and responses over multiple time points.
• NTVE is applicable to various cell types and biological investigations, including stem cell differentiation.