Characterization of single- and double-stranded DNA on gold surfaces

Single- and double-stranded deoxy ribonucleic acid (DNA) molecules attached to self-assembled monolayers (SAMs) on gold surfaces were characterized by a number of optical and electronic spectroscopic techniques. The DNA-modified gold surfaces were prepared through the self-assembly of 6-mercapto-1-hexanol and 5'-C(6)H(12)SH -modified single-stranded DNA (ssDNA). Upon hybridization of the surface-bound probe ssDNA with its complimentary target, formation of double-stranded DNA (dsDNA) on the gold surface is observed and in a competing process, probe ssDNA is desorbed from the gold surface. The competition between hybridization of ssDNA with its complimentary target and ssDNA probe desorption from the gold surface has been investigated in this paper using X-ray photoelectron spectroscopy, chronocoulometry, fluorescence, and polarization modulation-infrared reflection absorption spectroscopy (PM-IRRAS). The formation of dsDNA on the surface was identified by PM-IRRAS by a dsDNA IR signature at approximately 1678 cm(-)(1) that was confirmed by density functional theory calculations of the nucleotides and the nucleotides' base pairs. The presence of dsDNA through the specific DNA hybridization was additionally confirmed by atomic force microscopy through colloidal gold nanoparticle labeling of the target ssDNA. Using these methods, strand loss was observed even for DNA hybridization performed at 25 degrees C for the DNA monolayers studied here consisting of attachment to the gold surfaces by single Au-S bonds. This finding has significant consequence for the application of SAM technology in the detection of oligonucleotide hybridization on gold surfaces.     

Packing structures of single-stranded DNA and double-stranded DNA thiolates on Au(111): a molecular simulation study

The packing structures of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) thiolates on implicit gold surfaces were studied in explicit aqueous solutions of 1M NaCl using molecular dynamics simulations. The simulations were based on individual DNA chains placed in hexagonal simulation boxes of different sizes, representing various packing densities. The total potential energy per DNA chain was compared. The optimal packing structures were determined based on the minimal potential energy within the limits of the conditions that were evaluated in this study. The optimal packing density of ssDNA was found to be 0.19 DNA chains/nm(2), which is consistent with that determined experimentally. Furthermore, the optimal packing density of dsDNA was shown to be approximately 58% of the packing density for ssDNA, indicating that the packing of ssDNA should be approximately 58% of its optimal packing in order to achieve the best hybridization.     

Creation of mammalian single- and double-stranded DNA surfaces: a real-time QCM-D study

The quartz crystal microbalance with dissipation monitoring (QCM-D) is an excellent method for studying the creation of DNA-based surfaces and films. Previous studies have used QCM-D to focus on the construction of DNA surfaces composed of short synthetic DNA oligomers or plasmid DNA. Here, we have used QCM-D to monitor the creation of genomic single- and double-stranded calf thymus DNA surfaces on a polycation adsorbed to a SiO2 support. We have successfully monitored the hybridization between the ssDNA surfaces and their complementary strands in solution and have also shown that DNA multilayer formation can be observed using denatured calf thymus DNA. We have furthermore established that the ssDNA and dsDNA surfaces show different binding characteristics to ethidium bromide, a common dsDNA intercalator, demonstrating the potential use of such surfaces to identify possible DNA ligands.     

Immobilization of single-stranded deoxyribonucleic acid on gold electrode with self-assembled aminoethanethiol monolayer for DNA electrochemical sensor applications

A synthesized 24-mer single-stranded deoxyribonucleic acid (ssDNA) was covalently immobilized onto a self-assembled aminoethanethiol monolayer modified gold electrode, using water-soluble 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide (EDC). The covalently immobilized ssDNAs were hybridized with complementary ssDNA (cDNA) or yAL(3) gene in solution, forming double-stranded DNAs (dsDNA). Meanwhile, daunomycin as an electrochemical active intercalator in the hybridization buffer solution was intercalated into the dsDNA to form a dsDNA/daunomycin system on the gold electrode surface, which was used for DNA electrochemical sensor. The cathodic waves of daunomycin bound to the double-stranded DNA (dsDNA) by linear sweep voltammetry were utilized to detect the cDNA. The cathodic peak current (i(pc)) of duanomycin was linearly related to the concentrations of cDNA between 0.1 mug ml(-1) and 0.1 ng ml(-1). The detection limit was about 30 pg ml(-1).     

