Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds

Engelhardt, Floris A. S.; Praetorius, Florian; Wachauf, Christian; Brüggenthies, Gereon A.; Köhler, Fabian; Kick, Benjamin; Kadletz, Karoline L.; Pham, Phuong Nhi; Behler, Karl L.; Gerling, Thomas; Dietz, Hendrik

doi:10.1021/acsnano.9b01025

Cited by 117 publications

(137 citation statements)

References 66 publications

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“…10068013). Single stranded DNA with 4536, 6048, 7249 and 8064 bases was prepared as previously described (19). Single stranded DNA (155-mer) was synthesised on an Applied Biosystems 394 automated DNA/RNA synthesiser using a standard 0.2 μmole phosphoramidite cycle of acid-catalysed detritylation, coupling, capping, and iodine oxidation.…”

Section: Methodsmentioning

confidence: 99%

Single molecule mass photometry of nucleic acids

Struwe

Kukura

2020

Preprint

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Mass photometry is a recently developed methodology capable of detection, imaging and mass measurement of individual proteins under solution conditions. Here, we show that this approach is equally applicable to nucleic acids, enabling their facile, rapid and accurate detection and quantification using sub-picomoles of sample. The ability to count individual molecules directly measures relative concentrations in complex mixtures without need for separation. Using a dsDNA ladder, we find a linear relationship between the number of bases per molecule and the associated imaging contrast for up to 1200 bp, enabling us to quantify dsDNA length with 4 bp accuracy. These results introduce mass photometry as an accurate and rapid single molecule method complementary to existing DNA characterisation techniques. INTRODUCTIONSingle molecule analysis has had a tremendous impact on our ability to study DNA structure, function and interactions (1). Next generation sequencing heavily relies on single-molecule methods, be it using single molecule fluorescence (2, 3) or nanopore-based approaches (4, 5). Similarly, single molecule methods are now heavily used in a variety of incarnations to study DNA-protein interactions (6), with both DNA and proteins visualised by fluorescence labelling to reach single molecule sensitivity (7).Label-free detection and quantification would be highly desirable in this context due to the associated reduction in experimental complexity and minimisation of potential perturbations caused by the sample itself. While visualisation of single DNA molecules has been possible for decades using non-optical methods, such as electron microscopy (8) and atomic force microscopy (9), which can also be used to study mechanical properties (10), label-free optical detection has remained a considerable challenge.Label-free detection of single proteins has been reported for the first time in 2014 (11,12) in the context of increasing sensitivity of interferometric scattering microscopy (13,14). Further improvements to the detection methodology (15), recently lead to the development of mass photometry (MP), originally introduced as interferometric scattering mass spectrometry (16), which enables not only label-free detection and imaging of single molecules, but critically also their quantification through mass measurement with high levels of accuracy, precision and resolution at a lower detection limit on the order of 40 kDa. Given that biomolecules have broadly comparable optical properties in the visible range of the electromagnetic spectrum (17, 18), we therefore set out to investigate in this work to which degree the capabilities of MP translate to nucleic acids, which would enable not only their detection,

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Section: Methodsmentioning

confidence: 99%

Single molecule mass photometry of nucleic acids

Struwe

Kukura

2020

Preprint

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“…Lambda exonuclease is an exodeoxyribonuclease that digests the phosphorylated strand from the 5 to the 3 end (Figure 3b), so that only non-phosphorylated ssDNA remains in the system after digestion [113]. Although this method is a fast and efficient method for generating ssDNA with high efficiency and quality [114], it has fallen out of favor due to the fact that incomplete digestion of the PCR product results in the accumulation of dsDNA in the final products [74].…”

Section: Separation Of Ssdna From Dsdnamentioning

confidence: 99%

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

Hao

Qiao

2020

Genes

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Methods for synthesizing arbitrary single-strand DNA (ssDNA) fragments are rapidly becoming fundamental tools for gene editing, DNA origami, DNA storage, and other applications. To meet the rising application requirements, numerous methods have been developed to produce ssDNA. Some approaches allow the synthesis of freely chosen user-defined ssDNA sequences to overcome the restrictions and limitations of different length, purity, and yield. In this perspective, we provide an overview of the representative ssDNA production strategies and their most significant challenges to enable the readers to make informed choices of synthesis methods and enhance the availability of increasingly inexpensive synthetic ssDNA. We also aim to stimulate a broader interest in the continued development of efficient ssDNA synthesis techniques and improve their applications in future research.

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“…These methods have often been demonstrated for lattice-based designs, but some of them are equally available for wireframe structures [107]. Therefore, the combination of presented automated design methods with biotechnological mass production of DNA [9], various application-specific protection mechanisms, and the scaling-up capabilities of the assemblies [115][116][117][118] will undoubtedly pave the way for a plethora of applications and for the full commercialization of ready-to-use DNA nanostructure fabrication based on researcher/customer needs [7,119].…”

Section: Conclusion and Future Perspectivesmentioning

confidence: 99%

Increasing Complexity in Wireframe DNA Nanostructures

et al. 2020

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Structural DNA nanotechnology has recently gained significant momentum, as diverse design tools for producing custom DNA shapes have become more and more accessible to numerous laboratories worldwide. Most commonly, researchers are employing a scaffolded DNA origami technique by “sculpting” a desired shape from a given lattice composed of packed adjacent DNA helices. Albeit relatively straightforward to implement, this approach contains its own apparent restrictions. First, the designs are limited to certain lattice types. Second, the long scaffold strand that runs through the entire structure has to be manually routed. Third, the technique does not support trouble-free fabrication of hollow single-layer structures that may have more favorable features and properties compared to objects with closely packed helices, especially in biological research such as drug delivery. In this focused review, we discuss the recent development of wireframe DNA nanostructures—methods relying on meshing and rendering DNA—that may overcome these obstacles. In addition, we describe each available technique and the possible shapes that can be generated. Overall, the remarkable evolution in wireframe DNA structure design methods has not only induced an increase in their complexity and thus expanded the prevalent shape space, but also already reached a state at which the whole design process of a chosen shape can be carried out automatically. We believe that by combining cost-effective biotechnological mass production of DNA strands with top-down processes that decrease human input in the design procedure to minimum, this progress will lead us to a new era of DNA nanotechnology with potential applications coming increasingly into view.

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Custom-Size, Functional, and Durable DNA Origami with Design-Specific Scaffolds

Cited by 117 publications

References 66 publications

Single molecule mass photometry of nucleic acids

Single molecule mass photometry of nucleic acids

Current and Emerging Methods for the Synthesis of Single-Stranded DNA

Increasing Complexity in Wireframe DNA Nanostructures

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