A DNA tweezer-actuated enzyme nanoreactor

Liu, Minghui; Fu, Jinglin; Hejesen, Christian; Yang, Yuhe; Woodbury, Neal W.; Gothelf, Kurt V.; Liu, Yan; Yan, Hao

doi:10.1038/ncomms3127

Cited by 290 publications

(284 citation statements)

References 39 publications

(41 reference statements)

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“…The activity of each of the two enzymes was measured individually in bulk solution for the three swinging arm-enzyme distances (7, 14 and 21 nm) using a phenazine methosulfat-catalyzed resazurin fluorescence assay (more details in Supplementary Figs. S30 and S33-S35) 8,24 . As predicted, the 7-nm distance resulted in the highest activity for both G6pDH and MDH.…”

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confidence: 99%

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

et al. 2014

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The general design of the swinging arm nanostructure complex is shown in Fig. 1a, where a two-enzyme cascade consisting of glucose-6 phosphate dehydrogenase (G6pDH) 19 and malic dehydrogenase (MDH) 20 is displayed on a DNA double-crossover (DX) tile scaffold 21 (DNA sequences for all structures used in this study are shown in Supplementary Figs. S1-S7).G6pDH catalyzes the oxidation of glucose-6-phosphate and the reduction of NAD + to NADH.Subsequently, MDH catalyzes the reduction of oxaloacetate to malic acid using the NADH produced by G6pDH. To facilitate channeling of NADH between G6pDH and MDH, an NAD + -3 functionalized poly(T) 20 oligonucleotide was attached to the DNA tile surface halfway between G6pDH and MDH (see Supplementary Figs. S8-S21 for a detailed description of conjugation, assembly and purification of the nanostructured complex). Fig. 1b shows a native polyacrylamide gel electrophoresis (PAGE) analysis of the assembled enzyme complex, together with various sub-complexes. Both the gel results and the chromatogram resulting from sizeexclusion chromatography ( Supplementary Fig. S21) demonstrate assembly of the G6pDH-NAD + -MDH swinging arm cascade with >80% yield. The assembled mixture was further purified by size exclusion chromatography for enzyme activity assays. Assembly of the complete complex was also visualized by atomic force microscopy (AFM) ( Fig. 1c and Supplementary Fig. S57 for larger view images), where the presence of the enzymes on the structure is confirmed by differences in height ("brightness") compared to the surface of the DNA tile.To first explore the parameters and understand the kinetics and mechanism of the restricted diffusion mediated by the ssDNA swinging arm, we developed a simplified model system ( Supplementary Fig. S22). In this model, a Cy3 reporter dye takes the place of NAD + on the single-stranded poly(T) 20 arm, whereas a BHQ fluorescence quencher and a Cy5 energy transfer acceptor dye replace one or both enzymes on selected probe positions surrounding the swinging arm (Fig. 2a). An oligonucleotide sequence (5'-ATA GTG AAA) was extended from the 5' end of the poly(T) 20 sequence and positioned halfway between the quencher and acceptor, allowing the arm to transiently hybridize to the probes that each bear the complementary sequence (5'-TTT CAC TAT) in analogy to the binding of NAD + /NADH to the dehydrogenases. To characterize the distance dependence of the diffusive transport and binding mediated by the swinging arm using smFRET, we chose a design in which a single Cy5-labeled capture probe was placed at one of three topologically accessible distances from the Cy3-labeled arm: 7 nm (21 base pairs), 14 nm (42 base pairs) and 21 nm (63 base pairs). As shown in Fig. 2b, the most efficient hybridization to the capture probe was observed at 7 nm, where ~94% of all swinging arms were associated with the Cy5 probe at equilibrium (leading to high FRET). As the distance increased, the equilibrium fraction of captured swinging arms decreased to ~58% at 14 nm and only ...

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Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

et al. 2014

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“…In addition, DNA-based nanodevices can be utilized in controlling and actuating enzymatic reactions in sophisticated manner [56]. Liu et al [57] built a tweezerlike DNA device and placed an enzyme and its cofactor to the tips of the arms ( Figure 2B). They were able to control the enzymatic activity by opening and closing the tweezers using additional DNA oligonucleotides as fuel.…”

Section: Smart Dna Nanodevices and Molecular Platformsmentioning

confidence: 99%

“…[5]; Copyright (2009) [55]. (B) A switchable tweezer-like enzymatic nanoreactor [57]. (C) A tubular DNA origami-based enzyme cascade nanoreactor for biosensing [59].…”