Conformational change detection of DNA with the fluorogenic reagent of o-phthalaldehyde-beta-mercaptoethanol

o-Phthalaldehyde-beta-mercaptoethanol (OPAME) as a fluorogenic reagent has been found wide applications in the detection of amino acids based on its reaction with primary amino groups. In this contribution, we report our new findings concerning the reactions of OPAME with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), respectively. It has been found that ssDNA can react with OPAME easily as a result of giving rise to strong fluorescence emissions, while dsDNA, prepared by hybridizing ssDNA with its complementary target prior to the reaction, displays inert chemical activity and gives out weak fluorescence emission. Mechanism investigations have shown that the reaction activity between OPAME and DNA depends on the amino groups that are related to the conformation of uncoiled and exposed extent of DNA structure, and thus the inert chemical activity of dsDNA results from screening of the dsDNA bases in the interior of the double strands. Therefore, we could design a way to detect conformation change of DNA with OPAME and further develop a novel, simple label-free sequence detection method for complementary and single-base mismatched ssDNA in the hybridization of DNA.     

Tethered thiazole orange intercalating dye for development of fibre-optic nucleic acid biosensors

Single-stranded DNA (ssDNA) oligonucleotide in solution, or that is immobilized onto a surface to create a biosensor, can be used as a selective probe to bind to a complementary single-stranded sequence. Fluorescence enhancement of thiazole orange (TO) occurs when the dye intercalates into double-stranded DNA (dsDNA). TO dye has been covalently attached to probe oligonucleotides (homopolymer and mixed base 10mer and 20mer) through the 5′ terminal phosphate group using polyethylene glycol linker. The tethered TO dye was able to intercalate when dsDNA formed in solution, and also at fused silica surfaces using immobilized ssDNA. The results indicated the potential for development of a self-contained biosensor where the fluorescent label was available as part of the immobilized oligonucleotide probe chemistry. The approach was shown to be able to operate in a reversible manner for multiple cycles of detection of targeted DNA sequences.     

灿烂甲酚蓝在DNA修饰金电极上的电化学行为

利用自组装技术将巯基乙醇固定在金电极表面形成巯基乙醇自组装膜修饰金电极, 用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联试剂, 分别将鲱鱼精单链DNA(ssDNA)和双链DNA(dsDNA)固定于金电极表面形成ssDNA和dsDNA 修饰电极. 考察了灿烂甲酚蓝(BCB)在不同DNA 修饰电极上的电化学行为,结果表明, BCB 在ssDNA 和dsDNA 修饰电极上的吸附常数分别为1.67×10^4和3.22×10^4 L·mol-1, BCB 与ssDNA 主要以静电作用结合, 而与dsDNA作用存在静电和嵌插两种模式. dsDNA 对BCB 具有更高的亲和力, 使BCB 可以作为一种有效的电化学杂交指示剂.     

