Structure Of DNA Duplex Research Articles

Unveiling the complex pattern of intermolecular interactions responsible for the stability of the DNA duplex.

Herein, we provide new insights into the intermolecular interactions responsible for the intrinsic stability of the duplex structure of a large portion of human B-DNA by using advanced quantum mechanical methods. Our results indicate that (i) the effect of non-neighboring bases on the inter-strand interaction is negligibly small, (ii) London dispersion effects are essential for the stability of the duplex structure, (iii) the largest contribution to the stability of the duplex structure is the Watson–Crick base pairing – consistent with previous computational investigations, (iv) the effect of stacking between adjacent bases is relatively small but still essential for the duplex structure stability and (v) there are no cooperativity effects between intra-strand stacking and inter-strand base pairing interactions. These results are consistent with atomic force microscope measurements and provide the first theoretical validation of nearest neighbor approaches for predicting thermodynamic data of arbitrary DNA sequences.

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure.

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity.

Microsecond Equilibrium Dynamics of Hairpin-Forming Oligonucleotides Quantified by Two-Color Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy.

RNA and DNA play distinct roles in biological systems. However, the underlying physicochemical difference has been poorly understood, in particular, that in dynamical aspects. In this paper, we report on a comparative study of the formation-dissociation dynamics of a hairpin structure of RNA and DNA with development of two-color two-dimensional fluorescence lifetime correlation spectroscopy (two-color 2D FLCS). In this extension of 2D FLCS, we newly introduce the two-color detection scheme to analyze not only donor fluorescence photons but also acceptor fluorescence photons from a doubly labeled Förster resonance energy transfer (FRET) pair. This new 2D FLCS is utilized to resolve multiple species present in an equilibrated condition with a microsecond time resolution and enhanced sensitivity, and the combined use with the filtered fluorescence correlation spectroscopy (FCS) method enables a quantitative discussion on microsecond structural dynamics occurring in the equilibrium. This integrated approach is applied to FRET-labeled RNA/DNA oligonucleotides having analogous hairpin-forming sequences, and it was revealed that the hairpin dissociation rate of RNA is an order of magnitude slower than that of DNA while their hairpin-forming rates are comparable. This marked difference is attributable to the distinct duplex structure of RNA and DNA. The present study demonstrates that the integrated approach combining two-color 2D FLCS and filtered FCS has a high potential for quantifying microsecond kinetics at the single-molecule level, which allows us to experimentally construct a free energy landscape.

Dynamic Structure and Stability of DNA Duplexes Bearing a Dinuclear Hg(II)-Mediated Base Pair.

Quantum mechanical (QM) and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of a recently reported dinuclear mercury(II)-mediated base pair were performed aiming to analyse its intramolecular bonding pattern, its stability, and to obtain clues on the mechanism of the incorporation of mercury(II) into the DNA. The dynamic distance constraint was employed to find initial structures, control the dissociation process in an unbiased fashion and to determine the free energy required. A strong influence of the exocyclic carbonyl or amino groups of neighbouring base pairs on both the bonding pattern and the mechanism of incorporation was observed. During the dissociation simulation, an amino group of an adenine moiety of the adjacent base pair acts as a turnstile to rotate the mercury(II) ion out of the DNA core region. The calculations provide an important insight into the mechanism of formation of this dinuclear metal-mediated base pair and indicate that the exact location of a transition metal ion in a metal-mediated base pair may be more ambiguous than derived from simple model building.

Uniform palladium nanosheets for fluorimetric detection of circulating tumor DNA

Fluorescence quenching property of two-dimensional (2D) nanosheets (NSs) have received extensively attention in the construction of novel biosensing platform. However, the heterogeneity of the wide-size distribution and inefficient fluorescence quenching capacity limit its wide practical applications. Herein, for the first time, we report a novel fluorescent biosensor based on uniform palladium NSs (Pd NSs) with excellent fluorescence quenching efficiency and differential affinity toward ssDNA versus dsDNA and combination with a pair of DNA detection probes with fluorophore for detecting circulating tumor DNA (ctDNA). The DNA detection probes are facilitated to adsorbed to the surface of Pd NSs, leading to efficient fluorescence quench. In the presence of target DNA, it can be linked by T4 DNA ligase to form long DNA duplex structures, which display weak affinity toward Pd NSs, producing the fluorescence recovery. The remarkable fluorescence quenching efficiency and ssDNA/dsDNA differential affinity of Pd NSs make it have a good detection ability without signal amplification. The result indicates that this facile but cost-effective strategy holds great promise in bioanalysis.

