Publications by authors named "Arivazhagan Rajendran"

33 Publications

Implantable Microfluidic Device: An Epoch of Technology.

Curr Pharm Des 2021 Aug 25. Epub 2021 Aug 25.

School of Biotechnology, National Institute of Technology, Calicut, Kerala, India; b Institute of Advanced Energy, Kyoto University; c RIKEN, Nanomedical Engineering Laboratory. Japan.

Implantable microfluidic devices are milestones in developing devices that can either 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 utilizing on 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 procedure. These devices know no boundaries, and this article reviews these devices based on their design and applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1381612827666210825114403DOI Listing
August 2021

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

Nucleic Acids Res 2021 Aug;49(14):7884-7900

Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkab611DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8373134PMC
August 2021

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

Curr Protoc Nucleic Acid Chem 2019 03 12;76(1):e76. Epub 2019 Feb 12.

Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cpnc.76DOI Listing
March 2019

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

Curr Protoc Nucleic Acid Chem 2019 03 5;76(1):e72. Epub 2019 Feb 5.

Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cpnc.72DOI Listing
March 2019

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

Biochemistry 2018 05 10;57(20):2908-2912. Epub 2018 May 10.

Life Science Center of Tsukuba Advanced Research Alliance , University of Tsukuba , 1-1-1 Tennoudai , Tsukuba , Ibaraki 305-8577 , Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.biochem.8b00202DOI Listing
May 2018

Nucleic-Acid-Templated Enzyme Cascades.

Chembiochem 2017 04 22;18(8):696-716. Epub 2017 Mar 22.

Institute of Advanced Energy, Kyoto University, Uji, Kyoto, 611-0011, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.201600703DOI Listing
April 2017

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

Chem Commun (Camb) 2015 Jun;51(44):9181-4

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c5cc01678aDOI Listing
June 2015

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

Chem Commun (Camb) 2014 Aug 25;50(63):8743-6. Epub 2014 Jun 25.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c4cc02244kDOI Listing
August 2014

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

Angew Chem Int Ed Engl 2014 Apr 12;53(16):4107-12. Epub 2014 Mar 12.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502 (Japan); Current address: Faculty of Medicine and Life Science Center of TARA, University of Tsukuba, 1-1-1 Tennodai, Tsukuba-shi, Ibaraki-ken 305-8577 (Japan).

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201308903DOI Listing
April 2014

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

J Am Chem Soc 2013 Dec 27;135(49):18575-85. Epub 2013 Nov 27.

Department of Chemistry, Graduate School of Science, Kyoto University , Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja409085jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3898174PMC
December 2013

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

Chem Rev 2014 Jan 12;114(2):1493-520. Epub 2013 Nov 12.

Department of Chemistry, Graduate School of Science, Kyoto University , Kitashirakawa-oiwakecho Sakyo-ku, Kyoto 606-8502, Japan.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/cr300253xDOI Listing
January 2014

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

Nucleic Acids Res 2013 Oct 17;41(18):8738-47. Epub 2013 Jul 17.

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 and University of Bordeaux, INSERM, U869, ARNA Laboratory, 2 rue Robert Escarpit, Pessac, F-33607, France.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkt592DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3794576PMC
October 2013

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

J Am Chem Soc 2013 Jan 11;135(3):1117-23. Epub 2013 Jan 11.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja310454kDOI Listing
January 2013

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

Chem Commun (Camb) 2013 Jan;49(7):686-8

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c2cc37257fDOI Listing
January 2013

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

Chem Commun (Camb) 2012 Oct 17;48(84):10398-400. Epub 2012 Sep 17.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c2cc35625bDOI Listing
October 2012

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

Adv Protein Chem Struct Biol 2012 ;87:5-55

Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/B978-0-12-398312-1.00002-0DOI Listing
November 2012

DNA origami: synthesis and self-assembly.

Curr Protoc Nucleic Acid Chem 2012 Mar;Chapter 12:Unit 12.9.1-18

Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/0471142700.nc1209s48DOI Listing
March 2012

Single-molecule analysis using DNA origami.

Angew Chem Int Ed Engl 2012 Jan 25;51(4):874-90. Epub 2011 Nov 25.

Department of Chemistry, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/anie.201102113DOI Listing
January 2012

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

J Am Chem Soc 2011 Sep 29;133(37):14488-91. Epub 2011 Aug 29.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja204546hDOI Listing
September 2011

Two-dimensional DNA origami assemblies using a four-way connector.

Chem Commun (Camb) 2011 Mar 1;47(11):3213-5. Epub 2011 Feb 1.

Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan.

Two-dimensional self-assembly of DNA origami structures was carried out using a connector that has connection sites at all four edges. By utilizing this four-way connector, five and eight origami monomers were assembled to form a cruciate and a hollow square structure, respectively.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c0cc05306fDOI Listing
March 2011

Programmed two-dimensional self-assembly of multiple DNA origami jigsaw pieces.

ACS Nano 2011 Jan 28;5(1):665-71. Epub 2010 Dec 28.

