In Situ Imaging of Adjacent Biomolecules via Proximity Anchored Modules Assembly

Aberrant proximal biomolecular complexes are critical disease biomarkers. The precise in situ imaging of these complexes is essential for deciphering disease pathogenesis and precision diagnostics. However, current in situ analysis technologies are often constrained by limited resolution, diffusion-mediated false positives, or the requirements for rigid conjugation between recognition and amplification moieties, which hampers versatility and multiplexing. Here, we introduce Proximity Anchored Modules Assembly (PAMA), a versatile and multiplexed imaging strategy with a “plug-and-play” architecture. By decoupling target recognition from signal amplification via programmable DNA tracks synthesized by Primer Exchange Reaction (PER), PAMA triggers a polymerase-driven extension exclusively upon dual-recognition events. This mechanism ensures precise proximity-dependent activation, showing specific signal response to homologous and heterologous targets. We further validated PAMA as a versatile platform for detection of proximal biomarkers by visualizing HER2 receptor dimerization patterns in breast cancer cells and precise discrimination of BCR-ABL ^P210 fusion gene isoforms in clinical chronic myeloid leukemia (CML) samples. PAMA bridges molecular precision with spatial context through a rapid (approximately 2.5 h) and highly sensitive in situ profiling workflow. Ultimately, PAMA establishes a versatile, modular framework for the precise imaging of diagnostic biomolecular complexes, promising to accelerate both biological discovery and precision clinical diagnostics.