Novel multiple strand displacement reaction coupled hybridization chain reaction for label-free and ultrasensitive electrochemical Type b3a2 biosensing

Herein, a novel simple multiple strand displacement reaction (MSDR) coupled hybridization strand reaction (HCR) was proposed for label-free and ultrasensitive Type b3a2 assay. First, two kinds of messenger DNAs (mDNAs) were aligned on a long substrate strand DNA, which was immobilized on magnetic beads. Unlike traditional SDR, in the presence of target and a hairpin DNA, MSDR would be initiated and achieve target-recycling as well as the release of two mDNAs. Finally, one target input can convert into multiple mDNAs, which greatly increased the utility ratio of target. After magnetic separation, the released mDNAs can hybridize with the hairpin DNA immobilized on the electrode to trigger HCR and form a linear duplex-DNA structure. It can be used as an excellent template to in-situ grown silver nanoparticles (AgNPs) to generate electrochemical response directly. By taking advantage of MSDR as well as HCR, the electrochemical signal was amplified significantly. Under optimal conditions, this developed biosensor achieved a low detection limit of 0.56 fM (S/N = 3) and a wide linear range from 10 fM to 10 nM for sensitive detection of Type b3a2 with good selectivity and stability.

Long-Range Ordered Water Correlations between A–T/C–G Nucleotides

Summary The interactions between complementary base pairs are crucial to the helical duplex structure of DNA. Although base-base interactions have been widely reported, the study of long-range interactions in the base-pairing processes is still a major challenge in this field. Here, we first study the long-range interactions between the base pairs by atomic force microscopy-based single-molecule force spectroscopy (SMFS). The SMFS results of A–T/C–G imply that there are weak long-range interactions between them, which have a range of 15–25 nm. Raman spectroscopy results imply that these weak interactions can be attributed to multiplex hydrogen bond interaction of ordered water structure between A–T/C–G nucleotides. In addition, the theoretical calculations deduce that the ideal structure of these water molecules exhibits a specific helical structure. Our findings might open up a new understanding of biological assembly processes and provide helpful strategies for bio-nanotechnology.

A poly(thymine)-melamine duplex for the assembly of DNA nanomaterials.

The diversity of DNA duplex structures is limited by a binary pair of hydrogen-bonded motifs. Here we show that poly(thymine) self-associates into antiparallel, right-handed duplexes in the presence of melamine, a small molecule that presents a triplicate set of the hydrogen-bonding face of adenine. X-ray crystallography shows that in the complex two poly(thymine) strands wrap around a helical column of melamine, which hydrogen bonds to thymine residues on two of its three faces. The mechanical strength of the thymine-melamine-thymine triplet surpasses that of adenine-thymine base pairs, which enables a sensitive detection of melamine at 3 pM. The poly(thymine)-melamine duplex is orthogonal to native DNA base pairing and can undergo strand displacement without the need for overhangs. Its incorporation into two-dimensional grids and hybrid DNA-small-molecule polymers highlights the poly(thymine)-melamine duplex as an additional tool for DNA nanotechnology.

