Structure Of Double-stranded DNA Research Articles

Tying Up Loose Strands: Defining Equations of the Strand Symmetric Model

The strand symmetric model is a phylogenetic model designed to reflect the symmetry inherent in the double-stranded structure of DNA. We show that the set of known phylogenetic invariants for the general strand symmetric model of the three leaf claw tree entirely defines the ideal. This knowledge allows one to determine the vanishing ideal of the general strand symmetric model of any trivalent tree. Our proof of the main result is computational. We use the fact that the Zariski closure of the strand symmetric model is the secant variety of a toric variety to compute the dimension of the variety. We then show that the known equations generate a prime ideal of the correct dimension using elimination theory.

Resonant Infrared Multiple Photon Dissociation Spectroscopy of Anionic Nucleotide Monophosphate Clusters

We report mid-infrared spectra and potential energy surfaces of four anionic, 2'-deoxynucleotide-5'-monophosphates (dNMPs) and the ionic DNA pairs [dGMP-dCMP-H](1-), [dAMP-dTMP-H](1-) with a total charge of the complex equal to -1. We recorded IR action spectra by resonant IR multiple-photon dissociation (IRMPD) using the FELIX free electron laser. The potential energy surface study employed an on-the-fly molecular dynamics quenching method (MD/Q), using a semiempirical AM1 method, followed by an optimization of the most stable structures using density functional theory. By employing infrared multiple-photon dissociation (IRMPD) spectroscopy in combination with high-level computational methods, we aim at a better understanding of the hydrogen bonding competition between the phosphate moieties and the nucleobases. We find that, unlike in multimer double stranded DNA structures, the hydrogen bonds in these isolated nucleotide pairs are predominantly formed between the phosphate groups. This intermolecular interaction appears to exceed the stabilization energy resulting from base pairing and directs the overall cluster structure and alignment.

Hyperbranched Hybridization Chain Reaction for Triggered Signal Amplification and Concatenated Logic Circuits

A hyper-branched hybridization chain reaction (HB-HCR) is presented herein, which consists of only six species that can metastably coexist until the introduction of an initiator DNA to trigger a cascade of hybridization events, leading to the self-sustained assembly of hyper-branched and nicked double-stranded DNA structures. The system can readily achieve ultrasensitive detection of target DNA. Moreover, the HB-HCR principle is successfully applied to construct three-input concatenated logic circuits with excellent specificity and extended to design a security-mimicking keypad lock system. Significantly, the HB-HCR-based keypad lock can alarm immediately if the "password" is incorrect. Overall, the proposed HB-HCR with high amplification efficiency is simple, homogeneous, fast, robust, and low-cost, and holds great promise in the development of biosensing, in the programmable assembly of DNA architectures, and in molecular logic operations.

Identification of a pKa-regulating motif stabilizing imidazole-modified double-stranded DNA.

The predictable 3D structure of double-stranded DNA renders it ideally suited as a template for the bottom-up design of functionalized nucleic acid-based active sites. We here explore the use of a 14mer DNA duplex as a scaffold for the precise and predictable positioning of catalytic functionalities. Given the ubiquitous participation of the histidine-based imidazole group in protein recognition and catalysis events, single histidine-like modified duplexes were investigated. Tethering histamine to the C5 of the thymine base via an amide bond, allows the flexible positioning of the imidazole function in the major groove. The mutual interactions between the imidazole and the duplex and its influence on the imidazolium pKaH are investigated by placing a single modified thymine at four different positions in the center of the 14mer double helix. Using NMR and unrestrained molecular dynamics, a structural motif involving the formation of a hydrogen bond between the imidazole and the Hoogsteen side of the guanine bases of two neighboring GC base pairs is established. The motif contributes to a stabilization against thermal melting of 6°C and is key in modulating the pKaH of the imidazolium group. The general features, prerequisites and generic character of the new pKaH-regulating motif are described.

