Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT

Strauss, Sebastian; Jungmann, Ralf

doi:10.1038/s41592-020-0869-x

Cited by 130 publications

(200 citation statements)

References 25 publications

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“…Since its initial implementation in 2006, PAINT-based SMLM has seen many innovative applications. 1,2,5,[7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]36 The development of DNA-PAINT in 2010 represented a sea change, by providing a means to incorporate high specificity into the transient interaction. DNA-PAINT has since been optimized and extended in many ways.…”

Section: Discussionmentioning

confidence: 99%

“…DNA-PAINT has since been optimized and extended in many ways. [9][10][11][12][13][14][15][16][17][18][19][20][21][22] Recent work, which is the focus of this review, has demonstrated the potential for using peptide-protein interactions, rather than DNA duplex formation, to achieve transient labeling. Peptide-protein interactions are less straightforward to design than a complementary DNA duplex.…”

Section: Discussionmentioning

confidence: 99%

“…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%

“…19 Recently, however, Strauss and Jungmann have shown that on-rates can be increased about a hundred-fold by using multiple concatenated repeats of a short DNA sequence. 19 This modification of the method enables sufficient data for a 20 nm resolution image to be acquired in minutes. dye which is nonfluorescent in aqueous solution (grey star) but which fluoresces (red star) when it transiently interacts with the hydrophobic lipid membrane of the LUV (purple circles represent polar headgroups, yellow tails represent the aliphatic tails).…”

Section: Encoding Specificity Using Dna-paintmentioning

confidence: 99%

“…Background from unbound labeling protein is reduced by data acquisition in TIRF during a localization event to achieve "good" resolution. In DNA-PAINT experiments, exposure times of 100 ms are typically used 9,19 and in LIVE-PAINT it was 50 ms. 2 With exposure times of 50-100 ms, PAINT methods using currently available fluorophores, would benefit from using interactions with even higher off-rates, up to 10-20 s −1 . On-rates ideal for PAINT experiments are those that will enable rapid re-binding of fluorescent probes to molecules of interest.…”

Section: Important Requirements For a Good Protein-peptide Or Peptimentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Encoding Specificity Using Dna-paintmentioning

confidence: 99%

Section: Encoding Specificity Using Dna-paintmentioning

confidence: 99%

Section: Important Requirements For a Good Protein-peptide Or Peptimentioning

confidence: 99%

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

Mochrie

Horrocks

et al. 2020

Protein Science

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PCNA as Protein‐Based Nanoruler for Sub‐10 nm Fluorescence Imaging

Helmerich,

Budiarta,

Taban

et al. 2023

Advanced Materials

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Super‐resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single‐molecule localization microscopy methods achieve spatial resolutions in the one‐digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein‐based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super‐resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site‐specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6 nm distances. Bioorthogonal click labeling with tetrazine‐dyes and tetrazine‐functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time‐resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA‐based points accumulation for imaging in nanoscale topography (DNA‐PAINT) is used to resolve 6 nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super‐resolution imaging techniques, particularly in complex biological environments.

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Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

2022

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Photobleaching is the permanent loss of fluorescence after extended exposure to light and is a major limiting factor in super‐resolution microscopy (SRM) that restricts spatiotemporal resolution and observation time. Strategies for preventing or overcoming photobleaching in SRM are reviewed developing new probes and chemical environments. Photostabilization strategies are introduced first, which are borrowed from conventional fluorescence microscopy, that are employed in SRM. SRM‐specific strategies are then highlighted that exploit the on–off transitions of fluorescence, which is the key mechanism for achieving super‐resolution, which are becoming new routes to address photobleaching in SRM. Off states can serve as a shelter from excitation by light or an exit to release a damaged probe and replace it with a fresh one. Such efforts in overcoming the photobleaching limits are anticipated to enhance resolution to molecular scales and to extend the observation time to physiological lifespans.

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Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT

Cited by 130 publications

References 25 publications

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

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

PCNA as Protein‐Based Nanoruler for Sub‐10 nm Fluorescence Imaging

Bleaching‐Resistant Super‐Resolution Fluorescence Microscopy

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