Minor groove binding of a novel tetracationic diviologen

Novel tetracationic diviologen compounds of the general formula CH3(CH2)nV2+(CH2)6V2+(CH2)nCH3 (where V2+ = 4,4'-bipyridinium and n = 5 or 11) were investigated as electrochemical reporters of DNA duplex formation. These compounds bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) when the DNA is either present in solution or immobilized at electrode surfaces. Binding to thiolated ssDNA and dsDNA immobilized at Au electrodes was characterized using the electrochemical response for the reduction of the V2+ state to the V+ (viologen radical cation) state. An analysis of the charge for this reduction provided isotherms and binding constants for binding of these diviologens to both forms of immobilized DNA. Saturation of the binding is achieved at solution concentrations near 20 microM. For both the n = 5 and 11 diviologens, binding to ssDNA is driven by electrostatic charge neutralization. For the n = 11 case, the binding is cooperative. In the presence of dsDNA, the n = 11 diviologen exhibits a unique reduction potential for the V2+/+ redox couple that is shifted approximately 100 mV negative of that in the presence of ssDNA. This new electrochemical signature is attributed to the reduction of viologen groups bound in the minor groove of the DNA duplex. For dsDNA in solution, an increase in the thermal denaturation temperature (Tm) from 60 to 66 degrees C as a function of the n = 11 diviologen concentration confirmed its interaction with the duplex. Circular dichroism (CD) spectroscopy also was used to investigate the binding of both the V2+ and V+ redox states of the n = 11 diviologen to dsDNA in solution. For the V+ state, a CD signal was observed that is consistent with the presence of face-to-face pi dimers of the viologen groups. This unambiguously demonstrates the binding of this redox state of the diviologen in the dsDNA minor groove and the formation of such dimers in the minor groove.     

Optical properties of an immobilized DNA monolayer from 255 to 700 nm

The real (n) and imaginary (k) refractive indices of an immobilized monolayer of 27 nucleotide (nt) single stranded DNA (ssDNA) and the corresponding double stranded DNA (dsDNA) are measured in the 255-700 nm range. Multiple techniques are used to obtain consistent estimation. The coverage is approximately 6.5% with an average interchain distance of tethered ssDNA molecules of approximately 11.8 nm, which is significantly larger than the "footprint" of the chain on the surface. The measured increase in n by approximately 5% between the ssDNA and the dsDNA is 20% smaller than the expected change due to doubling of the molecular weight. The change in k is not significant, indicating that the electron delocalization effect expected in dsDNA due to base pair stacking is not important at optical frequencies.     

Specific Binding of Anionic Porphyrin and Phthalocyanine to the G-Quadruplex with a Variety of <em>i</em><em>n Vitro</em> and<em> </em><em>i</em><em>n Vivo</em> Applications

The G-quadruplex, a four-stranded DNA structure with stacked guanine tetrads (G-quartets), has recently been attracting attention because of its critical roles in vitro and in vivo. In particular, the G-quadruplex functions as ligands for metal ions and aptamers for various molecules. Interestingly, the G-quadruplex can show peroxidase-like activity with an anionic porphyrin, iron (III) protoporphyrin IX (hemin). Importantly, hemin binds to G-quadruplexes with high selectivity over single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), which is attributable to an electrostatic repulsion of phosphate groups in ssDNA and dsDNA. The G-quadruplex and hemin-G-quadruplex complex allow development of sensing techniques to detect DNA, metal ions and proteins. In addition to hemin, anionic phthalocyanines also bind to the G-quadruplex formed by human telomere DNA, specifically over ssDNA and dsDNA. Since the binding of anionic phthalocyanines to the G-quadruplex causes an inhibition of telomerase activity, which plays a role in the immortal growth of cancer cells, anionic phthalocyanines are promising as novel anticancer drug candidates. This review focuses on the specific binding of hemin and anionic phthalocyanines to G-quadruplexes and the applications in vitro and in vivo of this binding property.     

A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide

A fluorescent biosensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed based upon the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and double-stranded DNA. The fluorescence of FAM-labeled single-stranded DNA was quenched when it adsorbed on the surface of graphene oxide (GO). Upon addition of the target dsDNA, a homopyrimidine·homopurine part of dsDNA on the Simian virus 40 (SV40) (4424-4440, gp6), hybridization occurred between the dye-labeled DNA and the target dsDNA, which induced the dye-labeled DNA desorbed from the surface of GO, and turned on the fluorescence of the dye. Under the optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of target dsDNA in the range 40.0-260 nM, and the detection limit was found to be 14.3 nM alongside the good sequence selectivity.     

