Super-Resolution Imaging of Unlabeled Proteins on DNA

Abstract

Direct visualization of DNA-protein interactions at the single-molecule level is of ever increasing importance to unravel the salient details of a wide range of DNA-associated processes. Recent emergence of super-resolution imaging has the potential to further boost the impact of such investigations. However, current imaging methods heavily rely on (fluorescent) labeling strategies, which can be challenging and potentially even interfere with the molecular interactions under scrutiny. Here, we introduce a new label-free method to image the presence and location of DNA-bound proteins with super-resolution on optically manipulated DNA. The method is based on localization microscopy of DNA-intercalating dyes that locally bind to bare DNA but not to protein-bound DNA sections, yielding an inverted image that reveals the ‘shadows’ of the proteins on the DNA: inverse binding-activated localization microscopy (iBALM). We present the proof of principle of iBALM as well as of functional variations to this method and provide experimental and theoretical data that describe the spatial and temporal resolution that can be obtained. iBALM has the potential to become a valuable addition to the single-molecule toolkit and enable direct visualization of processes that were previously not possible due to limitations posed by labeling.