124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Wade, Orsolya K.; Woehrstein, Johannes B.; Nickels, Philipp C.; Strauss, Sebastian; Stehr, Florian; Stein, Jeremy C.; Schueder, Florian; Strauss, Mike; Ganji, Mahipal; Schnitzbauer, Joerg; Grabmayr, Heinrich; Yin, Peng; Schwille, Petra; Jungmann, Ralf

doi:10.1021/acs.nanolett.9b00508

Cited by 95 publications

(98 citation statements)

References 25 publications

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“…This technique enables to image a virtually infinite number of targets in high resolution in the same sample. 20,21 Additionally, we observed that pre-mixing 1.Ab and 2.Nb can save time in staining thick biological samples imaged under light-sheet microscopy, ensuring also a better sample penetration and homogenous staining. Finally, we systematically compared the probe-induced clustering of the target protein either using directly-labelled monovalent probes, like affibodies and single Fab' fragments, and conventional 1.Abs detected by polyclonal and bivalent 2.Abs or by monovalent 2.Nbs.…”

Section: Introductionmentioning

confidence: 82%

Circumvention of common labelling artefacts using secondary nanobodies

et al. 2020

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Section: Introductionmentioning

confidence: 82%

Circumvention of common labelling artefacts using secondary nanobodies

et al. 2020

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“…25 These kinetic fingerprints will allow for additional freedom when designing Ago-PAINT. 29,31 Furthermore, optimization of the imager sequence and imaging conditions allowed for an order of magnitude faster imaging for conventional DNA-PAINT 32 . We expect that optimization of the Furthermore, as the imager strand is loaded and protected inside the protein, degradation of the imager strand is less likely to occur over time, unlike oligos that are rapidly digested.…”

Section: Resultsmentioning

confidence: 99%

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

Filius

Cui

Ananth

et al. 2020

Preprint

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Super-resolution imaging allows for visualization of cellular structures on a nanoscale level. DNA-PAINT (DNA Point Accumulation In Nanoscale Topology) is a super-resolutionmethod that depends on the binding and unbinding of DNA imager strands. The current DNA-PAINT technique suffers from slow acquisition due to the low binding rate of the imager strands. Here we report on a method where imager strands are loaded into a protein, Argonaute (Ago), that allows for faster binding. Ago pre-orders the DNA imager strand into a helical conformation, allowing for 10 times faster target binding. Using a 2D DNA origami structure, we demonstrate that Ago-assisted DNA-PAINT (Ago-PAINT) can speed up the current DNA-PAINT technique by an order of magnitude while maintaining the high spatial resolution. We envision this tool to be useful not only for super-resolution imaging, but also for other techniques that rely on nucleic-acid interactions.

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“…DNA-PAINT has been widely used to image DNA origami type structures in vitro. [9][10][11][12][13][14][15][16][17][18][19][20] The enormous advantage of DNA-PAINT is that it is relatively straightforward to manipulate the specificity and affinity of the two interacting ssDNA strands. In more elaborate implementations, involving a ssDNA attached to a nanobody or aptamer for example, DNA-PAINT has been used to image proteins within fixed, permeabilized cells.…”

Section: Encoding Specificity Using Dna-paintmentioning

confidence: 99%

PAINT using proteins: A new brush for super‐resolution artists

Mochrie

Horrocks

et al. 2020

Protein Science

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PAINT (points accumulation for imaging in nanoscale topography) refers to methods that achieve the sparse temporal labeling required for super-resolution imaging by using transient interactions between a biomolecule of interest and a fluorophore. There have been a variety of different implementations of this method since it was first described in 2006. Recent papers illustrate how transient peptide-protein interactions, rather than small molecule binding or DNA oligonucleotide duplex formation, can be employed to perform PAINT-based single molecule localization microscopy (SMLM). We discuss the different approaches to PAINT using peptide and protein interactions, and their applications in vitro and in vivo. We highlight the important parameters to consider when selecting suitable peptide-protein interaction pairs for such studies. We also note the opportunities for protein scientists to apply their expertise in guiding the choice of peptide and protein pairs that are used. Finally, we discuss the potential for expanding super-resolution imaging methods based on transient peptide-protein interactions, including the development of simultaneous multicolor imaging of multiple proteins and the study of very high and very low abundance proteins in live cells.

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124-Color Super-resolution Imaging by Engineering DNA-PAINT Blinking Kinetics

Cited by 95 publications

References 25 publications

Circumvention of common labelling artefacts using secondary nanobodies

Circumvention of common labelling artefacts using secondary nanobodies

High-Speed Super-Resolution Imaging Using Protein-Assisted DNA-PAINT

PAINT using proteins: A new brush for super‐resolution artists

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