Enzyme Engineering via Rationally Designed Intermolecular Interactions: Applications towards Bioelectrocatalysis

In nature we find many examples of enzymes and multienzyme stuctures where catalysis is enhanced by well-designed molecular interactions between the enzymes and their substrates. Two compelling examples are the enzyme superoxide dismutase (SOD) and the bifunctional enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR). SOD, one of the fastest known enzymes (kcat » 1.5 ´ 109 M-1s-1), uses charge complementarity to produce substrate-enzyme interactions that enhance enzyme kinetics by directing the substrate to the enzyme’s active site. A positively charge patch on the surface TS-DHFR restricts diffusion of a negatively charged reaction intermediate to a pre-defined channel between two active sites, sequestering the intermediate along the enzyme’s surface and preventing diffusion to the bulk. This bounded diffusion promotes substrate channeling, enhancing pathway catalysis by protecting the intermediate from undesired side reactions. These examples are informative: They suggest that rational design of substrate-enzyme interactions can be used to enhance enzyme catalysis. In this work we engineer new enzyme structures with quantifiable binding interactions between the enzyme and its substrate. We hypothesize that the engineered molecular interactions will lead to increases in local substrate concentrations thereby enhancing enzyme catalysis. We confirm this hypothesis by demonstrating control over the apparent Michaelis constant (KM) of horseradish peroxidase (HRP) modified with a double stranded DNA structure that exhibits sequence dependent binding of phenolic HRP substrates. We extend this work to a second experimental system and demonstrate enhanced catalysis with an alcohol dehydrogenase (AdhD) through rationally designed molecular interactions between the NAD+ co-factor mimic nicotinamide mononucleotide (NMN+) and a double stranded DNA structure conjugated near the enzyme’s active site. Demonstration of rationally designed kinetic enhancements with enzyme co-factors such as NMN+ is an important demonstration with respect to bioelectrocatalysis as many such systems rely on this type of co-factor. We aim to extend this work towards other co-factor and substrates relevant to electrochemical biosensor and enzymatic biofuel cells.

Metal–organic framework MIL-101 as a low background signal platform for label-free DNA detection

A label-free and sensitive fluorescence method for recognition of sequence-specific DNA using DNA-intercalating dye and metal-organic frameworks (MOFs) is developed. Here, MIL-101 (Cr3F(H2O)2O[(O2C)-C6H4-(CO2)]3·nH2O) is introduced as a quenching platform to decrease the high background fluorescence of SYBR GreenI (SG)/probe DNA complex. Mechanism investigations show that MIL-101 can strongly adsorb the SG/probe DNA complex through π-π stacking and electrostatic interactions, and as a consequence, the fluorescence of the SG dye is greatly quenched. While in the presence of target DNA, the as-formed rigid double-stranded (ds) structure of DNA will be far away from the surface of MIL-101; meanwhile, the SG dye can be bound with the dsDNA in the mode of intercalation and minor groove binding, resulting in enhancement of the SG dye fluorescence. The increased signal-to-background ratio has a linear relationship with the concentration of target DNA in the range of 0.1-14 nM. It is confirmed that the detection limit is 73 pM (3σ), which is much lower than that based on the carbon nanotubes and graphene oxide platform. Moreover, one-base-mismatched target DNA can be discriminated effectively. With the introduction of MIL-101, the signal-to-background ratio has been improved ∼8-fold, demonstrating that MIL-101 is an efficient low-background signal platform.

Effect of Pressure on Thermal Stability of G-Quadruplex DNA and Double-Stranded DNA Structures

Pressure is a thermodynamic parameter that can induce structural changes in biomolecules due to a volumetric decrease. Although most proteins are denatured by pressure over 100 MPa because they have the large cavities inside their structures, the double-stranded structure of DNA is stabilized or destabilized only marginally depending on the sequence and salt conditions. The thermal stability of the G-quadruplex DNA structure, an important non-canonical structure that likely impacts gene expression in cells, remarkably decreases with increasing pressure. Volumetric analysis revealed that human telomeric DNA changed by more than 50 cm3 mol−1 during the transition from a random coil to a quadruplex form. This value is approximately ten times larger than that for duplex DNA under similar conditions. The volumetric analysis also suggested that the formation of G-quadruplex DNA involves significant hydration changes. The presence of a cosolute such as poly(ethylene glycol) largely repressed the pressure effect on the stability of G-quadruplex due to alteration in stabilities of the interactions with hydrating water. This review discusses the importance of local perturbations of pressure on DNA structures involved in regulation of gene expression and highlights the potential for application of high-pressure chemistry in nucleic acid-based nanotechnology.

