DNA‐Origami als Nanometerlineal für die superauflösende Mikroskopie

Steinhauer, Christian; Jungmann, Ralf; Sobey, Thomas L.; Simmel, Friedrich C.; Tinnefeld, Philip

doi:10.1002/ange.200903308

Cited by 22 publications

(7 citation statements)

References 34 publications

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“…[16][17][18][19][20] Each position of such an object can be addressed individually, since each of the staple strands can be functionalized with (bio)chemical groups or fluorescent dyes. [21][22][23][24]25] We attached Cy3-donor and Cy5-acceptor dyes to the surface of an origami structure so that linkers always point in the same direction, minimizing their static influence on FRET efficiencies ( Figure S1, Supporting Information). We varied the distances between the dyes and studied energy transfer efficiencies using single-molecule spectroscopy with alternating laser excitation of diffusing molecules.…”

Section: Introductionmentioning

confidence: 99%

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Stein

Schüller²,

Böhm³

et al. 2011

ChemPhysChem

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Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.

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Section: Introductionmentioning

confidence: 99%

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Stein

Schüller²,

Böhm³

et al. 2011

ChemPhysChem

Self Cite

128

120

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“…centrosomes comprising two orthogonally arranged centrioles, each consisting of nine triplet microtubules (Sillibourne et al, 2011;Lau et al, 2012;Lukinavičius et al, 2013), or pre-and post-synaptic proteins separated by the synaptic cleft (Dani et al, 2010), can also be used as natural biological calibration samples (Box 2). Artificial calibration samples for 2D and 3D localization microscopy include DNA origami (Steinhauer et al, 2009), single-molecule assembled patterns generated by cut-and-paste technology (SMCP) (Kufer et al, 2008;Cordes et al, 2010) and DNA bricks (Box 2) (Ke et al, 2012).…”

Section: Quantification and Reliability Of Localization Microcopymentioning

confidence: 99%

Localization microscopy coming of age: from concepts to biological impact

Sauer

2013

Journal of Cell Science

105

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SummarySuper-resolution fluorescence imaging by single-molecule photoactivation or photoswitching and position determination (localization microscopy) has the potential to fundamentally revolutionize our understanding of how cellular function is encoded at the molecular level. Among all powerful, high-resolution imaging techniques introduced in recent years, localization microscopy excels because it delivers single-molecule information about molecular distributions, even giving absolute numbers of proteins present in subcellular compartments. This provides insight into biological systems at a molecular level that can yield direct experimental feedback for modeling the complexity of biological interactions. In addition, efficient new labeling methods and strategies to improve localization are emerging that promise to achieve true molecular resolution. This raises localization microscopy as a powerful complementary method for correlative light and electron microscopy experiments.

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“…Few reports describe the use of the fluorescence-based techniques for this purpose. [28] Herein, we focus on the single-molecule analysis using DNA origami, which is a method used for the isolation of single molecules and for their analysis, in combination with the available unimolecular techniques.…”

Section: Introduction 875mentioning

confidence: 99%

Single‐Molecule Analysis Using DNA Origami

Rajendran¹,

Endo²,

Sugiyama³

2011

Angew Chem Int Ed

200

137

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During the last two decades, scientists have developed various methods that allow the detection and manipulation of single molecules, which have also been called "in singulo" approaches. Fundamental understanding of biochemical reactions, folding of biomolecules, and the screening of drugs were achieved by using these methods. Single-molecule analysis was also performed in the field of DNA nanotechnology, mainly by using atomic force microscopy. However, until recently, the approaches used commonly in nanotechnology adopted structures with a dimension of 10-20 nm, which is not suitable for many applications. The recent development of scaffolded DNA origami by Rothemund made it possible for the construction of larger defined assemblies. One of the most salient features of the origami method is the precise addressability of the structures formed: Each staple can serve as an attachment point for different kinds of nanoobjects. Thus, the method is suitable for the precise positioning of various functionalities and for the single-molecule analysis of many chemical and biochemical processes. Here we summarize recent progress in the area of single-molecule analysis using DNA origami and discuss the future directions of this research.

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DNA‐Origami als Nanometerlineal für die superauflösende Mikroskopie

Cited by 22 publications

References 34 publications

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Single‐Molecule FRET Ruler Based on Rigid DNA Origami Blocks

Localization microscopy coming of age: from concepts to biological impact

Single‐Molecule Analysis Using DNA Origami

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