The ability to image RNA identity and location with nanoscale precision in intact tissues is of great interest for defining cell types and states in normal and pathological biological settings. Here, we present a strategy for expansion microscopy (ExM) of RNA. We developed a small molecule linker that enables RNA to be covalently attached to a swellable polyelectrolyte gel synthesized throughout a biological specimen. Then, post-expansion, fluorescent in situ hybridization (FISH) imaging of RNA can be performed with high yield and specificity, with single molecule precision, in both cultured cells and intact brain tissue. Expansion FISH (ExFISH) de-crowds RNAs and supports amplification of single molecule signals (i.e., via hybridization chain reaction (HCR)) as well as multiplexed RNA FISH readout. ExFISH thus enables super-resolution imaging of RNA structure and location with diffraction-limited microscopes in thick specimens, such as intact brain tissue and other tissues of importance to biology and medicine.

Although the genome is generally thought to be transcriptionally silent during mitosis, technical limitations have prevented sensitive mapping of transcription during mitosis and mitotic exit. Thus, the means by which the interphase expression pattern is transduced to daughter cells have been unclear. We used 5-ethynyluridine to pulse-label transcripts during mitosis and mitotic exit and find that many genes exhibit transcription during mitosis, as confirmed by FITC-UTP labeling, RNA FISH, and RT-qPCR. The first round of transcription immediately following mitosis primarily activates genes involved in the growth and rebuilding of daughter cells, rather than cell type-specific functions. We propose that the cell’s transcription pattern is largely retained at a low level through mitosis, whereas the amplitude of transcription observed in interphase is re-established during mitotic exit.

Methods for detecting single nucleic acids in cell and tissues, such as fluorescence in situ hybridization (FISH), are limited by relatively low signal intensity and non-specific probe binding. Here we present click-amplifying FISH (clampFISH), a method for fluorescence detection of nucleic acids that achieves high specificity and high-gain (>400x) signal amplification. ClampFISH probes form a “C” configuration upon hybridization to the sequence of interest in a double helical manner. The ends of the probes are ligated together using bioorthogonal click chemistry, effectively locking the probes around the target. Iterative rounds of hybridization and click amplify the fluorescence intensity. We show that clampFISH enables the detection of RNA species with low magnification microscopy and in RNA-based flow cytometry. Additionally, we show that the modular design of clampFISH probes allows multiplexing of RNA and DNA detection, that the locking mechanism prevents probe detachment in expansion microscopy, and that clampFISH can be applied in tissue samples.

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Highlights d T cell identity genes are hyperconnected in 3D in C57BL/6 and NOD strains d Diabetes-associated regions are hyperconnected in 3D only in NOD strain d The 3D regulatory landscape in NOD mice is mediated in cis d KRAB-ZFP genes are highly expressed in the pancreas of humans with T1D