RNA sequencing is widely used to measure gene activity, but most methods require cells to be broken open or fixed before their RNA can be collected. This means researchers usually get only a single snapshot of gene expression from each cell population.

Researchers at Peking University have developed a method called POND-seq, short for protein nanocage-empowered non-destructive sequencing, that can collect RNA information from living cells without destroying them.

Using protein nanocages to export RNA

POND-seq uses engineered protein nanocages that cells release into their surroundings. These nanocages are fused to RNA-binding proteins, or RBPs, which interact with specific RNA molecules inside cells.

RNA associated with an RBP can be packaged into the nanocage and exported from the living cell. Researchers can then collect the nanocages from the culture medium, isolate the RNA, and analyze it by RNA sequencing.

Because the cells remain alive, this process can be repeated over time, allowing researchers to follow changes in the same cell population.

Tracking RNA interactions and gene expression

The researchers showed that POND-seq can identify RNA targets associated with cytoplasmic RBPs across multiple cell types. It can also track how these interactions change when cells are exposed to stress.

The method was also adapted to examine broader transcriptome changes by fusing the nanocage to PABPC1, a protein that binds the poly(A) tails of many messenger RNAs.

This version of POND-seq allowed researchers to monitor gene expression over time without destroying the cells.

Studying mixed cell populations

POND-seq can also selectively collect RNA from specific cell types growing within mixed populations.

Normally, researchers may need to separate cells before RNA sequencing. By engineering selected cells to release their RNA through nanocages, POND-seq can provide cell-type-specific transcriptome information without cell dissociation and sorting.

Examining disease-associated RNA-binding proteins

The researchers also used POND-seq to study mutations in FMR1, an RNA-binding protein associated with neurological disease.

By comparing different FMR1 variants, they were able to assess how individual mutations affected RNA association. This demonstrates how the method could support scalable analysis of disease-related protein variants.

Following living cells over time

Cells continually change as they respond to stress, drugs, differentiation, and disease. Conventional RNA sequencing can capture these changes only by analyzing different cells at each time point.

POND-seq provides a way to repeatedly measure RNA activity in living cells. By combining engineered protein nanocages with RNA-binding proteins, the method can track RBP-RNA interactions, transcriptome changes, cell-type-specific expression, and the effects of disease-associated mutations over time.