Direct detection of double-stranded DNA: Molecular methods and applications for DNA diagnostics

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics.     

Label-less fluorescence-based method to detect hybridization with applications to DNA micro-array

By coupling scattered light from DNA to excite fluorescence in a polymer, we describe a quantitative, label-free assay for DNA hybridization detection. Since light scattering is intrinsically proportional to number of molecules, the change in (scattering coupled) fluorescence is highly linear with respect to percent binding of single stranded DNA (ssDNA) target with the immobilized ssDNA probes. The coupling is achieved by immobilizing ssDNA on a fluorescent polymer film at optimum thickness in nanoscale. The fluorescence from the underlining polymer increases due to proportionate increase in scattering from double stranded DNA (dsDNA) (i.e., probe-target binding) compared to ssDNA (i.e., probe). Because the scattering is proportional to fourth power of refractive index, the detection of binding is an order of magnitude more sensitive compared to other label-free optical methods, such as, reflectivity, interference, ellipsometry and surface-plasmon resonance. Remarkably, polystyrene film of optimum thickness 30 nm is the best fluorescent agent since its excitation wavelength matches (within 5 nm) with wavelength for the maximum refractive index difference between ssDNA and dsDNA. A quantitative model (with no fitting parameters) explains the observations. Potential dynamic range is 1 in 10(4) at signal-to-noise ratio of 3:1.     

Electrochemical measurement of DNA hybridization using nanosilver as label and horseradish peroxidase as enhancer

A simple and sensitive electrochemical DNA biosensor based on in situ DNA amplification with nanosilver as label and horseradish peroxide (HRP) as enhancer has been designed. The thiolated oligomer single-stranded DNA (ssDNA) was initially directly immobilized on a gold electrode, and quartz crystal microbalance (QCM) gave the specific amount of ssDNA adsorption of 6.3 ± 0.1 ng/cm². With a competitive format, hybridization reaction was carried out via immersing the DNA biosensor into a stirred hybridization solution containing different concentrations of the complementary ssDNA and constant concentration of nanosilver-labeled ssDNA, and then further binding with HRP. The adsorbed HRP amount on the probe surface decreased with the increment of the target ssDNA in the sample. The hybridization events were monitored by using differential pulse voltammetry (DPV) with the adsorbed HRP toward the reduction of H₂O₂. The reduction current from the enzyme-generated product was related to the number of target ssDNA molecules in the sample. A detection of 15 pmol/L for target ssDNA was obtained with the electrochemical DNA biosensor. Additionally, the developed approach can effectively discriminate complementary from non-complementary DNA sequence, suggesting that the similar enzyme-labeled DNA assay method hold great promises for sensitive electrochemical biosensor applications.     

Segregation of viral double-stranded and single-stranded DNA molecules in nuclei of adenovirus infected cells as revealed by electron microscope in situ hybridization.

Formation of progeny viruses in the nuclei of HeLa cells infected with adenovirus type 5 was studied at the ultrastructural level by in situ hybridization techniques allowing specific detection of either viral double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA). Prior to the initiation of replication of viral genomes, infective DNA molecules which entered the nucleus of the target cell were randomly distributed among host chromatin fibers including nucleolus-associated chromatin. They were double-stranded, that is, without single-strand breaks. Such association of viral DNA with host condensed chromatin also occurred in mitosis. The initiation of viral genome replication occurred simultaneously with the appearance in the nucleoplasm of small fibrillar regions containing intermingled viral dsDNA and ssDNA. Later, at the intermediate stage of nuclear transformation, viral dsDNA and ssDNA molecules were almost entirely separated into two contiguous substructures. At this stage, viruses were observed occasionally in the vicinity of viral ssDNA accumulation sites. Still later, an additional substructure developed in the centre of the nucleus which consisted of large quantities of viral dsDNA, traces of viral ssDNA and abundant viruses. Portions of viral ssDNA were attached to some viruses even at late stage of nuclear transformation, an association which strongly suggests the occurrence of encapsidation of at least some of the viral genomes while they are still engaged in replication.     

