Proximity-Activated DNA Scanning Encoded Sequencing for Massive Access to Membrane Proteins Nanoscale Organization

Membrane proteins are integral to numerous cellular functions, thus understanding their spatial organization is crucial for elucidating disease mechanisms and developing effective therapeutic strategies. Despite advancements, current technologies for deciphering membrane proteins interaction in complex nanoenvironment remain hindered by limited resolution and modest coverage within interested regions. Herein, we demonstrate Track-Free DNA Walkers Indexed Proximity Sequencing (TRIP-seq), a novel technique that operates independently of track constraints within an adjustable nanoscale range, freely searching in random direction along arbitrary paths for proximal proteins. TRIP-seq leverages autonomous cyclic nicking reactions of flexible DNA strands to sequentially activate encoded probes distributed throughout the local area, initiating strand displacement amplification and producing a substantial number of amplicons embedded with protein and molecular barcodes for next-generation sequencing analysis. We validated TRIP-seq in vitro and across different breast cancer cell lines, showcasing its capacity in decoding multiplexed protein proximities at nanoscale. This approach provides a robust tool for high-resolution and comprehensive mapping of cellular molecular interactions, thereby opening new avenues for understanding complex biological processes and informing precision medicine.