Spatial barcoding reveals reaction radii and contact-dependent mechanism of proximity labeling

Proximity labeling techniques such as TurboID and APEX2 have become pivotal tools for studying protein interactions. However, the spatial patterns of labeling methods within the submicrometer range remain poorly understood. Here we used DNA nanostructure platforms to precisely measure the labeling radii of TurboID and APEX2 through in vitro assays. Our DNA nanoruler design enables the deployment of oligonucleotide-barcoded labeling targets with nanometer precision near the enzymes. By quantifying labeling yields using qPCR and mapping them against target distances, we uncovered surprising insights into the labeling mechanisms. Contrary to the prevailing diffusive labeling model, our results demonstrate that TurboID primarily operates through contact-dependent labeling. Similarly, APEX2 shows high labeling efficiency within its direct contact range. In parallel, it exhibits low-level diffusive labeling toward more distant phenols. These findings reframe our understanding in the mechanism of proximity labeling enzymes while highlighting the potential of DNA nanotechnology in spatially profiling reactive species.