Trojanowski, J.; Zilke, J.; Pak, V.; Geissen, E.-M.; Chalupska, R.; Reinhardt, R.; AlMehallawi, S.; Kishi, J. Y.; Heck, J.; Haase, K.; Saka, S. K.

Proximity detection methods facilitate contextual analysis of biomolecules by reporting their microenvironment, spatial organization and molecular interactions. To expand these detection capabilities towards highly sensitive, quantitative, and multiplexed interaction mapping, we introduce Proximity Primer Exchange Reaction (ProPER). ProPER is a novel ligation-free proximity extension and controlled DNA amplification strategy that enables spatially resolved in situ detection of molecular proximities. In ProPER, extension of DNA barcodes into linear concatemers is made conditional on the spatial proximity of primer and hairpin pairs split across two targets, thereby enabling efficient coincidence or proximity detection with isothermal signal amplification for high-sensitivity imaging. The method is readily integrated with mainstream in situ assays such as immunofluorescence, FISH and metabolic labeling, and supports high-resolution visualization of diverse molecular modalities, including RNA-RNA or protein-protein interactions, even at dense labeling sites. Leveraging pre-validated orthogonal sequence pairs, ProPER allows simultaneous multiplexed detection of molecular proximities within the same cell and on the same target molecule. We applied multiplexed ProPER to resolve the life-cycle stages of individual RNA molecules. Quantitative tracking of transcriptional, splicing and translational states enabled construction of a kinetic model that identifies key regulatory steps driving differential expression kinetics of two inflammatory response genes. Overall, we establish ProPER as a versatile and efficient framework for in situ multiplexed proximity detection, overcoming key limitations of existing approaches. The programmable DNA encoding scheme enables advanced capabilities, such as proximity cascades and multivalent interaction detection, and provides a basis for integrating molecular proximity measurements into future combinatorial barcoding or spatial omics workflows.