Combi-PAINT, a novel combinatorial DNA-PAINT multiplexing method integrated with RNA-FISH and MINFLUX microscopy, enables nanometer-precision three-dimensional visualization of mRNA ultrastructure in human cells, revealing that different mRNA species adopt distinct morphologies that correlate with genomic features such as intron density and are modulated by subcellular localization and processing events.

Messenger RNA (mRNA) is a central polymer of gene expression, whose ultrastructure and defining regulatory rules remain unclear. To visualize the ultrastructure of mRNA, we here develop Combi-PAINT, a powerful and generalizable method for combinatorial super-resolved DNA-PAINT multiplexing, which we combine with high-efficiency RNA labelling and MINFLUX microscopy. This approach enables the nanometer-precision tracing of mRNA in three dimensions in cells. We use Combi-PAINT to visualize multiple distinct mRNA species inside the human cell nucleus and cytoplasm, revealing their quantitative ultrastructures and transcript-specific molecular patterns. By bridging sequence specificity with nanometer resolution, we provide a new lens for studying the cellular life of mRNA.