Details of research

Molecular Switchboad


   In nature, multi-enzyme synthetic pathways regulate the cellular activities. Understanding the effect of spatial organization on the enzymatic activity in multi-enzyme systems is very important not only for revealing a principle that associates with the function of multi-enzyme system but also for constructing an effective material conversion system in vitro. However, only a few methods are available to systematically evaluate how spatial factors such as distance, orientation and ratio of enzymes influence the efficiency of the enzyme cascade.

Development of DNA binding adaptor for site-specific protein-of-interest positioning on DNA nanostructure based molecular switchboard

   Structural DNA nanotechnology, which includes DNA origami1, enables the rapid production of self-assembled nanostructures. One of the key features of this technology is that fully addressable nanoarchitectures of various shapes and geometries are easily designed and constructed. By taking advantage of their addressable nature, DNA nanostructures have been used as scaffolds for the site-directed assembly of functional entities, such as small molecules and nanoparticles. As well as these functional entities, proteins are a particularly interesting class of molecules to assemble because of their huge functional variability. We developed that different locations within DNA-origami structures are site-specifically and orthogonally targeted by using sequence-specific DNA-binding proteins as an adaptor, and demonstrate that adaptor-fused functional proteins are assembled at specific locations within DNA-origami structures.

Zinc finger protein as a monomeric protein adaptor to target a specific location within molecular switchboard2

   As the first trial, we choose the zinc-finger proteins (ZFPs), because it is one of the best-characterized classes of DNA-binding proteins and the artificial ZFPs can bind to a wide variety of DNA sequences. Two types of well-characterized ZFPs, each with an affinity for a unique sequence of ten base pairs in the low nanomolar range, were chosen as the orthogonal adaptors for specific locations in the DNA-origami structures. A rectangular DNA-origami structure that has five addressable cavities was designed. Each addressable cavity was designed to hold up to four ZFP-adaptor binding sites. The folding of an M13mp18 single-stranded DNA through the use of 159 staple strands was prepared. The mixture of DNA-origami and ZFP adaptor fused chimeric protein was adsorbed onto mica and analyzed by AFM at the single-molecule level. The AFM images indicated the selective and orthogonal binding of ZFP adaptors to their expected locations. Through these experiments, we have demonstrated that ZFPs are convenient and selective adaptors for targeting specific location within DNA-origami structures. The diversity of target DNA sequences and the semi-programmable design of ZFPs offers orthogonal adaptors, thereby enabling the placement of multiple engineered proteins at different locations onto DNA-origami structures. Nature uses multiple proteins and/or enzymes in close proximity to efficiently carry out chemical reactions and signal transductions. Such assemblies of multiple proteins may be realized in vitro by using DNA-origami structures that have defined binding sites and various kinds of ZFP adaptor-fused proteins.

Figure 1. Conjugation of DNA origami and ZFP adaptor fused functional domain as the molecular switchboard.

Basic-leucine zipper protein as a dimeric protein adaptor having orthogonality toward zinc finger adaptor3

   As described above, ZFPs were developed as a monomeric protein adaptor to target a specific location within molecular switchboard. Development of various types of adaptors with distinct sequence selectivity enables placing various adaptor-fused proteins on DNA origami at specific positions orthogonally, which will lead to construction of functional protein assemblies on molecular switchboard. Thus, we have developed a basic-leucine zipper (bZIP) class of proein GCN4 as a new adaptor to expand the range of target DNA sequences.
   Specific binding of GCN4 derivatives on DNA origami nanoarchitectures was successfully demonstrated. Analyses by AFM and gel electrophoresis confirmed specific binding of GCN4 to the address containing the specific binding site on DNA origami. And more, orthogonal targeting by GCN4 and zif268 adaptors at the respective sites on DNA origami was also confirmed by AFM and gel electrophoretic analyses. These results indicate that the adaptors for both monomeric, and homodimeric proteins of interests are now available to locate them at the specific sites on molecular switchboard with complete orthogonality.
   Furthermore, a GCN4-fused homodimeric enzyme was arranged on molecular switchboard. The GCN4 adaptor was applied for a homodimeric enzyme XDH. Conjugation of the GCN4 adaptor to the N-terminal of XDH afforded active adaptor-fused enzyme GCN4-XDH. The specific binding of GCN4-XDH on DNA origami was confirmed by AFM analyses. It should be noted that GCN4-XDH showed even higher activity than the wild type of XDH, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.

