Interaction between nile blue and immobilized single- or double-stranded DNA and its application in electrochemical recognition

The interactions of nile blue (NB) with calf-thymus double-stranded DNA (dsDNA) in solution, single-stranded DNA (ssDNA) and dsDNA adsorbed on gold electrodes were studied electrochemically. The binding of NB with dsDNA showed a mechanism containing an electrostatic interaction with the binding constant of (5.9 ± 0.2) × 10⁴ M⁻¹ in 0.01 M pH 7.4 Tris-HCl. The presence of adsorbed dsDNA resulted in a great increase in the peak currents of NB in comparison with those obtained at a bare or ssDNA adsorbed gold electrode. The electron transfer rate constants of nile blue adsorbed on bare gold and bound to ss- and dsDNA/Au electrodes were 1.4 ± 0.1, 1.2 ± 0.02 and 2.9 ± 0.2 s⁻¹, respectively. Nile blue showed an electrostatic action with adsorbed ssDNA and an action with adsorbed dsDNA by the mechanism containing both electrostatic and intercalative bindings. The Langmuir adsorption constants of NB at ssDNA/Au and dsDNA/Au electrodes were (1.6 ± 0.2) × 10⁵ and (4.2 ± 0.6) × 10⁵ M⁻¹, respectively. The difference between interactions of NB with adsorbed ss- and dsDNA has been used for hybridization recognition of hepatitis B virus DNA fragment. The interactions between NB and covalently immobilized hepatitis B virus ssDNA fragment and its hybridization product showed the mechanisms similar to those of adsorbed calf-thymus DNA.     

Electrooxidation of DNA at glassy carbon electrodes modified with multiwall carbon nanotubes dispersed in polyethylenimine

This work reports the electrochemical response of the complex between dsDNA and PEI formed in solution and at the surface of glassy carbon electrodes (GCE) modified with a dispersion of multi-walled carbon nanotubes in polyethylenimine (CNT-PEI). Scanning Electron Microscopy and Scanning Electrochemical Microscopy demonstrate that the dispersion covers the whole surface of the electrode although there are areas with higher density of CNT and, consequently, with higher electrochemical reactivity. The adsorption of DNA at GCE/CNT-PEI is fast and it is mainly driven by electrostatic forces. A clear oxidation signal is obtained either for dsDNA or a heterooligonucleotide of 21 bases (oligoY) at potentials smaller than those for the oxidation at bare GCE. The comparison of the behavior of DNA before and after thermal treatment demonstrated that the electrochemical response highly depends on the 3D structure of the nucleic acid.     

Direct electrochemistry of double strand DNA on ionic liquid modified screen-printed graphite electrode

An ionic liquid modified screen-printed graphite electrode (SPE) was used for direct electrochemistry of herring sperm double strand DNA (dsDNA) by voltammetry. Due to the high conductivity of ionic liquid n-octylpyridinum hexafluorophosphate (OPPF), this electrode exhibited excellent electrochemical activity for the oxidation of dsDNA. Two irreversible oxidation peaks were obtained at the developed electrode, which corresponded to the oxidation of guanine and adenine residues present in the dsDNA. The basic electrochemical behavior of dsDNA at the OPPF modified SPE was carefully investigated. Combined with the differential pulse voltammetry, this electrode exhibited a good linear range from 20 μg mL⁻¹ to 120 μg mL⁻¹ with a detection limit of 5 μg mL⁻¹ for the direct determination of dsDNA. Furthermore, the OPPF modified electrode displayed high reproducibility and stability for the dsDNA determination.     

Adsorption of double-stranded DNA to graphene oxide preventing enzymatic digestion

Investigation into the interactions between graphene oxide (GO) and biomolecules is very important for broad applications of GO in bioassay and bioanalysis. In this work, we describe the interactions between double-stranded DNA (dsDNA) and GO. We demonstrated that dsDNA can bind to GO forming complexes (dsDNA/GO) in the presence of certain salts, which protects dsDNA from being enzymatically digested. On the other hand, we found that a nonionic surfactant, such as triton X-100, can block the formation of dsDNA/GO complexes, so that the enzymatic digestion of dsDNA is restored. These results lead us to believe that the reason for GO protecting dsDNA from enzymatic digestion is the formation of dsDNA/GO complexes hindering the access of DNA enzymes to dsDNA, rather than direct inactivation of the DNA enzymes.     

