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

Abstract

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.

Evaluating the effect of ionic strength on PNA:DNA duplex formation kinetics

“…These approaches offer excellent advantages over bulk methods, as they not only give detailed kinetic and thermodynamic information about the process, but also provide insight into heterogeneity at the single-molecule level. 29,32,33 DNA-PAINT has undergone rapid development in the last 10 years, with reports focused on using this technology to quantitatively count molecules, 34,35 increasing the DNA binding rate to improve throughput, 36,37 and harnessing the unique kinetic signature of specific DNA sequences to multiplex measurements. 38 These advances have expanded the toolbox of single-molecule imaging techniques, making them an ideal method to quantify oligonucleotide binding kinetics.…”

Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

“…1j ). The 20% decrease in TH detection over time is likely due to photo-induced damage to the DNA caused by reactive oxygen species 21 , 23 during imaging (see Supplementary Note 1 ). To assess the number of imagers simultaneously bound to the TH, we analyzed the photon count distribution, which yielded distinct, equidistant single dye steps with the first step exhibiting the same photon count value in the TH and SD origami data sets (Supplementary Fig.…”

“…In this study, we focused on reconstituted 2D systems that (1) featured a very thin excitation volume (TIRF) and (2) showed very low levels of unspecific binding 21 . This enabled us to use rather high imager concentrations of up to 40 nM.…”

“…6, see Supplementary Fig. 2 ) imagers to bind simultaneously, maximizing their association rate ( k on ) 17 – 21 , and optimizing their dwell times ( k off ) (see Fig. 1b for schematic depiction).…”

Amphiphilic DNA nanostructures for bottom-up synthetic biology

“…By affording unprecedented control over the shape 11 , interactions 12,13 , and mechanical properties of nanostructures 14 , the DNA origami technology has opened further new avenues for the field, with concrete applications to biomolecular scaffolding [15][16][17] , single-molecule analysis 18,19 , biosensing 20 , nanomedicine 21 , imaging 22 , and the construction of advanced materials 23,24 . In parallel to structural control, our growing understanding of nucleic acid kinetics and thermodynamics has resulted in the ability to program dynamic responses 25 , marking the advent of DNA-based molecular computing [26][27][28] and the development of proof-of-concept actuable nanodevices 29,30 , biosensors 31,32 , and technologies for optical imaging [33][34][35][36][37] .…”