Comparison of RNA, single-stranded DNA and double-stranded DNA behavior during capillary electrophoresis in semidilute polymer solutions

We present a study of the separation of RNA, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in semidilute linear hydroxyethylcellulose (HEC) solution. Our results strive to provide a better understanding of the mechanisms of nucleic acid migration during electrophoresis in polymer solutions under native and denaturing conditions. From a study of the dependence of mobility on chain length and applied electric field, we found that RNA and ssDNA show better separation and higher resolution over a larger range of sizes compared to dsDNA. In addition, RNA reptation without orientation extends to longer chain lengths in comparison to ssDNA, possibly as a result of different type of short-lived secondary structure formations. Such a comparative study between nucleic acid capillary electrophoresis helps to optimize RNA separation and provides better understanding of RNA migration mechanisms in semidilute polymer solutions under denaturing conditions.     

Excited-state intramolecular proton transfer distinguishes microenvironments in single- and double-stranded DNA

Herein, the efficient interaction of an environment-sensitive fluorophore that undergoes excited-state intramolecular proton transfer (ESIPT) with DNA has been realized by conjugation of a 3-hydroxychromone (3HC) with polycationic spermine. On binding to a double-stranded DNA (dsDNA), the ratio of the two emission bands of the 3HC conjugates changes up to 16-fold, so that emission of the ESIPT product increases dramatically. This suggests an efficient screening of the 3HC fluorophore from the water molecules in the DNA complex, which is probably realized by its intercalation into dsDNA. In sharp contrast, the 3HC conjugates show only moderate changes in the dual emission on binding to a single-stranded DNA (ssDNA), indicating a much higher fluorophore exposure to water at the binding site. Thus, the 3-hydroxychromone fluorophore being conjugated to spermine discriminates the binding of this polycation to dsDNA from that to ssDNA. Consequently, ESIPT-based dyes are promising for monitoring the interaction of polycationic molecules with DNA and probing the microenvironment of their DNA binding sites.     

A simple fluorometric assay for DNA exonuclease activity based on graphene oxide

A new assay platform for DNA exonuclease activity is developed based on the preferential binding of single-stranded DNA (ssDNA) over double-stranded DNA (dsDNA) to graphene oxide. This approach allows a simple and quantitative activity measurement in a short time at low cost.     

Formation of a DNA layer on Langmuir-Blodgett films and its enzymatic digestion

Here, we report a system we have developed where long double-stranded DNAs (dsDNAs) are immobilized on a monolayer of Zn-arachidate. We have applied the Langmuir-Blodgett technique to form the monolayer of Zn-arachidate where Zn(II) is bound to arachidic acid through charge neutralization. Because tetrahedral Zn(II) participates in DNA recognition through coordination, we have been able to layer DNA over the Zn-arachidate monolayer. The DNA layer shows a typical compression and expansion cycle in a concentration-dependent fashion. Interestingly, the DNA monolayer is available for enzymatic degradation by DNaseI. The detection of DNA and its accessibility towards biological reaction is demonstrated by imaging through fluorescence microscopy. The conformation of the DNA, immobilized on the monolayer, was studied with the help of atomic force microscopy (AFM). We observed that the dsDNAs were aligned in a stretched manner on the surface. To investigate further, we also demonstrate here that the small single-stranded DNA (ssDNA) immobilized on the air-water interface can act as a target molecule for the complementary ssDNA present in the subphase. The study of DNA hybridization done with the help of fluorescence spectroscopy clearly supports the AFM characterization.     

