A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds

Nakata, Eiji; Dinh, Huyen; Ngo, Tien Anh; Saimura, Masayuki; Morii, Takashi

doi:10.1039/c4cc08167f

Cited by 42 publications

(47 citation statements)

References 29 publications

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“…Overexpression and purication of ZF-SNAP, AZ-SNAP, and AZ-CLIP were described in a previous report. 14,15 ZF-CLIP and CLIP-G were newly designed in this study. As a typical example, the preparation of ZF-CLIP is shown below.…”

Section: Preparation Of Protein-tag Derivatives (Zf-snap Az-snap Zfmentioning

confidence: 99%

“…Specic and nonspecic binding of modular adaptors was counted for only the modular adaptor bound to the perfectly folded DNA scaffold. The yield of the DNA-scaffold-assembled modular adaptor was calculated as described previously 14,18 and the results are shown in Table S14. †…”

Section: Afm Imaging and Statistical Analysismentioning

confidence: 99%

“…1a and b). 14,15 A modular adaptor consists of a DNA-binding protein (zif268 16 ) that functions as a DNA sequence-selective recognition module and self-ligating protein tag (SNAP-tag 17 ) which forms a chemoselective covalent linkage between the protein tag and its substrate O 2 -benzylguanine (BG) modied at the given DNA sequence (Fig. 1a).…”

Section: Introductionmentioning

confidence: 99%

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Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics

et al. 2019

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Section: Preparation Of Protein-tag Derivatives (Zf-snap Az-snap Zfmentioning

confidence: 99%

Section: Afm Imaging and Statistical Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics

et al. 2019

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“…We adopted an origami design that has three nanocavities into which the enzymes were attached. For site‐specific immobilization of the enzymes inside the origami cavities, a mutant XR with a preference for NADH over NADPH was fused to a modular adaptor (ZS) consisting of a DNA‐binding zinc finger protein (zif268) and a self‐ligating SNAP‐tag, to yield the ZS ⋅ XR (69.6 kDa) enzyme construct. The XDH was fused at the C terminus with basic leucine‐zipper protein (GCN4) to yield G ⋅ XDH homodimer (93.6 kDa).…”

Section: Scaffolded Dna Origami Templatesmentioning

confidence: 99%

Nucleic‐Acid‐Templated Enzyme Cascades

et al. 2017

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Cellular metabolism involves complex sequences of organized enzymatic reactions, known as metabolic pathways, that convert substrates into readily usable materials. In nature, these enzymatic complexes are organized in a well-defined manner so that the cascade reactions are more rapid and efficient than they would be if the enzymes were randomly distributed in the cytosol. Development of artificial enzyme cascades that resemble nature's organization of sequentially assembled enzymes is of current interest due to its potential applications, from diagnostics to the production of high-value chemicals. Nucleic acids and their nanostructures have been used to organize enzyme cascades and have been shown to enhance the efficiencies and rates of sequential reactions. Here we summarize the recent progress in the development of artificial enzyme cascades and sequential reactions by arranging enzymes on various DNA/RNA templates and discuss the future directions of this research endeavour.

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“…Notably,the simplicity and general applicability of our approachh as advantages over site-specific methods to improvep rotein loading of DONs,s uch as the fusion of Snaptagged POIs with az inc finger motif. [33] On the basis of our ini- Figure 2. Binding of A) BMR-HaloTag, B) BMR-HOB, and C) HOB-BMR with 22-mer oligonucleotides tCH-F1 (red) or iCH-F1 (blue), bearingt he CH ligand (green) at the terminal 5' or an internal thymine base position,respectively.…”

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confidence: 99%

A Rationally Designed Connector for Assembly of Protein‐Functionalized DNA Nanostructures

et al. 2016

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We report on the rational engineering of the binding interface of the self-ligating HaloTag protein to generate an optimized linker for DNA nanostructures. Five amino acids positioned around the active-site entry channel for the chlorohexyl ligand (CH) of the HaloTag protein were exchanged for positively charged lysine amino acids to produce the HOB (halo-based oligonucleotide binder) protein. HOB was genetically fused with the enzyme cytochrome P450 BM3, as well as with BMR, the separated reductase domain of BM3. The resulting HOB-fusion proteins revealed significantly improved rates in ligation with CH-modified oligonucleotides and DNA origami nanostructures. These results suggest that the efficient self-assembly of protein-decorated DNA structures can be greatly improved by fine-tuning of the electrostatic interactions between proteins and the negatively charged nucleic acid nanostructures.

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A modular zinc finger adaptor accelerates the covalent linkage of proteins at specific locations on DNA nanoscaffolds

Cited by 42 publications

References 29 publications

Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics

Rational design of a DNA sequence-specific modular protein tag by tuning the alkylation kinetics

Nucleic‐Acid‐Templated Enzyme Cascades

A Rationally Designed Connector for Assembly of Protein‐Functionalized DNA Nanostructures

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