F. Karlsson, M. Simonetti, C. Galonska, M. J. Karlsson, H. van Ooijen, D. Thiagarajan, T. Kallas, M. Schweitzer, L. Larsson, V. van Hoef, P. Tajvar, J. Dahlberg, F. De Temmerman, L. Leijonancker, V. Trombin, S. Geny, R. Forlin, E. Negrini, B. Wu, L. Xi, S. Petkov, L. Franzen, J. Bunz, C. Moge, H. Everberg, P. Brodin, A. Martinez Barrio, S. Fredriksson
Cellular functions depend on dynamic interactions of proteins and their spatial organisation. While transcriptomic and proteomic methods of molecular parts-lists have enabled single-cell profiling based on abundance, scalable technologies allowing high-resolution measurements of protein organization and interactions at scale are lacking. Here we present the Proximity Network Assay (PNA), a DNA-based method for constructing three-dimensional nanoscale maps of 155 plasma membrane proteins in single cells without the use of optics. PNA employs barcoded antibodies and in situ rolling circle amplification to generate >40,000 spatial nodes per cell, which are linked through proximity-dependent gap-fill ligation and decoded by DNA sequencing forming single cell Proximity Networks. PNA captures abundance, self-clustering, and ~12,000 pairwise colocalization relationships per single-cell, validating established protein interactions. This new modality provides a framework to uncover novel spatial biomarkers, reveal functional mechanisms, and advance translational studies in immunology, oncology, and cell therapy.