Comparative study on the electrocatalytic activities of ordered mesoporous carbons and graphene

In this work, a comparative study on the electrocatalytic activities of ordered mesoporous carbons (OMCs) and graphene (GR) is presented. Using voltammetry and amperometry as detection methods, four DNA bases, double-stranded DNA (dsDNA), six important electroactive compounds and various biomolecules were employed to investigate their electrochemical responses on OMC and GR modified glassy carbon electrodes (OMC/GCE and GR/GCE). The results show that OMC/GCE enhances the electron transfer kinetics of these compounds compared to GR/GCE. The discrepancy in electrochemical activities can be attributed to the different microstructures of OMC and GR, which were examined by transmission electron microscopy, X-ray photoelectron spectra, X-ray diffraction, Raman spectra and nitrogen adsorption–desorption.     

An artificial primosome: design, function, and applications

Double-stranded (ds) DNA is capable of the sequence-specific accommodation of an additional oligodeoxyribonucleotide strand by the peptide nucleic acid(PNA)-assisted formation of a so-called PD-loop. We demonstrate here that the PD-loop may function as an artificial primosome within linear, nonsupercoiled DNA duplexes. DNA polymerase with its strand displacement activity uses this construct to initiate the primer extension reaction at a designated dsDNA site. The primer is extended by several hundred nucleotides. The efficiency of dsDNA priming by the artificial primosome assembly is comparable to the single-stranded DNA priming used in various assays. The ability of the PD-loop structure to perform like an artificial primosome on linear dsDNA may find applications in biochemistry, molecular biology, and molecular biotechnology, as well as for DNA diagnostics. In particular, multiple labels can be incorporated into a chosen dsDNA site resulting in ultrasensitive direct quantification of specific sequences. Furthermore, nondenaturing dsDNA sequencing proceeds from the PD-loop. This approach opens the way to direct isothermal reading of the DNA sequence against a background of unrelated DNA, thereby eliminating the need for purification of the target DNA.     

Bright two-photon emission and ultra-fast relaxation dynamics in a DNA-templated nanocluster investigated by ultra-fast spectroscopy

Metal nanoclusters have interesting steady state fluorescence emission, two-photon excited emission and ultrafast dynamics. A new subclass of fluorescent silver nanoclusters (Ag NCs) are NanoCluster Beacons. NanoCluster Beacons consist of a weakly emissive Ag NC templated on a single stranded DNA ("Ag NC on ssDNA") that becomes highly fluorescent when a DNA enhancer sequence is brought in proximity to the Ag NC by DNA base pairing ("Ag NC on dsDNA"). Steady state fluorescence was observed at 540 nm for both Ag NC on ssDNA and dsDNA; emission at 650 nm is observed for Ag NC on dsDNA. The emission at 550 nm is eight times weaker than that at 650 nm. Fluorescence up-conversion was used to study the dynamics of the emission. Bi-exponential fluorescence decay was recorded at 550 nm with lifetimes of 1 ps and 17 ps. The emission at 650 nm was not observed at the time scale investigated but has been reported to have a lifetime of 3.48 ns. Two-photon excited fluorescence was detected for Ag NC on dsDNA at 630 nm when excited at 800 nm. The two-photon absorption cross-section was calculated to be ～3000 GM. Femtosecond transient absorption experiments were performed to investigate the excited state dynamics of DNA-Ag NC. An excited state unique to Ag NC on dsDNA was identified at ～580 nm as an excited state bleach that related directly to the emission at 650 nm based on the excitation spectrum. Based on the optical results, a simple four level system is used to describe the emission mechanism for Ag NC on dsDNA.     

