Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINT

Abstract

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.

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors

“…It has been demonstrated that small camelid single domain antibodies or nanobodies (15 kDa and ~3 nm in length) have the capacity to increase the accuracy of super-resolution microscopy for mapping POIs in a cellular context[15], [42]. Recently, nanobodies have been coupled to docking oligos to perform DNA-PAINT [25]. Unfortunately, only few nanobodies are currently available that work efficiently in immunoassay applications.…”

“…The recombinant production of the variable domain of these hcAbs result in a functional nanobody with only 2-3 nm size[24]. Recently, a significant improvement in spatial resolution, as compared to conventional antibody immunofluorescence, was demonstrated by using nanobodies for labeling[12], [25]. In addition to their small size, high specificity, and monovalent binding affinities, which make them an ideal tool for microscopy, the recombinant nature of nanobodies endows them with a great flexibility and allows introducing all types of modifications in a precise manner.…”

Directed manipulation of membrane proteins by fluorescent magnetic nanoparticles

“…Stably transfected GPI-GFP CV-1 cells were maintained with 50 μg ml −1 Geniticin (ThermoFisher Scientific) supplementing the culture medium. For transient transfection, CV-1 cells at 70-100% confluency were electroporated with the Neon 39 were recombinantly produced 40 and labeled. For the labeling reaction 41 , α-Biotin-ω-(succinimidyl propionate)-24(ethylene glycol) (Iris Biotech) was added to the nanobody at twofold molar excess.…”

Exploiting nanobodies and Affimers for superresolution imaging in light microscopy

“…Nanobodies have also been generated to a variety of proteins, including tubulin (Mikhaylova et al. , 2015), to allow direct rather than indirect labeling of microtubules and have been used successfully in SMLM approaches, including DNA PAINT (Fabricius et al. , 2018; Sograte-Idrissi et al.…”