Sex determination based on amelogenin DNA by modified electrode with gold nanoparticle

We have developed a simple and renewable electrochemical biosensor based on carbon paste electrode (CPE) for the detection of DNA synthesis and hybridization. CPE was modified with gold nanoparticles (AuNPs), which are helpful for immobilization of thiolated bioreceptors. AuNPs were characterized by scanning electron microscopy (SEM). Self-assembled monolayers (SAMs) of thiolated single-stranded DNA (SH–ssDNA) of the amelogenin gene was formed on CPE. The immobilization of the probe and its hybridization with the target DNA was optimized using different experimental conditions. The modified electrode was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The electrochemical response of ssDNA hybridization and DNA synthesis was measured using differential pulse voltammetry (DPV) with methylene blue (MB) as an electroactive indicator. The new biosensor can distinguish between complementary and non-complementary strands of amelogenin ssDNA. Genomic DNA was extracted from blood and was detected based on changes in the MB reduction signal. These results demonstrated that the new biosensor could be used for sex determination. The proposed biosensor in this study could be used for detection and discrimination of polymerase chain reaction (PCR) products of amelogenin DNA.     

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA‐based nanomaterials because of its direct recognition of natural double‐stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single‐strand DNA (ssDNA) fluorescent probes and fluorescent probe–double‐strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K_a) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20‐mer perfectly matched ssDNA probe and three single‐base mismatched ssDNA probes with 146‐mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single‐base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.     

Labeling of DNA probes with a photoactivatable hapten

A photoactivatable reagent for introducing haptens onto DNA probes has been prepared using a commercially available bifunctional linker arm reagent and amino-derivatized 2,4-dinitrophenyl (DNP). The resulting compound (photo-DNP) couples efficiently to DNA using an ordinary sunlamp. Under optimum conditions, about 7-23 DNP molecules per 1000 bases are incorporated into the DNA. Hybridization experiments demonstrate that as little as 1.5 x 10(5) copies of target DNA can be detected by filter hybridization with a photo-DNP-labeled probe and immunochemical detection.     

Mechanical measurement of single-molecule binding rates: kinetics of DNA helix-destabilization by T4 gene 32 protein

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein, and is essential for DNA replication, recombination and repair. While gp32 binds preferentially and cooperatively to ssDNA, it has not been observed to lower the thermal melting temperature of natural double-stranded DNA (dsDNA). However, in single-molecule stretching experiments, gp32 significantly destabilizes lambda DNA. In this study, we develop a theory of the effect of the protein on single dsDNA stretching curves, and apply it to the measured dependence of the DNA overstretching force on pulling rate in the presence of the full-length and two truncated forms of the protein. This allows us to calculate the rate of cooperative growth of single clusters of protein along ssDNA that are formed as the dsDNA molecule is stretched, as well as determine the site size of the protein binding to ssDNA. The rate of cooperative binding (ka) of both gp32 and of its proteolytic fragment *I (which lacks 48 residues from the C terminus) varies non-linearly with protein concentration, and appears to exceed the diffusion limit. We develop a model of protein association with the ends of growing clusters of cooperatively bound protein enhanced by 1-D diffusion along dsDNA, under the condition of protein excess. Upon globally fitting ka versus protein concentration, we determine the binding site size and the non-cooperative binding constants to dsDNA for gp32 and I. Our experiment mimics the growth of clusters of gp32 that likely exist at the DNA replication fork in vivo, and explains the origin of the "kinetic block" to dsDNA melting by gene 32 protein observed in thermal melting experiments.     

光标生物素HBV-DNA探针及其临床应用

本文报告了应用生物素交联光敏补骨酯素(Bp,Biotin-Psoralen)标记HBV DNA探针的动力学研究和临床应用结果。表明化学合成Bp经366nm光照与DNA发生加成反应。能标记dsDNA,也能标记ssDNA。加热可使Bp-dsDNA变性,强碱则不能使Bp-dsDNA变性。临床DNA检测结果表明,应用Bp-HBV探针杂交,DNA检出率与HBeAg符合率达99.08％,与~(32)P-HBV符合率达98.8％,提示光标记Bp-HBV探针灵敏度接近同位素探针。     

Electrochemical DNA biosensor for the detection of interaction between di[azino-di(5,6-azafluorene)-kappa2-NN'] dibromicoppers and DNA