Quenching the Chemiluminescence of Acridinium Ester by Graphene Oxide for Label-Free and Homogeneous DNA Detection

It was found that graphene oxide (GO) could effectively quench the chemiluminescence (CL) emission from a acridinium ester (AE)-hydrogen peroxide system. By taking advantage of this quenching effect, as a proof of concept, a label-free and homogeneous DNA assay was developed for the detection of Mycobacterium tuberculosis DNA. In the absence of target DNA, both probe DNA and AE were absorbed on the surface of GO, producing a weak CL emission owing to the CL quenching effect of GO. However, in the presence of target DNA, a double-stranded structure of DNA was generated, leading to the release of the oligonucleotide from the GO surface. AE favors binding with double-stranded DNA, which will be released from the GO surface; thus, the quenching effect of GO will be no longer effective and a strong CL signal can be observed. This assay can detect M. tuberculosis DNA with a detection limit of 0.65 nM. This sensitivity is lower than that of previously reported electrochemical detection.

Dynamical Treatment of Charge Transfer through Duplex Nucleic Acids Containing Modified Adenines

We address the issue of whether chemical alterations of nucleobases are an effective tool to modulate charge transfer through DNA molecules. Our investigation uses a multilevel computational approach based on classical molecular dynamics and quantum chemistry. We find yet another piece of evidence that structural fluctuations are a key factor to determine the electronic structure of double-stranded DNA. We argue that the electronic structure and charge transfer ability of flexible polymers is the result of a complex intertwining of various structural, dynamical and chemical factors. Chemical intuition may be used to design molecular wires, but this is not the sole component in the complex charge transfer mechanism through DNA.

A Targeted, Self-Delivered, and Photocontrolled Molecular Beacon for mRNA Detection in Living Cells

The spatiotemporal dynamics of specific mRNA molecules are difficult to image and detect inside living cells, and this has been a significant challenge for the chemical and biomedical communities. To solve this problem, we have developed a targeted, self-delivered, and photocontrolled aptamer-based molecular beacon (MB) for intracellular mRNA analysis. An internalizing aptamer connected via a double-stranded DNA structure was used as a carrier probe (CP) for cell-specific delivery of the MB designed to signal target mRNA. A light activation strategy was employed by inserting two photolabile groups in the CP sequence, enabling control over the MB's intracellular function. After the probe was guided to the target cell via specific binding of aptamer AS1411 to nucleolin on the cell membrane, light illumination released the MB for mRNA monitoring. Consequently, the MB is able to perform live-cell mRNA imaging with precise spatiotemporal control, while the CP acts as both a tracer for intracellular distribution of the MB before photoinitiation and an internal reference for mRNA ratiometric detection.

Structure of a double-stranded DNA (6–4) photoproduct in complex with the 64M-5 antibody Fab

