Duplex DNA Chain Research Articles

Enzyme-free electrochemical biosensor based on amplification of proximity-dependent surface hybridization chain reaction for ultrasensitive mRNA detection

The ability to recognize mRNA with high efficiency in cells would greatly facilitate the elucidation of mRNA-mediated cellular cascades and their disease associations. However, most traditional electrochemical strategies targeting nucleotides are always confronted with cumbersome interface operation and washing procedures, as well as the high cost of labeling and the strict reaction conditions of tool enzymes, limiting their potential applications. To address these issues, herein we reported, for the first time, a simple label-free, isothermal, non-enzymatic, and ultrasensitive homogeneous electrochemical biosensor based on autonomous proximity-dependent surface hybridization chain reaction (HCR), for sensitive signal amplification and highly specific detection of target survivin mRNA with a detection limit of 3 fM. The target triggers hybridization chain reaction and mRNA-fueled surface hybridization of ferrocene-tagged metastable DNA hairpin probes on proximity-dependent surface hybridization, resulting in the formation of multiple long-range duplex DNA chains which are immobilized onto the gold electrodes with a substantially stable ferrocene-mediated redox current. Thus, a significant electrochemical signal increase is observed dependent on the concentration of the target RNA, with a very low detection limit. Mo-reover, this molecular biosensor also exhibits excellent specificity to distinguish even single base mismatched, with strong reliability. The developed biosensor provides a novel promising tool for ultra-sensitive and selective detection, and it has great potential to be applied in mRNA-related biochemical research and clinical cancer diagnostics in more detail.

A fluorometric aptamer method for kanamycin by applying a dual amplification strategy and using double Y-shaped DNA probes on a gold bar and onmagnetite nanoparticles.

A simple and highly sensitive fluorometric method is described for the determination of the antibiotic kanamycin (Kana) in food. Dual signal amplification is accomplished by making use of double Y-shaped aptamer DNA probes acting as a capture probes and signal amplification probes. The DNA probes were immobilized on a gold bar and ona magnetic bar, respectively. On addition of Kana, the Y-shaped aptamer probe captures Kana and then is disassembled to release two single-stranded DNAs. These trigger target recycling and HCR between the two bars simultaneously. As a result, many long duplex DNA chains are formed in the supernatant. After pulling out the bars and adding the fluorescent intercalating probe SYBR Green I, strong fluorescence (with excitation/emission peaks at 497/525nm) is induced. The use of such double Y-shaped DNA probes obviously overcomes the unspecific signal amplification by HCR which increases selectivity and sensitivity. This is due to the fact that the hairpin of HCR is separated in being present in different arms of the Y-shaped probe. Under the optimal conditions, the assay has a limit of 0.45pg·mL-1 for Kana. It was applied to analyze spiked milk, fish and pork samples. Graphical abstract The scheme represents a sensitive fluorometric aptamer-based method to detect kanamycin (Kana). It is making use of a double stirring bar-assisted dual amplification strategy with zero background. Abbreviations: apt: aptamer, AuNPs: gold nanoparticles, HCR: hybridization chain reaction.

Microfluidic electrophoretic non-enzymatic kanamycin assay making use of a stirring bar functionalized with gold-labeled aptamer, of a fluorescent DNA probe, and of signal amplification via hybridization chain reaction.

The authors describe an enzyme-free aptamer-based assay for the determination of the model antibiotic kanamycin (Kana). The method is making use of (a) microfluidic chip electrophoresis; (b) a stirring bar carrying a gold-labeled aptamer probe, and (c) the hybridization chain reaction (HCR) for signal amplification. Firstly, a stirring bar (length: 1cm; diameter: 0.2mm) was modified with a large amount of duplex DNA and then hybridized with aptamer and its partially complementary chains (cDNA). In the presence of Kana, the binding between the Kana and aptamer unwinds the duplex structures and releases a corresponding amount of cDNA into the supernatant. The released cDNA triggers the HCR in the presence of H1 and H2 DNA hairpin to produce a large amount of duplex DNA chains with different lengths. At the same time, the amounts of H1 and H2 are reduced. The decreased signal of H1/H2 after several HCR cycles can be used to quantify kana in the 1pg·mL-1 to 10ng·mL-1, with a detection limit of 0.29pg·mL-1. The signal is generated by reading the fluorescence, best at excitation/emission maxima of 470/525nm. The whole detection process takes 3min only. The assay was employed to the detection of Kana in spiked milk and fish samples. Results are consistent with those of an enzyme linked immunosorbent assay. The assay has high throughput, high selectivity, and high amplification capability. Graphical abstract Schematic of a stirring bar functionalized with gold-labeled aptamer acting as the capture probe. It can capture the target and release primer simultaneously. The primer triggers the hybridization chain reaction inducing the consumption of H1 and H2. After a certain reaction time, the mixture is injected into the MCE platform for microfluidic electrophoretic separation and fluorometric detection.