Efficient Linear dsDNA Tagging Using Deoxyuridine Excision**

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3’ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3’ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5’ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.     

Electrochemical and AFM evaluation of hazard compounds-DNA interaction

The evaluation of the interaction of hazard compounds, divalent cations Pb, Cd, Ni and Pd, with double-stranded DNA (dsDNA), forming a metal-DNA complex, was studied by atomic force microscopy (AFM) and differential pulse voltammetry (DPV) on two carbon electrode surfaces, highly oriented pyrolytic graphite (HOPG) and glassy carbon (GC). The electrochemical behaviour of these metal-DNA complexes was related to the different adsorption patterns and conformational changes obtained by the AFM images on the HOPG surface. The dsDNA interaction was specific with each metal cation, inducing structural changes in the B-DNA structure, local denaturation of the double helix and oxidative damage. AFM images showed an increase of the electrode surface coverage for lead, cadmium and nickel DNA complexes. For cadmium and nickel-DNA complexes oxidative damage to DNA was electrochemically detected for the concentrations studied. Palladium interaction with dsDNA induced condensation of the dsDNA secondary structure, which led to the aggregation of helixes forming very compact and thick filaments in the network film. The voltammetric data for the palladium-DNA complex showed a sharp decrease of the guanosine and adenosine oxidation peak currents, consistent with the AFM results of DNA condensation in the presence of palladium, but no DNA oxidative damage was detected for the range of concentrations used.     

Synthesis and study of nucleic acids interactions of novel monomethine cyanine dyes

Six asymmetric monomethine cyanine dyes have been synthesized and their spectral characteristics and interaction with double stranded (ds)DNA have been investigated for their prospective use as fluorescent markers in molecular biology. Therefore, the non-covalent binding of the compounds with dsDNA was explored. Apart from the fluorescence spectroscopy, the study includes UV/Vis spectrophotometry and circular dichroism spectroscopy, as well as the thermal melting experiments. Although the compounds show relatively low binding affinity toward dsDNA and do not have intrinsic fluorescence, in the presence of dsDNA they exhibited considerable enhancement in fluorescence intensity. Therefore the studied dyes show interesting platform for future modifications directed toward more sequence selective derivatives. The compound with the highest affinity toward dsDNA showed interesting anti-proliferative potential and specificity.     

DNA Interaction Studies of Selected Polyamine Conjugates

The interaction of polyamine conjugates with DNA double helix has been studied. Binding properties were examined by ethidium bromide (EtBr) displacement and DNA unwinding/topoisomerase I/II (Topo I/II) activity assays, as well as dsDNA thermal stability studies and circular dichroism spectroscopy. Genotoxicity of the compounds was estimated by a comet assay. It has been shown that only compound 2a can interact with dsDNA via an intercalative binding mode as it displaced EtBr from the dsDNA-dye complex, with K_app = 4.26 × 10⁶ M⁻¹; caused an increase in melting temperature; changed the circular dichroism spectrum of dsDNA; converted relaxed plasmid DNA into a supercoiled molecule in the presence of Topo I and reduced the amount of short oligonucleotide fragments in the comet tail. Furthermore, preliminary theoretical study has shown that interaction of the discussed compounds with dsDNA depends on molecule linker length and charge distribution over terminal aromatic chromophores.     

Polyaza crown ethers as non-nucleosidic building blocks in DNA conjugates: synthesis and remarkable stabilization of dsDNA

The synthesis of amphiphilic polyaza crown ether monomers X (benzyl-substituted), Y (palmityl-substituted) and Z (cholesteryl-substituted) and their incorporation into oligonucleotides are described. Their effects on thermal duplex stability were investigated by UV melting curve analysis in different alkaline metal buffer solutions. Thermal-denaturation experiments showed remarkable stabilization of dsDNA by polyaza crown ether monomers when incorporated in opposite positions. The series of polyaza crown ether monomers (X, Y and Z) with different overall lipophilicities showed a trend of increased stability of the corresponding dsDNA with increasing lipophilicity of the polyaza crown ether monomer. Multiple incorporations of benzyl-substituted polyaza crown ether monomer X as dangling ends on both sides of dsDNA resulted in strongly increased stability of the corresponding duplex.     