Dual-output toehold-mediated strand displacement amplification for sensitive homogeneous electrochemical detection of specie-specific DNA sequences for species identification

The determination of specie-specific DNA sequences is a key factor for identification of animal species and detection of meat adulteration. Herein, a simple homogeneous electrochemical biosensor was developed for sensitive detection of specie-specific DNA sequences from meat products based on high efficient and specific dual-output toehold-mediated strand displacement (TMSD). After incubation with target DNA, large amount of methylene blue (electrochemical signal molecule) labeled probes (MB-P) were released from preformed DNA duplex structures by the process of dual-output TMSD amplification. The free MB-P could be further digested by Exonuclease I, and the enzymatic products contain little negative charge could diffuse to the surface of indium tin oxide electrode, generating significantly electrochemical signal. As a result, the designed biosensor showed a broad dynamic range from 0.01 pM to 100 pM, with a low detection limit of 8.2 fM, and ideal selectivity and reproducibility. Meanwhile, the approach exhibited acceptable accuracy for the detection of specie-specific DNA sequences, and possessed the potential application for the identification of animal species from meat products.

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

Life under Extreme Conditions: Aeropyrum pernix and Pernisine

2'-Alkynyl spin-labelling is a minimally perturbing tool for DNA structural analysis.

The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.

Opposite Effects of High-Valent Cations on the Elasticities of DNA and RNA Duplexes Revealed by Magnetic Tweezers.

We report that trivalent cobalt hexammine cations decrease the persistence length, stretching modulus, helical density, and size of plectonemes formed under torque of DNA but increase those of RNA. Divalent magnesium cations, however, decrease the persistence lengths, contour lengths, and sizes of plectonemes while increasing the helical densities of both DNA and RNA. The experimental results are explained by different binding modes of the cations on DNA and RNA in our all-atom molecular dynamics simulations. The significant variations of the helical densities and structures of DNA and RNA duplexes induced by high-valent cations may affect interactions of the duplexes with proteins.

The cooperative interaction of triplex forming oligonucleotides on DNA-triplex formation at electrode surface: Molecular dynamics studies and experimental evidences

An extensive study on cooperative interaction of Triplex Forming Oligonucleotides (TFOs) with a double strand DNA, to form a triplex-DNA structure at electrode surface, is here reported. The cooperative effect on triplex structure formation was assumed by the higher binding enthalpy value, calculated for the interaction between the duplex DNA structure and the TFO1 and TFO2 probes (-67.3 KJ/mol), respect the sum of the single duplex-TFO1 and duplex-TFO2 interactions (-47.0 kJ/mol). The formation of triplex-DNA structure was proven by kinetic modelling study performed using the Luzar and Chandler model. The results indicate that after 500 ns from interaction, H-bonds between the base pairs in the double strand DNA are weaken while new H-bonds between the TFOs and duplex DNA are formed. Molecular dynamic (MD) simulations indicate that the TFOs sequence distance (138 bps) and the amount of TA*T triplet units are the keystones for the effectiveness of the cooperative effect, reaching for the selected target a minimum of energy value of –19452.6 kJ/mol. The MD data were experimentally corroborated by electrochemical measurements, detecting a HBV-clone genome at TFOs modified electrode surface. The interaction was electrochemical transduced by an intercalative Osmium based compound. The Langmuir isotherm model reports for the forming triplex DNA an association constant value of about 2.9 × 1016M-1, this high value could be attributed to the synergic contribution of the TFOs cooperative effect and to the rigid circular duplex structure. Finally, AFM and SEM investigations suggest the formation of a triplex-DNA structure at electrode surface, consisting in circles of about 1.5 um in diameter with asymmetric stranded thickness. This finding data paving the way to future development of advanced miniaturized DNA computing and biosensors.

Donor/Acceptor-Induced Ratiometric Photoelectrochemical Paper Analytical Device with a Hollow Double-Hydrophilic-Walls Channel for microRNA Quantification.

