Stabilization and structural changes of 2D DNA origami by enzymatic ligation.

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.

Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions.

DNA minicircles exist in biological contexts, such as kinetoplast DNA, and are promising components for creating functional nanodevices. They have been used to mimic the topological features of nucleosomal DNA and to probe DNA-protein interactions such as HIV-1 and PFV integrases, and DNA gyrase. Here, we synthesized the topologically-interlocked minicircle rotaxane and catenane inside a frame-shaped DNA origami. These minicircles are 183 bp in length, constitute six individual single-stranded DNAs that are ligated to realize duplex interlocking, and adopt temporary base pairing of single strands for interlocking. To probe the DNA-protein interactions, restriction reactions were carried out on DNAs with different topologies such as free linear duplex or duplex constrained inside origami and free or topologically-interlocked minicircles. Except the free linear duplex, all tested structures were resistant to restriction digestion, indicating that the topological features of DNA, such as flexibility, curvature, and groove orientation, play a major role in DNA-protein interactions.

Topologically-Interlocked Minicircles as Probes of DNA Topology and DNA-Protein Interactions.

Invited for the cover of this issue are Prof. Takashi Morii and co-workers at Kyoto University and Ewha Womans University. The cover image depicts the graphical design and atomic force microscopic (AFM) images of the synthesized topologically-interlocked DNA catenane and rotaxanes inside a frame-shaped DNA origami. Read the full text of the article at 10.1002/chem.202200108.

Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation.

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.

One-Pot Isolation of a Desired Human Genome Fragment by Using a Biotinylated pcPNA/S1 Nuclease Combination.

Scission of the human genome at predetermined sites and isolation of a particular fragment are of great interest for the analysis of lesion/modification sites, in proteomics, and for gene therapy. However, methods for human genome scission and specific fragment isolation are limited. Here, we report a novel one-pot method for the site-specific scission of DNA by using a biotinylated pcPNA/S1 nuclease combination and isolation of a desired fragment by streptavidin-coated magnetic beads. The proof of concept was initially demonstrated for the clipping of plasmid DNA and isolation of the required fragment. Our method was then successfully applied for the isolation of a fragment from the cell-derived human genome.

Nucleic-Acid-Templated Enzyme Cascades.

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.

Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame.

Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G-G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex.

Direct and single-molecule visualization of the solution-state structures of G-hairpin and G-triplex intermediates.

We present the direct and single-molecule visualization of the in-pathway intermediates of the G-quadruplex folding that have been inaccessible by any experimental method employed to date. Using DNA origami as a novel tool for the structural control and high-speed atomic force microscopy (HS-AFM) for direct visualization, we captured images of the unprecedented solution-state structures of a tetramolecular antiparallel and (3+1)-type G-quadruplex intermediates, such as G-hairpin and G-triplex, with nanometer precision. No such structural information was reported previously with any direct or indirect technique, solution or solid-state, single-molecule or bulk studies, and at any resolution. Based on our results, we proposed a folding mechanism of these G-quadruplexes.

Near Quantitative Ligation Results in Resistance of DNA Origami Against Nuclease and Cell Lysate.

There have been limited efforts to ligate the staple nicks in DNA origami which is crucial for their stability against thermal and mechanical treatments, and chemical and biological environments. Here, two near quantitative ligation methods are demonstrated for the native backbone linkage at the nicks in origami: i) a cosolvent dimethyl sulfoxide (DMSO)-assisted enzymatic ligation and ii) enzyme-free chemical ligation by CNBr. Both methods achieved over 90% ligation in 2D origami, only CNBr-method resulted in ≈80% ligation in 3D origami, while the enzyme-alone yielded 31-55% (2D) or 22-36% (3D) ligation. Only CNBr-method worked efficiently for 3D origami. The CNBr-mediated reaction is completed within 5 min, while DMSO-method took overnight. Ligation by these methods improved the structural stability up to 30 °C, stability during the electrophoresis and subsequent extraction, and against nuclease and cell lysate. These methods are straightforward, non-tedious, and superior in terms of cost, reaction time, and efficiency.

Direct and real-time observation of rotary movement of a DNA nanomechanical device.

Analogous to the biologically abundant protein-based linear molecular machines that translocate along their target surface, we have recently constructed the DNA-based synthetic molecular motors that effect linear movement or navigate a network of tracks on a DNA origami substrate. However, a DNA-based molecular machine with rotary function, analogous to rotary proteins, is still unexplored. Here, we report the construction of a rotary motor based on the B-Z conformational transition of DNA and the direct and real-time observation of its function within a frame-shaped DNA origami. The motor can be switched off by introducing conditions that stabilize B-DNA, while it can be fueled by adding Z-DNA-promoting high-saline buffer. When MgCl(2) was used as external stimulus, 70% of the motors rotated, while 76% of the stators/controls exhibited no rotation. Such a motor system could be successfully applied to perform multiple actions aimed for our benefit. Moreover, for the first time we have directly observed the B-Z conformational transition of DNA in real-time, which shed light on the fundamental understanding of DNA conformations.

