Akinori Kuzuya

Precisely programmed and robust 2D streptavidin nanoarrays by using periodical nanometer-scale wells embedded in DNA origami assembly.

A new punched DNA origami assembly with periodic nanometer‐scale wells has been successfully designed and constructed. Through the attachment of two biotins at the two edges of each well, just one streptavidin (SA) tetramer (d=5 nm) was size‐selectively captured in each 6.8×12×2.0 nm well; this allowed formation of a 28 nm‐period SA nanoarray of individual molecules. The position of SA capture can be fully controlled by placement of biotins in the nanoarray well. Moreover, construction of a 2D nanoarray of individual SA tetramers through selective positioning of SA tetramers in any desired wells in a complex of such punched origami motifs is also possible. The stability of the SA captured by this fixation strategy (DNA wells and two biotin linkers) was directly compared on the same molecule with the stability of SA captured with other possible strategies that do not employ wells or two linkers. In this way, the robustness of this means of fixation was clearly established.

Discrete and active enzyme nanoarrays on DNA origami scaffolds purified by affinity tag separation.

Desired enzyme nanoarrays patterned on a DNA origami scaffold were selectively isolated by affinity tag purification from a pool of differently patterned nanoarrays, and their enzymatic activity was successfully confirmed. As few as 12 histidine residues were enough to hold a huge complex of DNA origami with multiple proteins, 260 nm in length and 5.2 MDa in molecular weight, to an immobilized metal affinity resin.

Conjugates of a Dinuclear Zinc(II) Complex and DNA Oligomers as Novel Sequence‐Selective Artificial Ribonucleases

Even in the presence of a large excess of ZnII ions, sequence-selective RNA hydrolysis is achieved by DNA conjugates involving a dinuclear ZnII complex (shown schematically). This is because the cooperation of two ZnII ions is essential for the RNA scission.

A DNA aptamer recognising a malaria protein biomarker can function as part of a DNA origami assembly

DNA aptamers have potential for disease diagnosis and as therapeutics, particularly when interfaced with programmable molecular technology. Here we have combined DNA aptamers specific for the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) with a DNA origami scaffold. Twelve aptamers that recognise PfLDH were integrated into a rectangular DNA origami and atomic force microscopy demonstrated that the incorporated aptamers preserve their ability to specifically bind target protein. Captured PfLDH retained enzymatic activity and protein-aptamer binding was observed dynamically using high-speed AFM. This work demonstrates the ability of DNA aptamers to recognise a malaria biomarker whilst being integrated within a supramolecular DNA scaffold, opening new possibilities for malaria diagnostic approaches based on DNA nanotechnology.

Accommodation of a Single Protein Guest in Nanometer-Scale Wells Embedded in a “DNA Nanotape”†

Remarkable progress in scanning probe microscope technology has enabled us to visualize various chemical or biological molecules at single-molecule resolutions. One attractive application of this technology is nanopatterning of proteins. Such protein nanoarrays would be essential tools in future diagnosis, proteome analysis, and many other biological research fields. Recently, more precise nanoarrays, in which each protein molecule is individually arrayed on a DNA scaffold, have been reported. Such DNA scaffolds are fruits of DNA nanotechnology, which is based on programmed assembly of branched DNA helices. Various DNA nanostructures, for example, 2D double crossover (DX) crystals, 1D triple crossover (TX) arrays, DNA nanogrids, kagome lattices, and DNA origami, have been used as the scaffold. The next important target of protein nanoarray study is to control the orientation of each protein molecule. Such nanoarrays of regularly ordered and oriented protein molecules in the nanometer range should realize more advanced systems for use in the above fields. However, previous fixation of proteins to the scaffold was carried out mostly by connecting the protein and DNA with a single flexible linker or by introducing an aptamer in a hairpin, and those proteins were placed on the surface of the scaffold. It is not easy to regulate precisely the orientation of such proteins. Herein, we propose a new strategy that leads to more robust and regulated protein nanoarrays: making nanometerscale wells embedded in a DNA sheet for the selective capture of a target protein in a single-molecule manner, by “anchoring” it with two linkers (Figure 1). We made a tapelike DNA scaffold (a “DNA nanotape”) by bundling nine DNA helices, and placed regularly arranged nanometerscale wells in it. By attaching two biotin residues at two edges of each well, we successfully and size-selectively captured just one streptavidin (SA) tetramer in a well, and formed 28-nmperiod SA nanoarrays. The structure of the DNA motifs used in this study is shown in Figure 2. Two types of U-shaped motif were designed (Figure 2a). Motif 1 consists of four helices each eight turns long (84 bp (base pairs)) and five helices each six turns long (63 bp). These helices are connected to the adjacent helices through immobile four-way junctions at two positions, which are two or four helical turns apart. Figure 2b shows the connection pattern of each strand in 1. Consequently, a flat, raftlike, nine-helix DNA bundle is formed. The longer helices are placed two by two in the edges of the motif, and thus 1 has a rectangular concavity two turns wide and five helices long at one side. The 5’ ends of the longer helices are sticky ends of five bases (dashed lines in Figure 2b). When the sticky ends on the same helix are complementary to each other, the resulting self-complementary 1 Figure 1. Strategy for the assembly of a protein nanoarray. a) Formation of a nanometer-scale well in a DNA scaffold. b) Introduction of two (or more) linkers to the edges of the well. c) Size-selective capturing of a single protein molecule in a well by “anchoring” the molecule with the linkers. In the present study, the DNA scaffold is a flat, nine-helix DNA bundle, the linker (red) is a biotin-TEG residue, and the protein (green) is a SA tetramer. TEG= triethylene glycol.

