Detecting Nanoscale Distribution of Protein Pairs by Proximity-Dependent Super-resolution Microscopy.

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy.

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA 'imagers' to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.

Responsive core-shell DNA particles trigger lipid-membrane disruption and bacteria entrapment.

Biology has evolved a variety of agents capable of permeabilizing and disrupting lipid membranes, from amyloid aggregates, to antimicrobial peptides, to venom compounds. While often associated with disease or toxicity, these agents are also central to many biosensing and therapeutic technologies. Here, we introduce a class of synthetic, DNA-based particles capable of disrupting lipid membranes. The particles have finely programmable size, and self-assemble from all-DNA and cholesterol-DNA nanostructures, the latter forming a membrane-adhesive core and the former a protective hydrophilic corona. We show that the corona can be selectively displaced with a molecular cue, exposing the 'sticky' core. Unprotected particles adhere to synthetic lipid vesicles, which in turn enhances membrane permeability and leads to vesicle collapse. Furthermore, particle-particle coalescence leads to the formation of gel-like DNA aggregates that envelop surviving vesicles. This response is reminiscent of pathogen immobilisation through immune cells secretion of DNA networks, as we demonstrate by trapping E. coli bacteria.