HIV-1 nucleocapsid proteins as molecular chaperones for tetramolecular antiparallel G-quadruplex formation.

HIV-1 nucleocapsid proteins (NCps) facilitate remodeling of nucleic acids to fold thermodynamically stable conformations, and thus called nucleic acid chaperones. To date only little is known on the stoichiometry, NCp-NCp interactions, chaperone activity on G-quadruplex formation, and so on. We report here the direct and real-time analysis on such properties of proteolytic intermediate NCp15 and mature NCp7 using DNA origami. The protein particles were found to predominantly exist in monomeric form, while dimeric and multimeric forms were also observed both in free solution and bound to the quadruplex structure. The formation and the dissociation events of the G-quadruplexes were well documented in real-time and the intermediate-like states were also visualized. We anticipate that this pioneering study will strengthen our understanding on the chaperone activity of HIV-1 proteins which in turn will be helpful for the drug design based on G-quadruplex and also for the development of drugs against AIDS.

Single-molecule analysis using DNA origami.

During the last two decades, scientists have developed various methods that allow the detection and manipulation of single molecules, which have also been called "in singulo" approaches. Fundamental understanding of biochemical reactions, folding of biomolecules, and the screening of drugs were achieved by using these methods. Single-molecule analysis was also performed in the field of DNA nanotechnology, mainly by using atomic force microscopy. However, until recently, the approaches used commonly in nanotechnology adopted structures with a dimension of 10-20 nm, which is not suitable for many applications. The recent development of scaffolded DNA origami by Rothemund made it possible for the construction of larger defined assemblies. One of the most salient features of the origami method is the precise addressability of the structures formed: Each staple can serve as an attachment point for different kinds of nanoobjects. Thus, the method is suitable for the precise positioning of various functionalities and for the single-molecule analysis of many chemical and biochemical processes. Here we summarize recent progress in the area of single-molecule analysis using DNA origami and discuss the future directions of this research.

Implantable Microfluidic Device: An Epoch of Technology.

Implantable microfluidic devices are milestones in developing devices that can measure parameters like ocular pressure and blood glucose level or deliver various components for therapeutic needs or behavioral modification. Researchers are currently focusing on the miniaturization of almost all its tools for a better healthcare platform. Implantable microfluidic devices are a combination of various systems including, but not limited to, microfluidic platforms, reservoirs, sensors, and actuators, implanted inside the body of a living entity (in vivo) with the purpose of directly or indirectly helping the entity. It is a multidisciplinary approach with immense potential in the area of the biomedical field. Significant resources are utilized for the research and development of these devices for various applications. The induction of an implantable microfluidic device into an animal would enable us to measure the responses without any repeated invasive procedures. Such data would help in the development of a better drug delivery profile. Implantable microfluidic devices with reservoirs deliver specific chemical or biological products to treat situations like cancers and diabetes. They can also deliver fluorophores for specific imaging inside the body. Implantable microfluidic devices help provide a microenvironment for various cell differentiation procedures. These devices know no boundaries, and this article reviews these devices based on their design and applications.

Effect of the bases flanking an abasic site on the recognition of nucleobase by amiloride.

BACKGROUND: We explain here the various non-covalent interactions which are responsible for the different binding modes of a small ligand with DNA. METHODS: The combination of experimental and theoretical methods was used. RESULTS: The interaction of amiloride with thymine was found to depend on the bases flanking the AP site and different binding modes were observed for different flanking bases. Molecular modeling, absorption studies and binding constant measurements support for the different binding patterns. The flanking base dependent recognition of AP site phosphates was investigated by (31)P NMR experiments. The thermodynamics of the ligand-nucleotide interaction was demonstrated by isothermal titration calorimetry. The emission behavior of amiloride was found to depend on the bases flanking the AP site. Amiloride photophysics in the context of AP-site containing DNA is investigated by time-dependent density functional theory. CONCLUSIONS: Flanking bases affect the ground and excited electronic states of amiloride when binding to AP site, which causes flanking base-dependent fluorescence signaling. GENERAL SIGNIFICANCE: The various noncovalent interactions have been well characterized for the determination of nucleic acid structure and dynamics, and protein-DNA interactions. However, these are not clear for the DNA-small molecule interactions and we believe that our studies will bring a new insight into such phenomena.

Photo-cross-linking-assisted thermal stability of DNA origami structures and its application for higher-temperature self-assembly.

Heat tolerance of DNA origami structures has been improved about 30 °C by photo-cross-linking of 8-methoxypsoralen. To demonstrate one of its applications, the cross-linked origami were used for higher-temperature self-assembly, which markedly increased the yield of the assembled product when compared to the self-assembly of non-cross-linked origami at lower-temperature. By contrast, at higher-temperature annealing, native non-cross-linked tiles did not self-assemble to yield the desired product; however, they formed a nonspecific broken structure.

Simultaneous recognition of nucleobase and sites of DNA damage: effect of tethered cation on the binding affinity.

