Pairwise Proximity‐Differentiated Visualization of Single‐Cell DNA Epigenetic Marks

Abstract Spatial positioning and proximity of relevant biomolecules such as DNA epigenetic marks are fundamental to a deeper understanding of life. However, it remains poorly explored and technically challenging. Here we report the pairwise proximity‐differentiated visualization of single‐cell 5‐formylcytosine (5fC) and 5‐hydroxymethylcytosine (5hmC). These two marks on chromatin in fixed cells are successively labeled and crosslinked with their DNA primer probes via click chemistry. Based on a pairwise proximity‐differentiated mechanism, proximal 5fC/5hmC sites and residual 5fC or 5hmC sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single‐molecule imaging. We thus demonstrate the differentiated visualization of 5fC or 5hmC spatial positioning and their pairwise proximity in single cells. Such multi‐level subcellular information may provide insights into regulation functions and mechanisms of chromatin modifications, and the spatial proximity can expose the potential crosstalk or interaction between their reader proteins.