Figure 2. Conjugation of DNA origami and GCN4-fused homo dimeric enzyme as the molecular switchboard.

A modular adaptor consists of zinc finger protein and self-ligating protein tag accelerates covalent linkage of proteins at specific locations on DNA nanoscaffolds4

   As described above, we have developed protein-based adaptors by utilizing the sequence-specific DNA binding proteins. These DNA binding proteins serve as the orthogonal adaptors to locate functional proteins at the specific sequences on DNA origami. However, the reversible nature of the complex between the DNA binding adaptor and DNA origami has the difficulty to saturate the target addresses on DNA origami with the protein binding adaptor fused a protein of interest. It should be noted that the same issue have been occurred in the case of oligodeoxynucleotide(ODN)-tethered protein, which have been often attempted for locating proteins on DNA origami through the hybridization of ODN. Formation of a covalent linkage between DNA and a protein of interest is a promising strategy for overcoming the issue. One of the solutions utilizes self-ligating protein tags, such as SNAP-tag,5 to crosslink a protein of interest onto DNA origami.6 The self-ligating protein tags showed chemo-selective cross-linking ability, but a major drawback for this approach is the crosslinking efficiency: at least a thousand fold of self-ligating protein tag and incubation for overnight were required to reach less than 60% of reaction. We hypothesized that combination of a sequence-specific DNA binding zinc finger adaptor and a self-ligating protein-tag could afford a new class of covalent cross-linking adaptors that would facilitate efficient loading of a protein of interest on DNA origami with fast kinetics.
The combined adaptor, named zif-SNAP, was designed by fusing the SNAP-tag,5 an engineered mutant of human O6-alkylguanine-DNA alkyltransferase (hAGT) that reacts specifically with benzylguanine (BG), to the C-terminal of zif268 derived zinc finger adaptor, reported by us as one of the sequence-selective zinc finger adaptors for delivering protein of interests to specific addresses on DNA origami.2 ODNs containing the zif268 binding sequence were designed to form a loop with four T nucleotides, and one of those T nucleotides was displaced by the BG-modified T derivative as the substrate for the SNAP-tag (ODN-zif-BG). Formation of a covalent linkage between ODN-zif-BG and ZF-SNAP was analyzed by denaturing polyacrylamide gel electrophoresis. The results indicated that ZF-SNAP induces the rapid and sequence-specific formation of a covalent linkage at the target DNA sequence. And the combined adaptor was applied to crosslink protein of interest at programmed addresses on the DNA origami, which were analyzed by atomic force microscopy.
We have demonstrated that a new adaptor consisting of a DNA-binding zinc finger adaptor and SNAP-tag, showed site-selective and efficient assembly of protein of interest at the programmed BG-modified addresses on DNA origami.

Figure 3. The design of new adaptor by combinating a zinc finger adaptor and a self-ligating protein-tag.


1) P. W. Rothemund, Nature 2006, 440, 297-302.
2) E. Nakata, F. F. Liew, C. Uwatoko, S. Kiyonaka, Y. Mori, Y. Katsuda, M. Endo, H. Sugiyama, T. Morii, Angew Chem. Int. Ed. 2012, 51, 2421-2424.
3) T. A. Ngo, E. Nakata, M. Saimura, T. Kodaki, T. Morii, Methods, 2014, 67, 142-150.
4) E. Nakata, D. Huyen, T. A. Ngo, M. Saimura, T. Morii, Chem. Commun., in press.
5) A. Keppler, S. Gendreizig, T. Gronemeyer, H. Pick, H. Vogel, K. Johnsson, Nat. Biotechnol., 2003, 21, 86-89.
6) B. Sacca, R. Meyer, M. Erkelenz, K. Kiko, A. Arndt, H. Schroeder, K. S. Rabe, C. M. Niemeyer, Angew. Chem. Int. Ed., 2010, 49, 9378-9383.


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