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation have been implicated in mutagenesis and cancer. The crystal structure of double-stranded DNA containing the (6-4) photoproduct in complex with the anti-(6-4)-photoproduct antibody 64M-5 Fab was determined at 2.5 Å resolution. The T(6-4)T segment and the 5'-side adjacent adenosine are flipped out of the duplex and are accommodated in the concave antigen-binding pocket composed of six complementarity-determining regions (CDRs). A loop comprised of CDR L1 residues is inserted between the flipped-out T(6-4)T segment and the complementary DNA. The separation of strands by the insertion of the loop facilitates extensive and specific recognition of the photoproduct. The DNA helices flanking the T(6-4)T segment are kinked by 87°. The 64M-5 Fab recognizes the T(6-4)T segment dissociated from the complementary strand, indicating that the (6-4) photoproduct can be detected in double-stranded DNA as well as in single-stranded DNA using the 64M-5 antibody. The structure and recognition mode of the 64M-5 antibody were compared with those of the DNA (6-4) photolyase and nucleotide-excision repair protein DDB1-DDB2. These proteins have distinctive binding-site structures that are appropriate for their functions, and the flipping out of the photolesion and the kinking of the DNA are common to mutagenic (6-4) photoproducts recognized by proteins.

Biophysical Modeling of Nucleic Acid Nanostructure Solution Shape and Stability

Programmed self-assembly of RNA and DNA provides a powerful approach to designing active, functional nanometer-scale structures for biomolecular science and technology. Scaffolded DNA Origami employs a long single-stranded DNA template to guide the hybridization of hundreds of shorter oligonucleotide staple strands to form twisted and bent double-stranded DNA structures of high precision and yield with well-defined mechanical properties. Biophysical models of nanostructure formation and stability are needed to inform the DNA Origami design process, which currently proceeds largely based on physical intuition and trial-and-error. Here we present a physics-based model for DNA Origami that incorporates double-stranded DNA mechanical properties in addition to screened electrostatic interactions on 3D solution shape and flexibility. Results are presented in detail for an eight-layer DNA Origami block designed on a square lattice (Ke et al., JACS 2009), whose 3D solution shape is computed under experimental folding conditions, revealing an undulating pattern of individual DNA double helices due to interhelix electrostatic repulsion. Effects of crossover spacing on solution shape and mechanical properties are also discussed.

Preparation of DNA–polyintercalator conjugate and its functional property

Psoralen immobilized polyvinyl alcohol (PVA-P) was synthesized from chloromethylmethoxypsoralen and polyvinyl alcohol. The psoralen part of PVA-P intercalated into the double-stranded DNA and formed covalent bonding between the psoralen and nucleic acid base after 365nm UV irradiation. As a result, DNA and PVA-P produced a water-insoluble conjugate. This DNA–PVA-P conjugate maintained the double-stranded structure of DNA and possessed the DNA's property, such as intercalation. Therefore, the DNA–PVA-P conjugate selectively accumulated the planar-structure containing chemical compounds, such as biphenyl and dibenzofuran, from an aqueous multi-component solution. These DNA–PVA-P conjugates may have the potential to be utilized as a separation material for the selective removal of harmful compounds.

ChemInform Abstract: Methods for Investigating G‐Quadruplex DNA/Ligand Interactions

AbstractReview: 58 refs.

Demonstration that Drug-targeted Down-regulation of MYC in Non-Hodgkins Lymphoma Is Directly Mediated through the Promoter G-quadruplex

Most transcription of the MYC proto-oncogene initiates in the near upstream promoter, within which lies the nuclease hypersensitive element (NHE) III(1) region containing the CT-element. This dynamic stretch of DNA can form at least three different topologies: single-stranded DNA, double-stranded DNA, or higher order secondary structures that silence transcription. In the current report, we identify the ellipticine analog GQC-05 (NSC338258) as a high affinity, potent, and selective stabilizer of the MYC G-quadruplex (G4). In cells, GQC-05 induced cytotoxicity with corresponding decreased MYC mRNA and altered protein binding to the NHE III(1) region, in agreement with a G4 stabilizing compound. We further describe a unique feature of the Burkitt's lymphoma cell line CA46 that allowed us to clearly demonstrate the mechanism and location of action of GQC-05 within this region of DNA and through the G4. Most importantly, these data present, as far as we are aware, the most direct evidence of intracellular G4-mediated control of a particular promoter.

