Bacterially Derived Antibody Binders as Small Adapters for DNA‐PAINT Microscopy

Abstract

Current optical super‐resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super‐resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial‐derived binders commonly used in antibody purification with DNA‐based point accumulation for imaging in nanoscale topography (DNA‐PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA‐PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.

Circumvention of common labelling artefacts using secondary nanobodies

“…On the other hand, a large amount of well validated monoclonal antibodies is available. Our data suggests that the localization of primary antibodies with recombinant secondary nanobodies or probably other small monovalent binder such as protein A, 33 can minimize the probe-induced clustering of targets, increase the localization accuracy in super-resolution microscopy, lower steric hindrance for detecting more target molecules, enhance the sample penetration, remove the species-limitation by pre-mixing allowing high multiplexing capabilities, and finally, increase the reproducibility of results with no needs of animals.…”

Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting

“…Recently, Jungmann and co-workers reported similar results when using ODN-functionalized protein A and G variants as secondary, non-covalent, labeling reagents in DNA-PAINT. 32 Since pG-ODN is covalently coupled to the antibody using UV light, we additionally investigated if the pG-ODN antibody coupling is reversed when the construct is exposed to high laser intensity. To validate the stability of the pG-ODN-antibody construct we quantified the number of localizations during image acquisition over the course of 8 minutes (Fig.…”

About samples, giving examples: Optimized Single Molecule Localization Microscopy

“…The transient interaction between the 9-basepair docking strand and imager strand can last up to several hundreds of milliseconds, so blinking is significantly slower than in STORM. Typical exposure times of PAINT images are in the range of 100-500 Ms, with the accumulation of tens of thousands of frames taking up to a few hours for a single image [25,80]. One way to alleviate this is to use FRET-PAINT [81,82] that allows to raise the imager concentration without obtaining an overwhelming background fluorescence.…”