Arivazhagan Rajendran

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.

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.

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.

State-of-the-art high-speed atomic force microscopy for investigation of single-molecular dynamics of proteins.

Investigation of Single-Molecular Dynamics of Proteins Arivazhagan Rajendran,†,⊥ Masayuki Endo,*,‡,§ and Hiroshi Sugiyama*,†,‡,§ †Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho Sakyo-ku, Kyoto 606-8502, Japan ‡Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan CREST, Japan Science and Technology Corporation (JST), Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

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.

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.

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.

G-quadruplex-binding ligand-induced DNA synapsis inside a DNA origami frame

Among the approaches for DNA-based drug targeting, G-quadruplex-binding ligands are of particular interest because of the high abundance of G-rich sequences in regions such as human chromosomal telomeres and promoters of several proto-oncogenes. A number of quadruplex-ligands have been reported, but their functions at single-molecule level have not been explored using direct and real-time methods. Here, we report on the direct observation of the formation of a four-stranded G-quadruplex induced by bisquinolinium pyridine dicarboxamide with a linker containing biotin at one end. We fabricated a DNA origami frame with incorporated duplex DNAs that contained 3–6 G–G mismatches in the middle. In the absence of ligand, the duplex DNAs of interest had no interaction, as visualized by their parallel-shape in high-speed atomic force microscopy (HS-AFM) images. Presence of ligand induced the formation of G-quadruplex structure, which was characterized by an X-shape. Addition of streptavidin to the ligand-induced quadruplex caused the protein to localize in the middle of the X-shape, indicating that the ligand is bound to the quadruplex. A sequence of real-time images of the ligand-induced formation of a quadruplex and its reverse conformational switching by removing the ligand was captured by HS-AFM. Unprecedented intermediate-like states were recorded in our real-time analysis.

DNA Origami: Synthesis and Self‐Assembly

DNA origami is an emerging technology for designing defined two‐ and three‐dimensional (2D and 3D) DNA nanostructures. Here, we report an introductory practical guide with step‐by‐step experimental details for the design and synthesis of origami structures, and their size expansion in 1D and 2D space by means of self‐assembly. Curr. Protoc. Nucleic Acid Chem. 48:12.9.1‐12.9.18.

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 natures 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.