Formation of 1D and 2D Gold Nanoparticle Arrays by Divalent DNA–Gold Nanoparticle Conjugates

Divalent DNA-AuNP (gold nanoparticle) conjugates comprising two DNA strands at diametrically opposed positions are prepared. Highly linear 1D and tetragonal lattice-like 2D AuNP arrays are constructed using the conjugates and DNA assemblies based on T- and double-crossover motifs and the Holliday junction.

Programmed nanopatterning of organic/inorganic nanoparticles using nanometer-scale wells embedded in a DNA origami scaffold.

DNA nanotechnology, which is based on programmed assembly of branched DNA helices, has been attracting great interest as a key technology in preparing scaffolds for nanopatterning of various nanomaterial. [ 1 , 2 ] DNA origami, in which long single-stranded DNA is folded into a designed planar nanostructure with the aid of many short staple strands, is one of the promising candidates for the scaffold. [ 3 , 4 ] The most popular way to fi x nanomaterial to a DNA scaffold to date is to use a single fl exible linker between a DNA component and the target nanomaterial, and to place the target on a surface of the DNA nanostructure. Recently, we proposed an effective new strategy to fi x nanomaterial to DNA nanostructures that leads to robust and precise nanoarrays. [ 5 , 6 ] This strategy is based on our previous fi nding that a nanometersized cavity embedded in a DNA nanostructure can serve as a well to selectively capture a single protein molecule and accommodate it quite stably. [ 5 ] To construct a streptavidin (SA) nanoarray, for example, we prepared a stick-like DNA origami scaffold equipped with nine periodical wells with dimensions of 7 nm × 14 nm × 2 nm. [ 6 ] When predetermined wells in the origami scaffold were modifi ed with two biotintriethylene glycol (TEG) residues by using appropriate combinations of biotinylated staple strands (anchor strands) placed at the two opposite edges of the wells, exactly one SA tetramer was selectively captured in each of the biotinylated wells. The captured SA showed remarkable stability against repetitive atomic force microscopy (AFM) scanning to provide consistent clear images of individual molecules, both with bidentate binding and a steric protection by a 2-nm deep DNA well. [ 6 , 7 ]

Nanomechanical DNA origami pH sensors.

Single-molecule pH sensors have been developed by utilizing molecular imaging of pH-responsive shape transition of nanomechanical DNA origami devices with atomic force microscopy (AFM). Short DNA fragments that can form i-motifs were introduced to nanomechanical DNA origami devices with pliers-like shape (DNA Origami Pliers), which consist of two levers of 170-nm long and 20-nm wide connected at a Holliday-junction fulcrum. DNA Origami Pliers can be observed as in three distinct forms; cross, antiparallel and parallel forms, and cross form is the dominant species when no additional interaction is introduced to DNA Origami Pliers. Introduction of nine pairs of 12-mer sequence (5′-AACCCCAACCCC-3′), which dimerize into i-motif quadruplexes upon protonation of cytosine, drives transition of DNA Origami Pliers from open cross form into closed parallel form under acidic conditions. Such pH-dependent transition was clearly imaged on mica in molecular resolution by AFM, showing potential application of the system to single-molecular pH sensors.

Blunt-ended DNA stacking interactions in a 3-helix motif

We demonstrate that intermolecular stacking is capable of forming one-dimensional arrays of a blunt-ended 3-helix DNA motif. The array can be visualized in the atomic force microscopy through conjugated streptavidin nanoparticles. We estimate the strength of the triple stacking interaction to be -8.6 kcal mol(-1).

Site-Selective Blocking of PCR by a Caged Nucleotide Leading to Direct Creation of Desired Sticky Ends in The Products

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative—4‐O‐[2‐(2‐nitrophenyl)propyl]thymine—was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5′→3′ direction) by polymerase was site‐selectively terminated at the 3′‐side of TNPP. Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light‐assisted cohesive‐ending PCR (LACE‐PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.