Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes

Lin, Peng; Dinh, Huyen; Morita, Yuki; Zhang, Zhengxiao; Nakata, Eiji; Kinoshita, Masahiro; Mutoh, T.

doi:10.1039/d1cc00276g

Cited by 14 publications

(28 citation statements)

References 35 publications

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“…The modular adaptor ( Nakata et al, 2015 ; Ngo et al, 2016 ; Nguyen et al, 2017 ; Nguyen et al, 2019 ) stably locates an enzyme of interest at the specific position on DNA scaffold with a covalent linkage between the protein and the scaffold. XDH was fused to the C-terminal of modular adaptor (HG) consisting of the basic leucine zipper protein GCN4 ( Ellenberger et al, 1992 ) and Halo-tag ( England et al, 2015 ) to construct a fusion enzyme HG-XDH as reported previously ( Lin et al, 2021 ). HG-XDH specifically reacts with the Halo-tag substrate 5-chlorohexane (CH) incorporated near the GCN4-binding DNA sequence ( Nguyen et al, 2017 ; Nguyen et al, 2019 ).…”

Section: Resultsmentioning

confidence: 99%

“…Enzyme HG-XDH (modular adaptor Halo-GCN4 fused xylitol dehydrogenase) was prepared as previously reported ( Lin et al, 2021 ). Briefly, a gene encoding HG-XDH was constructed via overlapping PCR using p4LZ vector containing GCN4-XDH gene ( Ngo et al, 2014 ) and pFN18A HaloTag® T7 Flexi® Vector containing a Halo-tag gene.…”

Section: Methodsmentioning

confidence: 99%

“…The sample was then purified by gel filtration (500 µL Sephacryl S-400) in an Ultrafree-MC-DV column with a buffer (pH 7.0) containing 40 mM Tris-HCl, 20 mM acetic acid, and 12.5 mM MgCl 2 to remove the excess staple strands. The concentration of DNA scaffold was quantified by the absorbance at 260 nm (Nanodrop, Thermo Fisher Scientific Inc.,) using the determined extinction coefficient of DNA scaffold (1.20 × 10 8 M −1 cm −1 ) ( Lin et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

“…10 nM DNA scaffold with the binding sites was incubated with 200 nM HG-XDH in a buffer (pH 7.0) containing 40 mM Tris-HCl, 20 mM acetic acid, 12.5 mM MgCl 2 , 5 mM β -mercaptoethanol, 0.002% Tween20, and 1 µM ZnCl 2 at 4°C for 1 h. The binding reaction mixture was purified by gel filtration (500 µL in volume of S-400) in an Ultrafree-MC-DV column with a buffer (pH 7.0) containing 40 mM Tris-HCl, 20 mM acetic acid, and 12.5 mM MgCl 2 to remove the unbound proteins. The concentration of DNA scaffold–protein assembly was estimated from the absorbance at 260 nm by using the extinction coefficient of DNA scaffold (1.20 × 10 8 M −1 cm −1 ) ( Lin et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

“…The total number of DNA scaffolds corresponded to the well-formed structures observed under AFM. The binding of HG-XDH was counted for only HG-XDH bound to the perfectly folded DNA scaffold, and the quantification result is shown in Supplementary Table S5 ( Lin et al, 2021 ).…”

Section: Methodsmentioning

confidence: 99%

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Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier

et al. 2021

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Structural programmability and accurate addressability of DNA nanostructures are ideal characteristics for the platform of arranging enzymes with the nanoscale precision. In this study, a three-dimensional DNA scaffold was designed to enable a dynamic shape transition from an open plate-like structure to its closed state of a hexagonal prism structure. The two domains in the open state were folded together to transform into the closed state by hybridization of complementary short DNA closing keys at both of the facing edges in over 90% yield. The shape transformation of the DNA scaffold was extensively studied by means of the fluorescence energy transfer measurement, atomic force microscope images, and agarose gel electrophoretic analyses. A dimeric enzyme xylitol dehydrogenase was assembled on the DNA scaffold in its open state in a high-loading yield. The enzyme loaded on the scaffold was subsequently transformed to its closed state by the addition of short DNA closing keys. The enzyme encapsulated in the closed state displayed comparable activity to that in the open state, ensuring that the catalytic activity of the enzyme was well maintained in the DNA nanocarrier. The nanocarrier with efficient encapsulation ability is potentially applicable for drug delivery, biosensing, biocatalytic, and diagnostic tools.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier

et al. 2021

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Dynamic Assembly of Cascade Enzymes by the Shape Transformation of a DNA Scaffold

Lin

Dinh

Morita

et al. 2023

Adv Funct Materials

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Within cells, the close spatial arrangement of cascade enzymes facilitates the channeling of intermediates and enhances cascade reaction efficiency. Reconfigurable DNA nanostructures, owing to their structural controllability and precise spatial addressability, are promising tools for mimicking such processes. In this study, a 3D DNA origami scaffold, with a dynamic shape transformation from its open boat form to a closed hexagonal prism induced by toehold‐mediated strand displacement, is designed to investigate the enzyme cascade reaction of xylose reductase and xylitol dehydrogenase from D‐xylose metabolic pathway. Enzymes are assembled on the DNA scaffold in its open state, which is subsequently closed by the assistance of DNA sequence‐specific closing keys. The enzyme cascade efficiency is much higher in the static encapsulated closed state than in the open state due not only to the enzyme proximity but also the environmental factors of 3D DNA structure. These results provide novel insights into controlling enzyme cascade reactions by inducing the shape transformation of DNA nanostructures and how environmental factors affect the action of multi‐enzyme complexes in the cell.

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Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

Kröll,

Niemeyer

2023

Angewandte Chemie

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The natural micro‐ and nanoscale organization of biomacromolecules is a remarkable principle within living cells, allowing for the control of cellular functions by compartmentalization, dimensional diffusion and substrate channeling. In order to explore these biological mechanisms and harness their potential for applications such as sensing and catalysis, molecular scaffolding has emerged as a promising approach. In the case of synthetic enzyme cascades, developments in DNA nanotechnology have produced particularly powerful scaffolds whose addressability can be programmed with nanometer precision. In this minireview, we summarize recent developments in the field of biomimetic multicatalytic cascade reactions organized on DNA nanostructures. We emphasize the impact of the underlying design principles like DNA origami, efficient strategies for enzyme immobilization, as well as the importance of experimental design parameters and theoretical modeling. We show how DNA nanostructures have enabled a better understanding of diffusion and compartmentalization effects at the nanometer length scale, and discuss the challenges and future potential for commercial applications.

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Evaluation of the role of the DNA surface for enhancing the activity of scaffolded enzymes

Abstract: The catalytic enhancements of enzymes loaded on DNA nanostructures have been attributed to the characteristics provided by highly negative charges on the surface of scaffold, such as modulation of the...

Cited by 14 publications

References 35 publications

Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier

Dynamic Shape Transformation of a DNA Scaffold Applied for an Enzyme Nanocarrier

Dynamic Assembly of Cascade Enzymes by the Shape Transformation of a DNA Scaffold

Nucleic Acid‐based Enzyme Cascades—Current Trends and Future Perspectives

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