A new Cu(II) complex CuL(2)Br(2) (L = azino-di(5,6-azafluorene)-kappa(2)-NN') was synthesized, and a new method of electrochemical probe has been proposed for the determination of hepatitis B virus (HBV) based on its interaction with [CuL(2)](2+). This ligand, containing functional groups, as well as planar aromatic domains, is capable of binding to double-stranded DNA (dsDNA) more efficiently than to single-stranded DNA (ssDNA). Emphasis has been placed on the elucidation of the nature of the interaction by electrochemical techniques. The electroactive [CuL(2)](2+) could be employed as an electrochemical indicator to detect hybridization events in DNA biosensors. These biosensors have been constructed by immobilization of a probe DNA sequence from HBV onto glassy carbon electrode (GCE). After hybridization with the complementary target sequence, [CuL(2)](2+) was accumulated within the dsDNA layer. Electrochemical detection was performed by differential pulse voltammetry over the potential range. Using this approach, complementary target sequences of HBV can be quantified over the range of 1.74 x 10(-9) to 3.45 x 10(-7) M, with a detection limit of 8.32 x 10(-10) M and a linear correlation coefficient of 0.9936.In addition, this approach is capable of detecting hybridization of complementary sequences containing one or three mismatched bases.     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Methods for assessing DNA hybridization of peptide nucleic acid-titanium dioxide nanoconjugates

We describe the synthesis of peptide nucleic acid (PNA)-titanium dioxide (TiO₂) nanoconjugates and several novel methods developed to investigate the DNA hybridization behaviors of these constructs. PNAs are synthetic DNA analogs resistant to degradation by cellular enzymes that hybridize to single-stranded DNA (ssDNA) with higher affinity than DNA oligonucleotides, invade double-stranded DNA (dsDNA), and form different PNA/DNA complexes. Previously, we developed a DNA-TiO₂ nanoconjugate capable of hybridizing to target DNA intracellularly in a sequence-specific manner with the ability to cleave DNA when excited by electromagnetic radiation but susceptible to degradation that may lower its intracellular targeting efficiency and retention time. PNA-TiO₂ nanoconjugates described in the current article hybridize to target ssDNA, oligonucleotide dsDNA, and supercoiled plasmid DNA under physiological-like ionic and temperature conditions, enabling rapid, inexpensive, sequence-specific concentration of nucleic acids in vitro. When modified by the addition of imaging agents or peptides, hybridization capabilities of PNA-TiO₂ nanoconjugates are enhanced, providing essential benefits for numerous in vitro and in vivo applications. The series of experiments shown here could not be done with either TiO₂-DNA nanoconjugates or PNAs alone, and the novel methods developed will benefit studies of numerous other nanoconjugate systems.     

A toolbox for generating single‐stranded DNA in optical tweezers experiments

Essential genomic transactions such as DNA‐damage repair and DNA replication take place on single‐stranded DNA (ssDNA) or require specific single‐stranded/double‐stranded DNA (ssDNA/dsDNA) junctions (SDSJ). A significant challenge in single‐molecule studies of DNA–protein interactions using optical trapping is the design and generation of appropriate DNA templates. In contrast to dsDNA, only a limited toolbox is available for the generation of ssDNA constructs for optical tweezers experiments. Here, we present several kinds of DNA templates suitable for single‐molecule experiments requiring segments of ssDNA of several kilobases in length. These different biotinylated dsDNA templates can be tethered between optically trapped microspheres and can, by the subsequent use of force‐induced DNA melting, be converted into partial or complete ssDNA molecules. We systematically investigated the time scale and efficiency of force‐induced melting at different ionic strengths for DNA molecules of different sequences and lengths. Furthermore, we quantified the impact of microspheres of different sizes on the lifetime of ssDNA tethers in optical tweezers experiments. Together, these experiments provide deeper insights into the variables that impact the production of ssDNA for single molecules studies and represent a starting point for further optimization of DNA templates that permit the investigation of protein binding and kinetics on ssDNA. © 2013 Wiley Periodicals, Inc. Biopolymers 99:611–620, 2013.     

The stretched DNA geometry of recombination and repair nucleoprotein filaments

The RecA protein of Escherichia coli plays essential roles in homologous recombination and restarting stalled DNA replication forks. In vitro, the protein mediates DNA strand exchange between single-stranded (ssDNA) and homologous double-stranded DNA (dsDNA) molecules that serves as a model system for the in vivo processes. To date, no high-resolution structure of the key intermediate, comprised of three DNA strands simultaneously bound to a RecA filament (RecA x tsDNA complex), has been elucidated by classical methods. Here we review the systematic characterization of the helical geometries of the three DNA strands of the RecA x tsDNA complex using fluorescence resonance energy transfer (FRET) under physiologically relevant solution conditions. Measurements of the helical parameters for the RecA x tsDNA complex are consistent with the hypothesis that this complex is a late, poststrand-exchange intermediate with the outgoing strand shifted by about three base pairs with respect to its registry with the incoming and complementary strands. All three strands in the RecA x tsDNA complex adopt extended and unwound conformations similar to those of RecA-bound ssDNA and dsDNA.     