Spectroscopic and electrochemical studies on the interaction between luteolin and DNA

Journal of Solid State Electrochemistry - The interactions of luteolin (LUT) with DNA, including double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA), were investigated in detail by...     

ssDNA binding reveals the atomic structure of graphene

We used AFM to investigate the interaction of polyelectrolytes such as ssDNA and dsDNA molecules with graphene as a substrate. Graphene is an appropriate substrate due to its planarity, relatively large surfaces that are detectable via an optical microscope, and straightforward identification of the number of layers. We observe that in the absence of the screening ions deposited ssDNA will bind only to the graphene and not to the SiO(2) substrate, confirming that the binding energy is mainly due to the π-π stacking interaction. Furthermore, deposited ssDNA will map the graphene underlying structure. We also quantify the π-π stacking interaction by correlating the amount of deposited DNA with the graphene layer thickness. Our findings agree with reported electrostatic force microscopy (EFM) measurements. Finally, we inspected the suitability of using a graphene as a substrate for DNA origami-based nanostructures.     

Synthesis of Ag@carbonized Polymer Dots and their Electrochemical Sensing of miRNA

Polyethyleneimine (PEI) is a cationic polymer with a lot of amino groups in its molecular structure that is secure, sterile, and biocompatible. However, it is difficult for PEI molecules to be directly applied in electroanalysis due to their poor electroactivity. Herein, the PEI-based carbonized polymer nanodots (CPDs) with core-shell structure were prepared by hydrothermal method using PEI as the precursors. The results showed that the as-prepared PEI-based CPDs can not only exhibit the unique properties of carbon nanodots but also inherit the properties of PEI. Subsequently, based on the strong coordination interaction between the abundant amino groups on the surface of PEI and silver ions, the electroactive Ag@carbonized polymer dots (Ag@CPDs) were prepared. Furthermore, to investigate the electrochemical behaviors and application of Ag@CPDs, the DNA molecule (double-stranded and single-stranded DNA) was taken as an example. It was found that the interaction of DNA with Ag@CPDs had a significant influence on the current intensity of Ag@CPDs. Based on this phenomenon, a sensitive electrochemical technique for miRNA detection was established and effectively employed to detect miRNA in human serum samples based on the discriminating effect of Ag@CPDs towards ssDNA probes and ssDNA probe/miRNA complex (dsDNA).     

Interaction of DNA with echinomycin at the mercury electrode surface as detected by impedance and chronopotentiometric measurements

The capacitance measurement (dependence of the differential capacitance C of the electrode double layer on potential E, C-E curves), electrochemical impedance spectroscopy (frequency response of the impedance Z of the electrode double layer-EIS) and constant current chronopotentiometry (dependence of dt/dE on potential at constant current, chronopotentiometric stripping analysis-CPSA) have been used for electrochemical study of echinomycin and its interaction with single-stranded (ss) and double-stranded (ds) DNA at the hanging mercury drop electrode (HMDE). The capacitance measurement showed that echinomycin gives a pseudocapacitance redox peak strongly dependent on the a.c. voltage frequency at the potential of -0.53 V. This peak is observed with dsDNA-echinomycin complex as well, but not with ssDNA treated by echinomycin. Similar results were obtained using CPSA measurements. Thus capacitance measurements and CPSA can distinguish with the aid of the bis-intercalator echinomycin the single-stranded and double helical form of DNA adsorbed at the mercury electrode surface. Impedance measurement in connection with adsorptive transfer technique can find the differences between ssDNA and dsDNA, which promise to use this technique for detection of dsDNA in hybridisation reactions.     

自组装DNA吸附取向的表面增强拉曼光谱法研究

对金基体上自组装寡聚核苷酸探针杂交前后进行电化学非现场及现场表面增强拉曼光谱(SERS)研究.非现场SERS研究表明,杂交形成的dsDNA在基体表面以A型和B型两种构象同时存在,杂交过程可能伴随DNA链在基体表面吸附取向的变化.根据现场SERS研究结果可知,ssDNA及dsDNA的大多数SERS谱带强度随电极电位正移而降低,尤其是归属于碱基A的两种面外振动模式,谱带变化更为明显.利用SERS表面选择定则判断出随着电极电位由负向正变化,ssDNA及dsDNA螺旋吸附取向由垂直吸附向平躺吸附于金基体表面变化.