Abstract
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, 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 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.
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