Section: Shape-complementarity-based Constructionmentioning

confidence: 99%

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices

Linko

Ora

Kostiainen

2015

Trends in Biotechnology

227

166

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Emerging DNA nanotechnologyThe field of structural DNA nanotechnology started around 30 years ago when Ned Seeman performed pioneering research with DNA junctions and lattices [1]. To date, a cornucopia of different DNA nanostructures and shapes based on WatsonCrick basepairing [2] have been designed and demonstrated, and the whole research area has enjoyed a rapid progress especially during the past decade [3]. The key player in the fast development of DNA nanotechnology has been the invention of DNA origami in 2006 [4]. The DNA origami method is based on folding a long single-stranded "DNA scaffold strand" into a customized shape with a set of short synthetic staple strands. The robustness of the technique has made it widely accessible.Since then, the method has been generalized to 3D-fabrication [5][6][7][8][9][10], and it enables shapes with custom curvatures, twists and bends [11,12]. Very recently, techniques for modular scaffold-free fabrication [13,14], 3D meshing and wireframe-based approaches [15][16][17], as well as shape-complementarity-based construction [18] of DNA objects have been introduced. In addition, powerful computational tools for designing and analyzing DNA nanostructures have been developed [19][20][21], which appreciably help researchers to create their own DNA nanoarchitectures for any conceivable application. Table 1 lists and 2 describes the novel design strategies that can be used for fabricating diverse DNA origami nanostructures and other complex DNA-based shapes for various nanotechnological purposes.The platonic DNA nanostructures and DNA origamis are by all means remarkably impressive examples of precise engineering and constructing at the nanoscale. However, the attractiveness of the origami method lies in the fact that one can add any desired functionality to the tailored DNA shapes by assembling other biomolecules and molecular components to them with nanometer precision. Importantly, DNA is inherently biocompatible, and its stability can be further tuned by the optional functionalization [20,[22][23][24]. Aforementioned superior features can be eventually exploited in designing artificial DNA-based biomachinery, such as nucleic acid devices [25] and protein-DNA hybrid structures [26] for nanomedicine and biosensing. In this focused review, we discuss the recent progress of the nanomedical applications of self-assembled DNA systems; especially the drug delivery vehicles and nanomachines based on the DNA origami technique. Towards DNA-based drug delivery vehicles and advanced therapeuticsThe tremendous self-assembly properties of DNA can be exploited in creating various programmable shapes and larger assemblies for cellular delivery for e.g. cancer and enzyme replacement therapy. The very first DNA origami nano-objects proposed to work as molecular containers for drug delivery applications were single-layer origamis, which were further assembled into 3D shapes -a tetrahedron [5] or hollow cubes [6,7] (Fig. 1A). Since then, cellular uptake for various DNA structures (based o...

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“…381 To each of the arms of the tweezer, a glucose-6-phosphate dehydrogenase and its cofactor NAD + were covalently bound. When the tweezer took the open form, the enzyme/cofactor pair was spatially separated, and thus no enzymatic reaction occurred.…”

Section: Determination Of Dna Nanostructures In Solutionsmentioning

confidence: 99%

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

Komiyama

Yoshimoto

Sisido

et al. 2017

Bulletin of the Chemical Society of Japan

275

131

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In this review, we introduce two kinds of bio-related nanoarchitectonics, DNA nanoarchitectonics and cellmacromolecular nanoarchitectonics, both of which are basically controlled by chemical strategies. The former DNA-based approach would represent the precise nature of the nanoarchitectonics based on the strict or "digital" molecular recognition between nucleic bases. This part includes functionalization of single DNAs by chemical means, modification of the main-chain or side-chain bases to achieve stronger DNA binding, DNA aptamers and DNAzymes. It also includes programmable assemblies of DNAs (DNA Origami) and their applications for delivery of drugs to target sites in vivo, sensing in vivo, and selective labeling of biomaterials in cells and in animals. In contrast to the digital molecular recognition between nucleic bases, cell membrane assemblies and their interaction with macromolecules are achieved through rather generic and "analog" interactions such as hydrophobic effects and electrostatic forces. This cell-macromolecular nanoarchitectonics is discussed in the latter part of this review. This part includes bottom-up and top-down approaches for constructing highly organized cell-architectures with macromolecules, for regulating cell adhesion pattern and their functions in twodimension, for generating three-dimensional cell architectures on micro-patterned surfaces, and for building synthetic/natural macromolecular modified hybrid biointerfaces.

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A DNA tweezer-actuated enzyme nanoreactor

Cited by 290 publications

References 39 publications

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

DNA Nanostructures as Smart Drug-Delivery Vehicles and Molecular Devices

Chemistry Can Make Strict and Fuzzy Controls for Bio-Systems: DNA Nanoarchitectonics and Cell-Macromolecular Nanoarchitectonics

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