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

Abstract

DNA-PAINT is a rapidly developing fluorescence super-resolution technique which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20-25 nm, thus limiting the resolving power.Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using 3 nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the 3 targets with 20 nm resolution, and within only 35 minutes acquisition time. Introduction 1 Super-resolution light microscopy is developing rapidly, and a growing number of cell 2 biologists are embracing this technology to study proteins of interest (POI) at the nanoscale. Single 3 molecule localization techniques like PALM[1], (d)STORM[2], [3], and others[4] achieve resolutions 4 that allows for distinguishing molecules that are separated by only a few nanometers. Among these 5 localization techniques, DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-6 PAINT)[5] has demonstrated to achieve a resolution below 5 nm on DNA origami structures[6], [7] 7 and offers the possibility to detect multiple POIs within the same sample[8]. A special feature of 8 DNA-PAINT is that it is not limited by photobleaching of the fluorophore, due to the constant 9 replenishment of fluorophores from solution. In fact, a target site carries one or more single stranded 10 DNA oligonucleotides (commonly referred to as the docking strand or handle) instead of a single 11 fluorophore, while a second single stranded DNA molecule with a complementary sequence to the 12 docking strand bears a fluorophore (referred to as the imager strand). In a DNA-PAINT experiment, 13 the imager strands continuously bind to the docking strands and unbind due to thermal fluctuations.14 The continuous transient binding of the imager strands results in sparse "blinking-like" fluorescence 15 detection events. Similar to PALM or STORM, these events are then precisely localized to reconstruct 16 a super-resolved image. The localization precision depends on the number of photons collected in a 17 single event, whereas the total number of events recorded affects the quality of the final super-18 resolved image. Importantly, DNA-PAINT benefits from the orthogonality of DNA hybridization 19(with different sequences). DNA docking strands with different nucleotide sequences can be 20 associated with different targe...

Profiling of Exosomal Biomarkers for Accurate Cancer Identification: Combining DNA‐PAINT with Machine‐ Learning‐Based Classification

“…The continuous fluorescence on and off in the focus plane can be detected by total internal reflection fluorescence (TIRF) microscopy, thus creating a “blinking‐like” event . Similar to SMLM, the apparent “switching” events between dark and bright states can be used for stochastic super‐resolved reconstruction with ≈25 nm spatial resolution . By introducing orthogonal imager strands sequentially, multiplex imaging with a same excitation laser is available, without the requirement of spectrally distinct dyes .…”

Circumvention of common labeling artifacts using secondary nanobodies

“…Additionally, using recombinantly produced 2.Nbs not only increases the reproducibility of biomedical experiments, but importantly it eliminates the ethically controversial use of animals for 2.Abs (donkey, goat, sheep, etc). Moreover, their recombinant production allows a high level of flexibility to modify them in a controlled manner, for example with site-specific and quantitative conjugation of flurophores 9 or docking strands for DNA-PAINT 26 .…”

“…DNA-PAINT uses affinity reagents attached to short DNA oligonucleotides, so we first coupled the 2.Nbs to a single stranded DNA oligo (termed docking strand) as described previously 26 . We performed an assay which has been used as gold standards in the field to assess linkage error 27 .…”

“…The pre-mixing feature seems to be ideal for a technique that allows the detection of multiple targets (multiplexing). Therefore, we turned once again to DNA-PAINT, this time we used an extension termed Exchange-PAINT which can, in theory, image an unlimited number of POIs on the same sample with a few nanometer precision 24,26 . We stained primary hippocampal neurons with two mouse monoclonal 1.Abs and each was pre-mixed with 2.Nbs conjugated to DNA docking strands with orthogonal sequences.…”

“…Secondary nanobodies (obtained from NanoTag Biotechnologies GmbH) were coupled to docking oligonucleotide strands (Biomers GmbH, Ulm, Germany) functionalized with an azide group at the 5ʹ-end and an Atto488 fluorophore at the 3ʹ-end following the protocol described by Sograte-Idrissi et al 26 strand sequences were obtained from Agasti el al. 24 and can be found in Supp.…”