Enhanced gene expression promoted by the quantized folding of pDNA within polyplex micelles

Selective packaging of plasmid DNA (pDNA) into folded rod or collapsed sphere structures in polyplex micelles was demonstrated by modulating the PLys segment length of poly(ethylene glycol)- block- poly( l-lysine) (PEG-PLys) block catiomers used for micelle formation. The two basic packaging structures correlated well to the integrity of double-stranded DNA contained within the micelles. Rod structures formed by the quantized folding mechanism, which results in dissociation of double-stranded DNA only at each fold. Collapsed sphere structures formed by substantial random disruption of the double-stranded DNA structure. Analysis of gene expression in a cell-free transcription/translation system, cultured cells and also skeletal muscle of mice showed that micelles containing pDNA packaged by quantized folding exhibited higher gene expression than naked pDNA and micelles containing collapsed pDNA. These results indicate that controlled packaging of pDNA into an appropriate structure is critical for achieving effective gene expression. Improved gene transfection and expression resulting from the quantized folding of pDNA within polyplex micelles is promising for application in therapeutic gene delivery systems.

On the Structure and Stretching of Microhydrated DNA

The structure of short double-stranded DNA oligomers in complex with varied amounts of water is investigated with classical molecular dynamics. Free simulations are performed first and mechanical stress is switched on afterward, resembling the conditions of single-molecule conductivity experiments. Water as well as counterions are seen to contribute to the stabilization of double-stranded DNA structure, and a collapse of the native DNA structure is observed upon the removal of a certain amount of water. Pulling with a moderate external force provides additional support to the double-stranded DNA structure, whereas larger forces lead to the overstretching transition followed by the separation of DNA strands, in a manner similar to that observed in fully hydrated DNA.

Aptamer-quantum dots conjugates-based ultrasensitive competitive electrochemical cytosensor for the detection of tumor cell

A novel competitive electrochemical cytosensor was reported by using aptamer (Apt)-quantum dots (Qdots) conjugates as a platform for tumor cell recognition and detection. The complementary DNA (cDNA), aptamer and Qdots could be assembled to the gold electrode surface. When the target cells existed, they could compete with cDNA to bind with Apt-Qdots conjugates based on the specific recognition of aptamer to MUC1 protein overexpressed on the cell surface, which resulted in the denaturation of double-stranded DNA structure and the release of the Apt-Qdots conjugates from the electrode. Electrochemical stripping measurement was then employed to determine the Cd2+ concentration in Qdots left at the electrode. The peak current was inversely proportional to the logarithmic value of cell concentration ranging from 1.0×102 to 1.0×106cellsmL−1 with a detection limit of 100cellsmL−1. Meanwhile, the recognition of aptamer to the target cells could be clearly observed through the strong fluorescence from Qdots. This is an example of the combination of aptamer and nanoparticles for the application of cell analysis, which is essential to cancer diagnosis and therapy.

Secondary structure of double-stranded DNA under stretching: Elucidation of the stretched form

Almost two decades ago, measurements of force versus extension on isolated double-stranded DNA molecules revealed a force plateau. This unusual stretching phenomenon in DNA suggests that the long molecules may be extended from the usual B form into a new conformation. Different models have been proposed to describe the nature of DNA in its stretched form, S-DNA. Using atomic force microscopy combined with a molecular combing method, we identified the structure of λ-phage DNA for different stretching values. We provide strong evidence for the existence of a first-order transition between B form and S form. Beyond a certain extension of the natural length, DNA molecules adopt a new double-helix conformation characterized by a diameter of 1.2 nm and a helical pitch of 18 nm.

Nanomechanics of Canonical Double Stranded DNA Structures Probed with Single-Molecule Force Spectroscopy

Elasticity is a key property of DNA. Throughout the cell cycle, DNA is subjected to mechanical loadings that deform its original structure. Beyond the biological significance of DNA elasticity, in DNA nanotechnology, a solid understanding of DNA reaction to force is necessary to create functional nanostructures. We characterize the elastic profile of different canonical DNA double helical structures, specifically the A and Z helices, by stretching with atomic force microscope (AFM) based single-molecule force spectroscopy.