BACKGROUND: The 3,5-diamino-N-(3-aminopropyl)-6-chloropyrazine-2-carboxamide (DCPC-NH(2)) has been synthesized and characterized by Mass and (1)H NMR. The selective binding of the ligand to thymine (T) target base is investigated by the melting temperature (T(m)) and fluorescence measurements. METHODS: Thermal denaturation study of DNA duplex containing T target base revealed the DeltaT(m) of 5.1 degrees C, while least influence was observed for other target bases. The fluorescence of the ligand DCPC-NH(2) is quenched only upon adding the DNA containing T target base. RESULTS: The binding constant for the interaction of the ligand to T target base containing DNA duplex was determined to be 4.7 (+/-0.3)x10(6) M(-1). The tethered cation in the ligand is found to enhance the binding constant. The ligand binds to both a target nucleotide and an AP site on the complimentary strand for the target strand in a DNA duplex. GENERAL SIGNIFICANCE: Interestingly, the electronic behavior of the ligand depends on the bases flanking the AP site. Its fluorescence is quenched with guanine flanking bases, while it is enhanced with DNA duplex containing T bases flanking an AP site. Finally, the binding modes were visualized by molecular modeling.

Folding pathways of human telomeric type-1 and type-2 G-quadruplex structures.

We have investigated new folding pathways of human telomeric type-1 and type-2 G-quadruplex conformations via intermediate hairpin and triplex structures. The stabilization energies calculated by ab initio methods evidenced the formation of a hairpin structure with Hoogsteen GG base pairs. Further calculations revealed that the G-triplet is more stable than the hairpin conformation and equally stable when compared to the G-tetrad. This indicated the possibility of a triplex intermediate. The overall folding is facilitated by K(+) association in each step, as it decreases the electrostatic repulsion. The K(+) binding site was identified by molecular dynamics simulations. We then focused on the syn/anti arrangement and found that the anti conformation of deoxyguanosine is more stable than the syn conformation, which indicated that folding would increase the number of anti conformations. The K(+) binding to a hairpin near the second lateral TTA loop was found to be preferable, considering entropic effects. Stacking of G-tetrads with the same conformation (anti/anti or syn/syn) is more stable than mixed stacking (anti/syn and vice versa). These results suggest the formation of type-1 and type-2 G-quadruplex structures with the possibility of hairpin and triplex intermediates.

NBD-based green fluorescent ligands for typing of thymine-related SNPs by using an abasic site-containing probe DNA.

The binding behavior of green fluorescent ligands, derivatives of 7-nitrobenzo-2-oxa-1,3-diazole (NBD), with DNA duplexes containing an abasic (AP) site is studied by thermal denaturation and fluorescence experiments. Among NBD derivatives, N(1)-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)propane-1,3-diamine (NBD-NH(2)) is found to bind selectively to the thymine base opposite an AP site in a DNA duplex with a binding affinity of 1.52 x 10(6) M(-1). From molecular modeling studies, it is suggested that the NBD moiety binds to thymine at the AP site and a protonated amino group tethered to the NBD moiety interacts with the guanine base flanking the AP site. Green fluorescent NBD-NH(2) is successfully applied for simultaneous G>T genotyping of PCR amplification products in a single cuvette in combination with a blue fluorescent ligand, 2-amino-6,7-dimethyl-4-hydroxypteridine (diMe-pteridine).

Effect of substituents of alloxazine derivatives on the selectivity and affinity for adenine in AP-site-containing DNA duplexes.

Using the DNA duplex containing an AP site (5'-TCC AGX GCA AC-3'/3'-AGG TCN CGT TG-5', X = AP site, N = A, T, C, or G), we have found that 2-amino-4-hydroxypteridine (pterin) selectively binds to guanine (G), and that the enhanced binding affinity for G is obtained by its methylated derivative 2-amino-6,7-dimethyl-4-hydroxypteridine (diMe pteridine). Similarly, among the cytosine (C)-selective ligands, i.e. derivatives of 2-amino-1,8-naphthyridine, a trimethyl-substituted derivative (2-amino-5,6,7-trimethyl-1,8-naphthyridine) selectively binds to C with a strong binding affinity of 1.9 × 10(7) M(-1). In the case of lumazine derivatives, pteridine-2,4(1H,3H)-dione (lumazine) binds to adenine (A), and its methylated derivative, 6,7-dimethylpteridine-2,4(1H,3H)-dione (diMe lumazine) strongly binds to A with enhanced binding affinity, keeping the same base-selectivity. On the other hand, the benzo-annelated (with phenyl ring, 2.4 Å) derivative of lumazine, benzo[g]pteridine-2,4(1H,3H)-dione (alloxazine), can bind to A selectively, whereas its methylated ligand, 7,8-dimethylbenzo[g]pteridine-2,4(1H,3H)-dione (lumichrome) selectively binds to thymine (T) over A, C and G. Methyl-substituted lumichrome derivatives show moderate binding affinities for target nucleobases. The changes in the base-selectivity and binding affinities are discussed in detail with respect to the substituents of these ligands, considering hydrogen-bonding patterns, size of AP site and stacking interactions.