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.

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.

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.

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.

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.

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.

Affinity Isolation of Defined Genomic Fragments Cleaved by Nuclease S1-based Artificial Restriction DNA Cutter.

The human genome is highly susceptible to various modifications, lesions, and damage. To analyze lesions and proteins bound to a defined region of the human genome, the genome should be fragmented at desired sites and the region of interest should be isolated. The few available methods for isolating a desired region of the human genome have serious drawbacks and can only be applied to specific sequences or require tedious experimental procedures. We have recently developed a novel method to isolate a desired fragment of the genome released by site-specific scission of DNA using a pair of pseudo-complementary peptide nucleic acids (pcPNAs) and S1 nuclease. When conjugated to biotin, one of the pcPNAs can be used to affinity purify the cleavage product. Here we report a detailed protocol to isolate defined kilobase-length DNA fragments that can be applied to plasmid or genomic DNA and is not limited by sequence. © 2019 by John Wiley & Sons, Inc.

Artificial Restriction DNA Cutter Using Nuclease S1 for Site-Selective Scission of Genomic DNA.

By combining a pair of pseudo-complementary peptide nucleic acids (pcPNAs) with S1 nuclease, a novel tool to cut DNA at a predetermined site can be obtained. Complementary pcPNAs invade the DNA duplex and base pair to each strand of a target site, creating single-stranded regions that are cleaved by S1 nuclease. The scission site can be freely modulated by the design of pcPNAs. This method can be used to cleave a single site in the human genome. This protocol presents experimental details for site-selective scission using this versatile new tool. © 2019 by John Wiley & Sons, Inc.

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.

Small molecule binding to a G-hairpin and a G-triplex: a new insight into anticancer drug design targeting G-rich regions.

To gain new insights into G-quadruplex-drug interactions, we captured the solution-state structures of the complexes between a drug-like small molecule and a G-hairpin/G-triplex. Our results indicated that the ligand initially binds to the intermediates and induces stepwise folding into a quadruplex.

A lock-and-key mechanism for the controllable fabrication of DNA origami structures.

Controllable fabrication of DNA origami structures was achieved using cationic comb-type copolymers (CCCs) as locks and polyvinyl sulphonic acid (PVS) as a key. A CCC binds to the phosphate backbone of either M13mp18/staples alone or both together and restricts origami folding, while PVS unlocks the CCC, restoring the formation of origami structures.

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.

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.

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 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.

Control of the two-dimensional crystallization of DNA origami with various loop arrangements.

We have developed a new strategy to control the two-dimensional (2D) crystallization of DNA origami by introducing loops on the surface and aligning them in various orientations. Among the orientations tested, vertically connected loops successfully produced the 2D crystal lattice on a micrometer scale, while all other orientations failed.

Deciphering DNA-based asymmetric catalysis through intramolecular Friedel-Crafts alkylations.

We describe asymmetric intramolecular Friedel-Crafts alkylations with a DNA-based hybrid catalyst and propose a plausible binding model. This study shows promise for studying relationships between the helical chirality of DNA and enantioselectivity of the chemical reaction.

Structural and functional analysis of proteins by high-speed atomic force microscopy.

Proteins are dynamic in nature, work at the single-molecule level, and facilitate several biological functions. The structure of a protein is closely associated with its function; thus, a large number of structural analyses of proteins were performed using techniques such as X-ray crystallography and NMR. Although these methods provide structural information, they often fail because of difficulties in crystallizing the proteins that are complexed with other biomolecules. Moreover, these techniques do not allow the observation of structural changes in the active form of the molecule. Single-molecule fluorescence techniques have been used for the direct observation of protein functions; however, they only reveal the dynamics of individual fluorescent spots, rather than the structural changes that occur over the entire protein. The recent development of high-speed atomic force microscopy (HS-AFM) overcame this problem and allowed the observation of the structural dynamics of proteins and other biomacromolecules directly and in real time. In this chapter, we describe the HS-AFM analysis of the dynamic molecular processes in photoactivated bacteriorhodopsin, membrane-mediated protein-protein interactions, ATP-induced conformational changes in purinergic receptors, the two-dimensional crystal structure of streptavidin, the nature of FtsZ polymers, the role of ClpX in the regulation of FtsZ polymer dynamics, the function of restriction enzymes, the action of motor proteins, the movement of TrCel7A on crystalline cellulose substrates, and the antimicrobial peptide activity on individual bacterial cells.