Sequence‐Specific Molecular Lithography Towards DNA‐Templated Electronics

Recent advances in the realization of individual molecular‐scale devices [1,2] highlight the integration of individual devices into large‐scale functional circuits as the major challenge. DNA‐programmed assembly is a promising avenue in that direction due to the large amount of information that can be coded into the molecules and the ability to translate that information into physical constructs [3]. Large‐scale DNA‐templated electronics require, however, complex manipulation of double‐stranded DNA (dsDNA) molecules, as well as patterning of the electrical properties instilled to them by, e.g., metallization. To that end, sequence‐specific molecular lithography on single DNA molecules has been developed [4]. This was achieved by harnessing the exquisite homologous recombination process of the RecA protein. Sequence‐specific patterning of the metal coating of DNA molecules, localization of arbitrary labeled molecular objects at any desired dsDNA address without prior modifications, and generation of molecularly accurate stable dsDNA‐dsDNA junctions are demonstrated. The information encoded in the DNA molecules directs the lithographic process in analogy to the masks used in conventional microelectronics. The RecA protein provides the assembling capabilities, as well as the resist function.     

Oxidative DNA Cleavage with Clip-Phenanthroline Triplex-Forming Oligonucleotide Hybrids

A systematic study of several new types of hybrids of Cu-chelated clamped phenanthroline artificial metallonuclease (AMN) with triplex-forming oligonucleotides (TFO) for sequence-specific cleavage of double-stranded DNA (dsDNA) is reported. The synthesis of these AMN–TFO hybrids is based on application of the alkyne–azide cycloaddition click reaction as the key step. The AMN was attached through different linkers at either the 5′- or 3′-ends or in the middle of the TFO stretch. The diverse hybrids efficiently formed triplexes with the target purine-rich sequence and their copper complexes were studied for their ability to cleave dsDNA in the presence of ascorbate as a reductant. In all cases, the influence of the nature and length of the AMN–TFO, time, conditions and amounts of ascorbate were studied, and optimum conjugates and a procedure that gave reasonably efficient (up to 34 %) cleavage of the target sequence, while rendering an off-target dsDNA intact, were found. The footprint of cleavage on PAGE was identified only in one case, with low conversion; this means that cleavage does not proceed with single nucleotide precision. On the other hand, these AMN–TFO hybrids are useful for the selective degradation of target dsDNA sequences. Future improvements to this design may provide higher resolution and selectivity.     

N-terminal Cationic Modification of Linear Pyrrole-Imidazole Polyamide Improves Its Binding to DNA

Pyrrole-imidazole polyamides (PIPs) bind to double-stranded DNA (dsDNA) with varied sequence selectivity. We synthesized linear PIPs that can bind to narrow minor grooves of polypurine/polypyrimidine sequences and target long recognition sequences but have lower molecular weights than commonly used hairpin PIPs. We modified the N-terminus of linear PIPs using several groups, including β-alanine extension and acetyl capping. Melting curve analysis of dsDNA demonstrated that cationic modifications improved the binding affinity of the PIPs to the targeted dsDNA. In addition, circular dichroism assays revealed the characteristic spectra depending on the binding stoichiometry of the N-cationic linear PIP and dsDNA (1 : 1, monomeric; 2 : 1, dimeric). Surface plasmon resonance assays confirmed the high binding affinities of linear PIPs. These findings may aid in the design of effective linear PIPs.     

Electrochemical determination of anticancer drug fulvestrant at dsDNA modified pencil graphite electrode

The interaction of fulvestrant with double-stranded DNA (dsDNA) was studied by means of differential pulse voltammetry (DPV) at dsDNA modified pencil graphite electrode (PGE). The decrease in intensity of the guanine oxidation signals was used as an indicator for the interaction mechanism and sensitive determination of fulvestrant in pH 4.80 acetate buffer (30% ethanol). A linear dependence of the guanine oxidation signals was observed within the range of 1.00–20.00 μg/mL fulvestrant, with a detection limit of 0.41 μg/mL. The determination method was validated according to the standard validation procedure. The proposed method was applied to fulvestrant pharmaceutical dosage form.     