Single-strandedness of the majority of DNA sequences complementary to mRNA-coding sites isolated from rat hepatoma tissue cultured cells

Single-stranded DNA (ssDNA), separated from bulk double-stranded DNA (dsDNA) of HTC by an improved method of hydroxyapatite chromatography, exhibited the same characteristics as ssDNA previously found in various cell species. It amounted to 1.5–2% of the total nuclear DNA. Only 24–26% could be self-reassociated, but the greatest part hybridized to non-repetitious DNA fraction and about 30% hybridized to homologous mRNA.Other results tend to prove that the complementary sequences of HTC-ssDNA probably consist of non-base-paired segments attached to double helical regions of dsDNA. In effect, after hydroxyapatite chromatography, a small portion of HTC-dsDNA (2–3%) was found to be rapidly digestible by S1 nuclease and this limited digestion was sufficient to reduce markedly the hybridization rates of dsDNA with both DNA and cell-free synthesised cDNA copies of polyadenylated RNAs. Furthermore, these ³H-cDNA copies could not be annealed to ssDNA under conditions that allowed their reassociation with total nuclear DNA. These findings complete the demonstration that the greatest part of ssDNA appears to be formed via selective nicks, probably enzymatic, in the coding strand of actively transcribed DNA regions.     

Surface Plasmon Resonance Studies of the Hybridization Behavior of DNA-Modified Gold Nanoparticles with Surface-Attached DNA Probes

Colloidal gold nanoparticles (AuNPs) have been extensively investigated as amplification tags to improve the sensitivity of surface plasmon resonance (SPR) biosensors. When using the so-called AuNP-enhanced SPR technique for DNA detection, the density of single-stranded DNA (ssDNA) on both the AuNPs and planar gold substrates is of crucial importance. Thus, in this work, we carried out a systematical study about the influence of surface ssDNA density onto the hybridization behavior of various DNA-modified AuNPs (DNA-AuNPs) with surface-attached DNA probes by using surface plasmon resonance spectroscopy. The lateral densities of the ssDNA on both the AuNPs and planar gold substrates were controlled by using different lengths of oligo-adenine sequence (OAS) as anchoring group. Besides SPR measurements, the amount of the captured DNA-AuNPs after the hybridization was further identified via atomic force microscope (AFM). SPR and AFM results clearly indicated that a higher ssDNA density on either the AuNPs or the gold substrates would give rise to better hybridization efficiency. Moreover, SPR data showed that the captured DNA-AuNPs could not be removed from SPR sensor surfaces using various dehybridization solutions regardless of surface ssDNA density. Consequently, it is apparent that the hybridization behavior of DNA-AuNPs was different from that of solution-phase ssDNA. Based on these data, we hypothesized that both multiple recognitions and limited accessibility might account for the hybridization of DNA-AuNPs with surface-attached ssDNA probes.

     

Salt dependent binding of T4 gene 32 protein to single and double-stranded DNA: single molecule force spectroscopy measurements

Bacteriophage T4 gene 32 protein (gp32) is a well-studied representative of the large family of single-stranded DNA (ssDNA) binding proteins, which are essential for DNA replication, recombination and repair. Surprisingly, gp32 has not previously been observed to melt natural dsDNA. At the same time, *I, a truncated version of gp32 lacking its C-terminal domain (CTD), was shown to decrease the melting temperature of natural DNA by about 50 deg. C. This profound difference in the duplex destabilizing ability of gp32 and *I is especially puzzling given that the previously measured binding of both proteins to ssDNA was similar. Here, we resolve this apparent contradiction by studying the effect of gp32 and *I on the thermodynamics and kinetics of duplex DNA melting. We use a previously developed single molecule technique for measuring the non-cooperative association constants (K(ds)) to double-stranded DNA to determine K(ds) as a function of salt concentration for gp32 and *I. We then develop a new single molecule method for measuring K(ss), the association constant of these proteins to ssDNA. Comparing our measured binding constants to ssDNA for gp32 and *I we see that while they are very similar in high salt, they strongly diverge at [Na+] < 0.2 M. These results suggest that intact protein must undergo a conformational rearrangement involving the CTD that is in pre-equilibrium to its non-cooperative binding to both dsDNA and ssDNA. This lowers the effective concentration of protein available for binding, which in turn lowers the rate at which it can destabilize dsDNA. For the first time, we quantify the free energy of this CTD unfolding, and show it to be strongly salt dependent and associated with sodium counter-ion condensation on the CTD.     

Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.