Single molecule localization-based approaches to super-resolution microscopy (SMLM) create images that resolve features smaller than the diffraction limit of light by rendering them from the sequentially measured positions of thousands of individual molecules. New SMLM approaches based on the transient binding of very bright dyes via DNA-DNA interaction (DNA-PAINT) allow the resolution of dyes only a few nanometers apart in vitro. This imaging of cellular structures requires the specific association of dyes to their targets, which results in an additional 'linkage error'. This error can be minimized by using extremely small, singledomain antibody-based binders such as nanobodies, but the DNA-oligomers used in DNA-PAINT are of significant size in comparison to nanobodies and may interfere with binding. We have developed an optimized procedure based on enzymatic labeling and click-chemistry for the coupling of DNA oligomers to the nanobody C-terminus, which is located on the opposite side of the epitope-binding domain. Our approach allows for straightforward labeling, purification and DNA-PAINT imaging. We performed high efficiency labeling of two different nanobodies and show dual color multiplexed SMLM to demonstrate the general applicability of our labeling scheme.
Single molecule localization-based approaches to superresolution microscopy (SMLM) create images that resolve features smaller than the diffraction limit of light by rendering them from the sequentially measured positions of thousands of individual molecules. New SMLM approaches based on the transient binding of very bright dyes via DNA-DNA interaction (DNA-PAINT) allow the resolution of dyes only a few nanometers apart in vitro. This imaging of cellular structures requires the specific association of dyes to their targets, which results in an additional "linkage error". This error can be minimized by using extremely small, single-domain antibody-based binders such as nanobodies, but the DNA-oligomers used in DNA-PAINT are of significant size in comparison to nanobodies and may interfere with binding. We have here developed an optimized procedure based on enzymatic labeling and click-chemistry for the coupling of DNA oligomers to the nanobody C-terminus, which is located on the opposite side of the epitope-binding domain. Our approach allows for straightforward labeling, purification and DNA-PAINT imaging. We performed high efficiency labeling of two different nanobodies and show dual color multiplexed SMLM to demonstrate the general applicability of our labeling scheme.
DNA point accumulation in nanoscale topography (DNA-PAINT) advances super-resolution microscopy with superior resolution and multiplexing capabilities. However, cellular DNA may interfere with this single-molecule localization technique based on DNA-DNA hybridization. Here, we introduce lefthanded DNA (L-DNA) oligomers that do not hybridize to naturally present R-DNA and demonstrate that L-DNA PAINT has the same specificity and multiplexing capability as R-DNA PAINT, but greatly improves specific visualization of nuclear target molecules.Novel super-resolution microscopy techniques facilitate the resolution of cellular structures down to the molecular detail. In single molecule localization superresolution microscopy (SMLM) techniques, nanometer resolution is achieved by sequential imaging of a large number of single molecules at nanometer scale resolution, a few molecules at a time 1-3 . Different approaches have been developed to keep the majority of molecules dark while a small fraction can be imaged as single molecules. The commonly used fluorescent proteins and organic dyes are physically or chemically switched between dark and bright states to separate detection events. Recently, DNA point accumulation in nanotopography (DNA-PAINT 4 ) was developed as a novel approach that relies on transient hybridization of fluorophore-coupled DNA-oligomer imagers to target-associated reverse-complement DNA-oligomer binders. Since single molecule detection occurs here through DNA hybridization and is uncoupled from dye photophysics, DNA-PAINT allows the use of bright, photostable organic dyes to obtain highest single molecule localization resolution 4,5 . Furthermore, different oligomer sequences enable multiplexing in a single wavelength 4,6 , thereby avoiding chromatic aberration 7 . Finally, the well-understood chemical kinetics of DNA hybridization 3 facilitates quantitative imaging 8,9 . In the light of these advantages, DNA-PAINT is a significant advancement in single molecule localization microscopy.However, any DNA-nonamer 4 has statistically on average 22'000 complementary binding sites in the diploid ~ 3 gigabase human genome that can contribute to false positive hybridization events. In addition, the transcriptome may as well contribute to undesired DNA-RNA hybridizations. This is a significant problem in a super-resolution technique based on single molecule localizations, which is one of the most promising approaches to advance our understanding of the nanoscopic functional organization of the nucleus, an area of intensive research in cell biology 10-12 . To overcome this problem, we here employed oligomers synthesized from L-DNA for DNA-PAINT. L-DNA has identical physico-chemical properties to the R-DNA common to life (see Figure 1a and Supp. Fig. 1), but does not naturally occur and cannot hybridize with R-DNA ( Figure 1b) 13 . We hypothesized that L-DNA PAINT would perform better in superresolution imaging of nuclear targets than traditional R-DNA-PAINT. 4 Figure 1: Comparison of DNA-PAINT with left-handed ...
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