A cyclometalated iridium(III) complex used as a conductor for the electrochemical sensing of IFN-\u03b3

A novel iridium(III) complex was prepared and used as a conductor for sensitive and enzyme-free electrochemical detection of interferon gamma (IFN-γ). This assay is based on a dual signal amplification mechanism involving positively charged gold nanoparticles ((+)AuNPs) and hybridization chain reaction (HCR). To construct the sensor, nafion (Nf) and (+)AuNPs composite membrane was first immobilized onto the electrode surface. Subsequently, a loop-stem structured capture probe (CP) containing a special IFN-γ interact strand was modified onto the (+)AuNP surface via the formation of Au-S bonds. Upon addition of IFN-γ, the loop-stem structure of CP was opened, and the newly exposed “sticky” region of CP then hybridized with DNA hairpin-1 (H1), which in turn opened its hairpin structure for hybridizing with DNA hairpin-2 (H2). Happen of HCR between H1 and H2 thus generated a polymeric duplex DNA (dsDNA) chain. Meanwhile, the iridium(III) complex could interact with the grooves of the dsDNA polymer, producing a strong current signal that was proportional to IFN-γ concentration. Thus, sensitive detection of IFN-γ could be realized with a detection limit down to 16.3 fM. Moreover, satisfied results were achieved by using this method for the detection of IFN-γ in human serum samples.

Label-Free and Enzyme-Free Homogeneous Electrochemical Biosensing Strategy Based on Hybridization Chain Reaction: A Facile, Sensitive, and Highly Specific MicroRNA Assay.

Homogenous electrochemical biosensing strategies have attracted substantial attention, because of their advantages of being immobilization-free and having rapid response and improved recognition efficiency, compared to heterogeneous biosensors; however, the high cost of labeling and the strict reaction conditions of tool enzymes associated with current homogeneous electrochemical methods limit their potential applications. To address these issues, herein we reported, for the first time, a simple label-free and enzyme-free homogeneous electrochemical strategy based on hybridization chain reaction (HCR) for sensitive and highly specific detection of microRNA (miRNA). The target miRNA triggers the HCR of two species of metastable DNA hairpin probes, resulting in the formation of multiple G-quadruplex-incorporated long duplex DNA chains. Thus, with the electrochemical indicator Methylene Blue (MB) selectively intercalated into the duplex DNA chain and the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target miRNA. Thus, using this "signal-off" mode, a simple, label-free and enzyme-free homogeneous electrochemical strategy for sensitive miRNA assay is readily realized. This strategy also exhibits excellent selectivity to distinguish even single-base mismatched miRNA. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Therefore, the as-proposed label-free and enzyme-free homogeneous electrochemical strategy may become an alternative method for simple, sensitive, and selective miRNA detection, and it has great potential to be applied in miRNA-related clinical diagnostics and biochemical research.

3P125 DNAを湾曲するHMG-1/2は塩基配列非特異的に長鎖DNAを折り畳み(05B. 核酸:相互作用・複合体,ポスター,第52回日本生物物理学会年会(2014年度))

3P125 DNAを湾曲するHMG-1/2は塩基配列非特異的に長鎖DNAを折り畳み(05B. 核酸:相互作用・複合体,ポスター,第52回日本生物物理学会年会(2014年度))

Twist–radial normal mode analysis in double-stranded DNA chains

We study the normal modes of a duplex DNA chain at low temperatures. We consider the coupling between the hydrogen-bond radial oscillations and the twisting motion of each base pair within the Peyrard–Bishop–Dauxois model. The coupling is mediated by the stacking interaction between adjacent base pairs along the helix. We explicitly consider different mass values for different nucleotides, extending previous works. We disclose several resonance conditions of interest, determined by the fine-tuning of certain model parameters. The role of these dynamical effects on the DNA chain charge transport properties is discussed.

3C0924 長鎖DNAの1分子内折り畳みはカチオン性ポリマー凝縮剤の長さによってモードが変わる(核酸,口頭発表,日本生物物理学会第50回年会(2012年度))

3C0924 長鎖DNAの1分子内折り畳みはカチオン性ポリマー凝縮剤の長さによってモードが変わる(核酸,口頭発表,日本生物物理学会第50回年会(2012年度))

Weak Interaction Induces an ON/OFF Switch, whereas Strong Interaction Causes Gradual Change: Folding Transition of a Long Duplex DNA Chain by Poly-l-lysine

A large-scale conformational change in genomic DNA is an essential feature of gene activation in living cells. Considerable effort has been applied to explain the mechanism in terms of key-lock interaction between sequence-specific regulatory proteins and DNA, in addition to the modification of DNA and histones such as methylation and acetylation. However, it is still unclear whether these mechanisms can explain the ON/OFF switching of a large number of genes that accompanies differentiation, carcinogenesis, etc. In this study, using single-molecule observation of DNA molecules by fluorescence microscopy with the addition of poly-L-lysine with different numbers of monomer units (n = 3, 5, 9, and 92), we found that an ON/OFF discrete transition in the higher-order structure of long duplex DNA is induced by short poly-L-lysine, whereas a continuous gradual change is induced by long poly-L-lysine. On the other hand, polycations with a lower positive charge have less potential to induce DNA compaction. Such a drastic difference in the conformational transition of a giant DNA between short and large oligomers is discussed in relation to the mechanisms of gene regulation in a living cell.

