Location‐Differentiated DNA Hierarchical Cyclic Construction Demarcating Identical Biomolecules Across Cell Membrane Microenvironments

Abstract Local molecular composition across cell membrane microenvironments exerts profound effects on cellular physiology and pathology. Yet spatial demarcation of identical biomolecules inside and outside lipid raft microdomains has not been determined. Here we report location‐differentiated DNA hierarchical cyclic construction for lipid raft‐differentiated spatial profiling of identical biomolecules in living cells. It contains two sequential DNA cyclic construction reactions: primary one triggered by total target molecules and secondary one activated by lipid raft's location markers proximal to target molecules. Using this method, we differentially visualize identical biomolecules localized in lipid raft and non‐lipid raft microdomains with different signals. We demonstrate the robustness and universality of this method by testing cell surface RNAs and proteins in diverse cell types. We reveal their diverse spatial demarcation with intrinsic heterogeneity. In both lipid raft‐disrupting cells and senescent cells, MUC1 protein is dynamically organized between these two microdomains. Our strategy provides a promising avenue to investigate physiological and pathological functions of dynamic molecular compositions across cellular microenvironments.