Integrating ratiometric photoelectrochemical (PEC) techniques with paper microfluidics to construct a ratiometric PEC paper analytical device for practical application is often restricted by the grave dependence of ratiometric assay on photoactive materials and low mass-transfer rates of the paper channel. Herein, a universal donor/acceptor-induced ratiometric PEC paper analytical device with a hollow double-hydrophilic-walls channel (HDHC) was fabricated for high-performance microRNA-141 (miRNA-141) quantification. Concretely, a photoanode and photocathode were integrated on the paper-based sensing platform in which the photocathode served as a biosensing site for the pursuit of higher selectivity. For formulation of a cascading signal amplification strategy, a unique duplex-specific nuclease-induced target recycling reaction was engineered for the output of a double amount of all useful DNA linkers instead of conventional output of only one available DNA product, which could guarantee the output of abundant DNA linkers with the initiation of a cascade of hybridization chain reaction on both the trunk and branch in the presence of miRNA-141. Then the formed dendriform polymeric DNA duplex structures were further decorated with glucose oxidase (GOx)-mimicking gold nanoparticles by the electrostatic interaction to form a branchy gold tree (BGT). Profiting from the perfect GOx-mimicking activity of BGT and high mass-transfer rates of HDHC, the cathodic photocurrent from Ag2S/Cu2O hybrid structure was in a "signal off" state while the anodic photocurrent from graphene quantum dots (GQDs) and Ag2Se QDs cosensitized ZnO nanosheets was in a "signal on" state because BGT-catalyzed glucose oxidation reaction evoked the consumption of dissolved O2 as an electron acceptor and the generation of H2O2 as an electron donor. With calculation of the ratio of two photocurrent intensities, the quantitative detection of miRNA-141 was achieved with high sensitivity, accuracy, and reliability.

Electrochemiluminescence biosensor for miRNA-21 based on toehold-mediated strand displacement amplification with Ru(phen)32+ loaded DNA nanoclews as signal tags

A novel electrochemiluminescence (ECL) biosensor was developed for high sensitive and selective detection of miRNA-21 based on the efficient and specific toehold-mediated strand displacement (TMSD) amplification with Ru(phen)32+ loaded DNA nanoclews (NCs–Ru(phen)32+) as signal tags. The stable DNA nanoclews, synthesized by a simple rolling circle amplification reaction, were employed to load with Ru(phen)32+ efficiently as ECL signal tags to amplify the signals. As for TMSD, the substrate strand (Sub) was initially hybridized with P1 and P2 to form DNA duplex structures with a toehold 1. miRNA-21 could hybridize with the toehold 1 and trigger the TMSD amplification with the help of assist strand, releasing lots of P1 stands from DNA duplex structures. The TMSD technique realized the converting and amplification of the single miRNA-21 input to lots of output DNA (namely P1) with good selectivity, simultaneously. Output P1 were designed to expand the stem-locked region of HP, which were immobilized on the Au electrodes firstly. Subsequently, the opened HP could hybridize with the Ru(phen)32+, capturing the ECL signal tags closed to the sensing surface. The ECL intensity of the system had a linear relationship with the logarithm of the miRNA-21 concentration in the range of 1.0 fM to 100 pM with a limit of detection of 0.65 fM. The strategy was further applied to detect miRNA-21 in complex samples, and the result was consistent with the qRT-PCR.

Mechanism of stimulation of DNA binding of the transcription factors by human apurinic/apyrimidinic endonuclease 1, APE1

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state.

Stimuli‐Responsive DNA‐Gated Nanoscale Porous Carbon Derived from ZIF‐8

AbstractStimuli‐responsive nanoscale porous carbon derived from ZIF‐8 (NCZIF) gated by DNA capping units is reported. The NCZIF is first obtained by calcination of nano‐ZIF‐8 crystals under an inert atmosphere. It is further conjugated with amine‐modified single‐stranded DNA after carboxylation (DNA/NCZIF). The guest molecules are sealed in the pore of NCZIF by the formation of a DNA duplex structure on the surface of NCZIF. As proof of principle, two systems that can be, respectively, used for controlled drug delivery and biosensing are introduced. In the first system, the drug model (rhodamine 6G, Rh6G) is locked in the NCZIF by the DNA capping units composed of rich‐G sequences and its complementary DNA strand. The in vitro cellular experiments reveal that DNA/NCZIF has good biocompatibility and can controllably release Rh6G upon the K+‐stimuli in cells. In the second system, the signal probe (methylene blue, MB) is locked in the NCZIF and then released after the unlocking of the pores triggered by the dissociation of the aptamer‐hybrid capping units. The MB‐loaded DNA/NCZIF can linearly respond to target molecules in the range from 1 × 10−9 to 10 × 10−6 m and has good specificity.