FOLDING DYNAMICS IN A SEMIFLEXIBLE POLYMER AS A MODEL OF DNA

We studied the folding transition of a semiflexible homopolymer chain using a Brownian dynamics simulation. A duplex DNA chain exhibits a discrete transition between an elongated coil state and a folded state with toroidal morphology. We show that these folding properties are understood with the stiffness of DNA chains. Semiflexible chains fold through various paths into collapsed toroidal states stochastically. Some chains fold directly by winding around a ring along a chain. Others fold through rod states, which are kinetically trapped metastable states.

Various Morphology with Collapse Transition in a Homopolymer Chain

We have studied the collapse transition of a single homopolymer chain with “multicanonical Monte Carlo simulation”. As a chain becomes stiffer, the manner of the transition changes from continuous to discontinuous. In addition to this, it is found that the morphology in the collapsed state changes from the state only with spherical globule, through the coexistence of both rod and toroidal morphology, into the state only with toroid. The morphological change obtained thus theoretically reproduces the essential features in the experimental collapsed structures from single duplex DNA chains observed with electron microscopy.

Folding and unfolding of a giant duplex-DNA in a mixed solution with polycations, polyanions and crowding neutral polymers

To understand the conformational behavior of a giant duplex-DNA chain in a mixed solution with various biopolymers with different state of ionization, the higher-order structure of the DNA chain was analyzed with a fluorescence microscope in the presence of polycations (poly-arginine), polyanions (poly-glutamic acid), and neutral polymers (poly-ethylene glycol) as a model for cellular environment. Concentrated medium with neutral polymer induced the discrete folding transition of the DNA. At the threshold condition for the transition, addition of small amounts of either the polycation or the polyanion caused marked structural changes in the folded DNAs. Based on thermodynamic considerations on the experimental results, profile of free energy of a single giant DNA chain was depicted with respect to the size, or the expansion factor α, in the three-dimensional structure of the DNA. The effect of the neural crowding polymer on the degree of folding of a single giant DNA chain is discussed in a semi-quantitative manner.

Folding transition in single long duplex DNA chain

Folding transition in single long duplex DNA chain

高分子をみる 蛍光顕微鏡 長鎖ＤＮＡの構造をみる

高分子をみる 蛍光顕微鏡 長鎖ＤＮＡの構造をみる

Self-organized nanostructures constructed with a single polymer chain

A Monte Carlo simulation was performed on the folding transition of a single stiff polymer chain. We have adapted a polymer model: neighboring beads are connected by a stick and the length of each stick is taken as 1 8 of the Kuhn length. Among the products of the folding from the single chain, the toroidal structure is found to be the most stable. Rod and fused structures between rod and toroid are also generated as metastable states with almost infinite lifetime. These morphologies resemble the experimental structures observed with electron microscopy for the products of collapse from single duplex DNA chains.

The folded state of long duplex-DNA chain reflects its solution history

The higher-order structure of compacted single giant DNA induced by complexation with polypeptide (poly-Arg) in NaCl solution was investigated using fluorescence microscopy. As the poly-Arg concentration increased, the mean size of extended DNA chains gradually decreased. In the presence of excess poly-Arg, individual DNA chains collapsed into compact globules, and the degree of collapse of the DNA chains depended not only on the concentration of poly-Arg, but also on the time course of the addition of poly-Arg and NaCl, indicating that the structure of the collapsed DNA is not determined simply according to the minimum free energy. We discuss theoretically the presence of multiple-stationary states based on a consideration of simple kinetics in the process of binding. Depending on the past history, the number of poly-Arg and Na+ that bind to each DNA changes markedly. This interesting characteristic of long DNA is discussed in relation to the possible mechanism of self-regulation of gene expression in living cells.

Energy Flow Considerations and Thermal Fluctuational Opening of DNA Base Pairs at a Replicating Fork: Unwinding Consistent with Observed Replication Rates

The effect of an open loop of various sizes on the thermal stability of the adjoining intact base pairs in a duplex DNA chain is studied in a lattice model of Poly(dG).Poly(dC). We find that for a Y-shaped fork configuration the thermal fluctuation at the fork is so enhanced that the life time of the adjoining base pair is much smaller than the 1 millisecond time scale associated with helicase separation of a base pair in some systems. Our analysis indicates that thermal fluctuational base pair opening may be of importance in facilitating the enzyme unwinding process during chain elongation of a replicating DNA. It is most likely that the thermal fluctuational opening of the base pair at the junction of a replicating fork is fast enough so that a DNA unwinding enzyme can encounter an unstacked base pair with reasonable probability. This conclusion can explain several experimental observations regarding the temporal relationship between ATP hydrolysis by accessory proteins and primer elongation by a holoenzyme complex in ssDNA. We also discuss a mechanism by which the energy associated with ATP hydrolysis may enhance the thermal driven base opening mechanism.