Site‐Specific Labeling of Affimers for DNA‐PAINT Microscopy

Schlichthaerle, Thomas; Eklund, Alexandra S.; Schueder, Florian; Strauss, Maximilian T.; Tiede, Christian; Curd, Alistair; Ries, Jonas; Peckham, Michelle; Tomlinson, Darren C.; Jungmann, Ralf

doi:10.1002/anie.201804020

Cited by 74 publications

(86 citation statements)

References 33 publications

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“…GFP nanobodies were DNA-labeled as previously reported 32 . Nanobodies were concentrated via Amicon 10 kDa spin filters and buffer exchanged into 5 mM TCEP in 1× PBS + 3 mM EDTA at pH 7.5 mM TCEP in 1× PBS + 3 mM EDTA was then added to the GFP Nanobody and was incubated for 2 h at 4 °C on a shaker.…”

Section: Methodsmentioning

confidence: 99%

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

Strauss

Schueder

Auer

et al. 2019

Preprint

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The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions,s erves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active researchtopic for decades with recent cryogenic electron microscopya nd super-resolution studies advancing our understanding of the architecture of the NPC complex. However,the specific and direct visualization of single copies of NPC proteins is thus far elusive.H erein, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We resolve single copies of nucleoporins in the human Y-complex in three dimensions with ap recision of circa 3nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.Super-resolution techniques allow diffraction-unlimited fluorescence imaging [1] and with recent advancements,t rue biomolecular resolution is well within reach. [2] One implementation of single-molecule localization microscopy (SMLM) is called DNAp oints accumulation in nanoscale topography [2b] (DNA-PAINT), where dye-labeled DNA strands (called "imager" strands) transiently bind to their complements (called "docking" strands) on at arget of interest, thus creating the typical "blinking" used in SMLM to achieve super-resolution. While localization precisions down to approximately one nanometer (basically the size of as ingle dye molecule) are now routinely achievable from at echnology perspective,t his respectable spatial resolution has yet to be translated to cell biological research. Currently, this is mainly hampered by the lack of small and efficient protein labels.R ecent developments of nanobody-or aptamer-based tagging approaches [3] are providing an attractive route ahead, however both approaches are not yet deploying their full potential either due to limited binder availability (in

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Section: Methodsmentioning

confidence: 99%

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

Strauss

Schueder

Auer

et al. 2019

Preprint

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“…However, for application of DNA‐PAINT to a question in cell biology, the docking strands need to be “linked” to the target of interest through DNA‐conjugated affinity reagents . The optimal labeling probe should ideally be smaller than the target protein under investigation, allow for quantitative labeling (1:1 stoichiometry), be available for a large variety of targets, and ultimately be cost‐effective.…”

Section: Figurementioning

confidence: 99%

“…Different methods have been introduced to conjugate DNA docking strands to protein‐based affinity reagents. Typically, a bifunctional chemical crosslinker harboring a reactive moiety (which can subsequently react with a modified DNA oligonucleotide) was used to react with amino groups or reduced thiols . To produce DNA‐modified secondary labeling reagents, protein A from Staphylococcus aureus and protein G from Streptococcus sp.…”

Section: Figurementioning

confidence: 99%

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

et al. 2019

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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.

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“…The best results for SMLM of actin are obtained using fluorescent phalloidin, because it allows for a very high density of labeling and results in crisp reconstruction of filamentous actin (K. Xu et al, 2012). As an alternative, PAINT of actin has been performed using phalloidin coupled to a DNA docking strand or actin-targeting affimers (Schlichthaerle et al, 2018a). It is also possible to use fluorescent LifeAct that transiently binds to actin in fixed samples to generate an SMLM image, a technique called Integrating exchangeable single-molecule localization (IRIS) (Ashdown et al, 2017;Kiuchi et al, 2015;Tas et al, 2018).…”

Section: Actin Stainingmentioning

confidence: 99%

About samples, giving examples: Optimized Single Molecule Localization Microscopy

Jimenez

Friedl

Leterrier

2019

Preprint

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Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery.

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Site‐Specific Labeling of Affimers for DNA‐PAINT Microscopy

Cited by 74 publications

References 33 publications

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

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

About samples, giving examples: Optimized Single Molecule Localization Microscopy

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