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

We demonstrate a novel strategy of self-assembly to scale up origami structures in two-dimensional (2D) space using multiple origami structures, named "2D DNA jigsaw pieces", with a specially designed shape. For execution of 2D self-assembly along the helical axis (horizontal direction), sequence-programmed tenon and mortise were introduced to promote selective connections via π-stacking interaction, sequence-complementarity, and shape-complementarity. For 2D self-assembly along the helical side (vertical direction), the jigsaw shape-complementarity in the top and bottom edges and the sequence-complementarity of single-stranded overhangs were used. We designed and prepared nine different jigsaw pieces and tried to obtain a 3 × 3 assembly. The proof of concept was obtained by performing the assembly in four different ways. Among them, the stepwise self-assembly from the three vertical trimer assemblies gave the target 2D assembly with ∼35% yield. Finally, the surfaces of jigsaw pieces were decorated with hairpin DNAs to display the letters of the alphabet, and the self-assembled 2D structure displayed the word "DNA JIG SAW" in nanoscale. The method can be expanded to create self-assembled modules carrying various functional molecules for practical applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/nn1031627DOI Listing
January 2011

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

J Am Chem Soc 2010 Oct;132(42):14910-8

Department of Chemistry, Graduate School of Science, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/ja105806uDOI Listing
October 2010

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

Org Biomol Chem 2010 Nov 31;8(21):4949-59. Epub 2010 Aug 31.

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c0ob00057dDOI Listing
November 2010

Molecular crowding of the cosolutes induces an intramolecular i-motif structure of triplet repeat DNA oligomers at neutral pH.

Chem Commun (Camb) 2010 Feb 14;46(8):1299-301. Epub 2010 Jan 14.

Frontier Institute for Biomolecular Engineering Research, Konan University, 7-1-20 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan.

We now present the first example in which triplet repeat DNAs adopt the i-motif structure at neutral pH by molecular crowding. Crowding stabilized the i-motif and the pK(a) of N3 of cytosine was raised in such a microenvironment. Molecular crowding is known to accelerate the formation of the multi-stranded i-motif while the triplet repeats adopt the single-strand structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/b922050jDOI Listing
February 2010

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

Biochim Biophys Acta 2010 Jun 20;1800(6):599-610. Epub 2010 Mar 20.

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2010.03.007DOI Listing
June 2010

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

Chembiochem 2010 Jan;11(1):94-100

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai, Japan.

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).
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/cbic.200900530DOI Listing
January 2010

Effect of methyl substitution in a ligand on the selectivity and binding affinity for a nucleobase: a case study with isoxanthopterin and its derivatives.

Bioorg Med Chem 2009 Jan 31;17(1):351-9. Epub 2008 Oct 31.

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.

Isoxanthopterin (IX) has two edges with hydrogen bond-forming sites suitable for binding to thymine (T) and cytosine (C). The binding affinity of IX for T or C is stronger than for adenine (A) and guanine (G), whereas the base selectivity of IX for T over C (and vice versa) is moderate. In order to improve both the binding affinity and base selectivity for T over C or C over T, a methyl group is introduced respectively at the N-3 or N-8 position of IX. This leads to the known ligands 3-methyl isoxanthopterin (3-MIX) and 8-methyl isoxanthopterin (8-MIX), and the binding affinity for C or T is expected to be tuned and improved by methyl substitution. Indeed, 3-MIX selectively binds to T more strongly than IX with a binding constant of 1.5 x 10(6) M(-1) and it loses its binding affinity for C. In contrast, 8-MIX selectively binds to C over T with a binding constant of 1.0 x 10(6) M(-1) and the binding affinity is greatly improved compared to the parent ligand IX. The thermodynamics of the ligand-nucleotide interaction is analyzed by isothermal calorimetric titrations, and the results show that the interaction follows a 1:1 stoichiometry and is enthalpy-driven. The introduction of methyl groups at both N-3 and N-8 positions results in an increase in enthalpy of the ligand-nucleotide interaction, which leads to the improved binding affinity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bmc.2008.10.062DOI Listing
January 2009

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

Biochim Biophys Acta 2009 Feb 25;1790(2):95-100. Epub 2008 Sep 25.

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2008.09.003DOI Listing
February 2009

A pyrazine-based fluorescence-enhancing ligand with a high selectivity for thymine in AP site-containing DNA duplexes.

Anal Sci 2008 Jun;24(6):693-5

Department of Chemistry, Graduate School of Science, Tohoku University, Aoba, Sendai, Japan.

A fluorescent pyrazine derivative, 3,5-diamino-6-chloro-2-pyrazine carbonitrile (DCPC), is presented as a promising light-up ligand for single-nucleotide polymorphisms (SNPs) typing. In solutions buffered to pH 7.0 (I = 0.11 M, at 5 degrees C), DCPC can bind to thymine selectively over other nucleobases opposite an abasic site in DNA duplexes (5'-GTGTG CGTTG ANA TGGAC GCAGA-3'/3'-CACAC GCAAC TXT ACCTG CGTCT-5', X = abasic site, N = target nucleotide) with a dissociation constant of 2.6 microM. The binding of DCPC is accompanied by a significant enhancement of its fluorescence (lambda(max), 412 nm), and the response is highly selective to thymine base. These binding and sensing properties allow a clear detection of thymine-related mutations present in polymerase chain reaction (PCR) amplification products.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2116/analsci.24.693DOI Listing
June 2008
-->