A rapid and simple ratiometric fluorescent sensor for patulin detection based on a stabilized DNA duplex probe containing less amount of aptamer-involved base pairs

In this study, a sensor is described for determination of patulin by using ratiometric fluorescence measurement and strand displacement strategy. In the presence of patulin, the ratiometric fluorescence response decreases, owing to disassembly of DNA duplex structure and target-mediated release of TAMRA-labeled complementary DNA sequence2 (cDNA2). While, in the absence of target, the fluorescence resonance energy transfer (FRET) phenomenon happens between FAM and TAMRA under excitation at 490 nm, resulting in the enhancement of ratiometric signal. The use of ratiometric fluorescence signal with different signal indicators avoids the problem of environmental interference and improves the sensitivity of the aptasensor. Also, the DNA duplex structure contains minimum aptamer-involved base pair sequence, resulting in further improvement of the aptasensor sensitivity. This sensing platform provided a wide linear range from 15 ng/L to 35 μg/L and a detection limit of 6 ng/L for patulin. The aptasensor was used to determine patulin in spiked apple juice samples and showed satisfactory results.

Cationic comb-type copolymer as an artificial chaperone

Accurate folding of biopolymers and assembly of complexes containing these molecules are essential for biological activities. We designed cationic comb-type copolymers that act as an artificial chaperone to assist in the folding of biopolymers such as nucleic acids and peptides. The copolymers consist of a polycation backbone grafted with a high density of hydrophilic chains that allow the formation of soluble and soft interpolyelectrolyte complexes between the copolymer and oppositely charged biopolymers. The copolymers can chaperone DNA assembly into duplex, triplex, and quadruplex structures and accelerate strand exchange reactions while stabilizing DNA complexes. This chaperone activity of the copolymer is useful in nucleic acid-based nanotechnological applications such as DNA nanomachines, DNA logic gates, and DNA enzymes. The cationic comb-type copolymers also assisted in the folding of functional peptides. The basic E5 peptide folds into an alpha-helical structure and exhibits a stronger membrane-disrupting activity in the presence of the copolymer than in its absence. The cationic comb-type copolymers, as an artificial chaperone, were shown to be beneficial for enhancing the functions and facilitating the application of biopolymers in nanotechnology and medicine. The cationic comb-type copolymers, which consisted of a polycation backbone grafted with high density of hydrophilic chains, form soluble and soft interpolyelectrolyte complexes with biopolymers and act as an artificial chaperone to assist in the folding of nucleic acids and peptides. The copolymers stabilize DNA duplex, triplex, and quadruplex structures and accelerate strand exchange reactions as well as assist in the folding of functional peptides into the active conformation.

Ligand-Based Stability Changes in Duplex DNA Measured with a Microscale Electrochemical Platform.

Development of technologies for rapid screening of DNA secondary structure thermal stability and the effects on stability for binding of small molecule drugs is important to the drug discovery process. In this report, we describe the capabilities of an electrochemical, microdevice-based approach for determining the melting temperatures (Tm) of electrode-bound duplex DNA structures. We also highlight new features of the technology that are compatible with array development and adaptation for high-throughput screening. As a foundational study to exhibit device performance and capabilities, melting-curve analyses were performed on 12-mer DNA duplexes in the presence/absence of two binding ligands: diminazene aceturate (DMZ) and proflavine. By measuring electrochemical current as a function of temperature, our measurement platform has the ability to determine the effect of binding ligands on Tm values with high signal-to-noise ratios and good reproducibility. We also demonstrate that heating our three-electrode cell with either an embedded microheater or a thermoelectric module produces similar results. The ΔTm values we report show the stabilizing ability of DMZ and proflavine when bound to duplex DNA structures. These initial proof-of-concept studies highlight the operating characteristics of the microdevice platform and the potential for future application toward other immobilized samples.