杨树菇中一种脱氧核糖核酸酶的纯化及其性质

目的分离纯化杨树菇子实体中脱氧核糖核酸酶,并对其性质进行分析。方法通过80%饱和(NH4)2SO4沉淀、Blue Sepharose 6 Fast Flow、DEAE-Sepharose Fast Flow和SP-Sepharose Fast Flow层析方法,从杨树菇子实体中分离纯化一种脱氧核糖核酸酶。SDS-PAGE和活性SDS-PAGE测定相对分子质量,采用琼脂糖凝胶电泳和紫外分光光度法分析其酶学性质,并检测pH、温度、Mg2+和EDTA浓度对酶活性的影响。结果经纯化得到了一种脱氧核糖核酸酶,其相对分子质量为31000。该酶对超螺旋质粒DNA、λDNA、ssDNA和dsDNA均具有降解活性,对dsDNA的降解活性略高于ssDNA,且酶活性依赖于二价金属阳离子。该酶的最适pH值范围为7.5～9.6,50℃时相对酶活性最高。结论从杨树菇子实体中纯化的脱氧核糖核酸酶属于一种非限制性脱氧核糖核酸内切酶。     

Interaction between promethazine hydrochloride and DNA and its application in electrochemical detection of DNA hybridization

The interactions of promethazine hydrochloride (PZH) with thiolated single-stranded DNA (HS-ssDNA) and double-stranded DNA (HS-dsDNA) self-assembled on gold electrodes have been studied electrochemically. The binding of PZH with ssDNA shows a mechanism containing an electrostatic interaction, while the mode of PZH interaction with dsDNA contains both electrostatic and intercalative bindings. The redox system belongs to the category of diffusion control approved by cyclic voltammetry (CV). The diffusion coefficients of PZH at the bare, HS-dsDNA and HS-ssDNA modified gold electrodes decrease regularly as 1.34 × 10⁻³ cm² s⁻¹, 1.04 × 10⁻³ cm² s⁻¹, 7.47 × 10⁻⁴ cm² s⁻¹, respectively. The electron transfer standard rate constant k_s of PZH at bare gold, HS-ssDNA and HS-dsDNA modified electrodes are 0.419 s⁻¹, 0.131 s⁻¹, and 0.154 s⁻¹, respectively. The presence of adsorbed dsDNA results in a great increase in the peak currents of PZH in comparison with those obtained at a bare or ssDNA adsorbed gold electrode. The difference between interactions of PZH with HS-ssDNA and HS-dsDNA has been used for hybridization recognition of 14-mer DNA oligonucleotide. The peak current (i_pa) of PZH is linearly proportional to the logarithmic concentration of complementary target DNA in the range from 2.0 × 10⁻⁹ mol L⁻¹ to 5.0 × 10⁻⁷ mol L⁻¹ with the detection limit of 3.8 × 10⁻¹⁰ mol L⁻¹.     

Light-up Hoechst-DNA aptamer pair: generation of an aptamer-selective fluorophore from a conventional DNA-staining dye

We have designed a strategy to generate a light-up fluorophore-aptamer pair based on a down-modification of a conventional DNA-staining dye to suppress its affinity to the original dsDNA targets, followed by reselection of aptamers that would bind to the modified dye. Following this line, we prepared a micropolarity-sensitive Hoechst derivative possessing two tBu groups with low affinity to the usual AT-rich dsDNA targets. DNA aptamers selected in vitro from a random pool worked as triggers to enhance the fluorescence of an otherwise nonfluorescent Hoechst derivative, and the shortened 25-mer sequence showed remarkable enhancement (light-up). The 25-mer sequence was split into binary aptamer probes, thus enabling us to detect a target nucleic acid sequence with a single-nucleotide resolution by use of unmodified DNA as a probe.