Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.     

Sensitive voltammetric detection of DNA damage at carbon electrodes using DNA repair enzymes and an electroactive osmium marker

This paper presents a new approach to electrochemical sensing of DNA damage, using osmium DNA markers and voltammetric detection at the pyrolytic graphite electrode. The technique is based on enzymatic digestion of DNA with a DNA repair enzyme exonuclease III (exoIII), followed by single-strand (ss) selective DNA modification by a complex of osmium tetroxide with 2,2'-bipyridine. In double-stranded DNA possessing free 3'-ends, the exoIII creates ss regions that can accommodate the electroactive osmium marker. Intensity of the marker signal measured at the pyrolytic graphite electrode responded well to the extent of DNA damage. The technique was successfully applied for the detection of (1) single-strand breaks (ssb) introduced in plasmid DNA by deoxyribonuclease I, and (2) apurinic sites generated in chromosomal calf thymus DNA upon treatment with the alkylating agent dimethyl sulfate. The apurinic sites were converted into the ssb by DNA repair endonuclease activity of the exoIII enzyme. We show that the presented technique is capable of detection of one lesion per approximately 10(5) nucleotides in supercoiled plasmid DNA.     

Topology effect for DNA structure of cisplatin: topological transformation of cisplatin-closed circular DNA adducts by DNA topoisomerase I.

The reaction of cis-Pt(NH3)2Cl2(cis-DDP)-closed circular DNA adducts with DNA topoisomerse I(topo I) were studied by electron microscopy. We identified unique topoisomers such as a singly-linked catenane (2(1)2), trefoil (3(1), and dimetric catenane (2(1)2), etc., by analysis with electron micrographs. These unique recombination products resulted from cis-DDP-intra-twisting looped DNA adducts by DNA topo I, and the products could be explained a new mechanism based on an odd-even number rule. Our results suggest a new model on the working mechanisms for DNA topology of cis-DDP which enhances the recombination of DNA. Based on our results, we propose the topological idea that the yields of a mini closed circular DNA and pseudo trefoil DNA, etc., can be expected by reaction of cis-DDP-DNA-histone complexes with DNA topo I in the body.     

Hybridization of oligonucleotide by using DNA self-assembled monolayer

The interaction between DNA immobilized on surface and oligonucleotides at the interface is important in detection and diagnostic processes. However, it is difficult to immobilize DNA with maintaining its activity and to realize an efficient hybridization in previous methods. Here, to establish a novel DNA-functionalized surface, the DNA self-assembled monolayer (SAM) was constructed on a gold substrate using thiolated DNA composed of double-stranded (ds) and single-stranded (ss) portion. The DNA SAM was characterized by surface plasmon resonance (SPR), XPS. The hybridization of ss portion of DNA was attempted using the SAM, and in situ monitored by SPR. XPS measurement indicated that the thiolated DNA could form a stable monolayer on a gold substrate through sulfur–gold interaction. SPR measurement implied that the long axis of the DNA standing on the substrate. These results indicated formation of the DNA SAM on the substrate. Hybridization of target DNA containing a complementary sequence for the probe portion was observed by SPR. Moreover, one mismatch of oligonucleotide could be distinguished using the DNA SAM. The SPR result indicates that hybridization of target DNA and probe DNA on the DNA SAM occurs on the DNA SAM.     

High-density DNA alignment on an Au(111) surface starting from folded DNA

High-density uniform DNA alignment on a metal substrate is essential for creating sensitive DNA devices. We develop a self-sensing DNA alignment process starting from folded DNA to achieve high-density, uniform DNA alignment on an Au(111) surface. We demonstrate that folded DNA plays a critical role in avoiding DNA aggregation and distributing the DNA uniformly on an Au(111) surface at the greatest density and quality ever attained. We also verify that the distributed, folded DNA can be stimulated to align only when the appropriate buffer flow is applied. This selective self-sensing DNA alignment on an Au surface will be a key technology for creating dynamic DNA sensors and switches.     

Liposome-mediated conformation transition of DNA detected by molecular probe: methyl green

Recent studies have focused on the structural features of DNA-lipid assemblies. In this paper, we take methyl green (MG) as a probe molecule to detect the conformational change of DNA molecule induced by dimethyldioctadecylammonium bromide (DDAB) liposomes before the condensation process of DNA begins. DDAB-induced DNA topology changes were investigated by cyclic voltammetry (CV), circular dichroism (CD) and UV-VIS spectrometry. We find that upon binding to DNA, positively charged liposomes induce a conformational transition of DNA molecules from the native B-form to the C motif. Conformational transition in DNA results in the binding modes of MG to DNA, changing and being isolated from DNA to the solution. More stable complexes are formed between DNA and DDAB. That is also proved by the melting study of DNA.     

Complexation of DNA with cationic surfactants as studied by small-angle X-ray scattering

The phase behaviors of the complex formed by didodecyldimethylammonium bromide(DDAB)and cetyltrimethylammonium bromide(CTAB)interacting with three different types of DNAs,salmon testes DNA(~2000 bp),21-bp double-stranded oligonucleotides(oligo-ds DNA),and 21-nt single-stranded oligonucleotides(oligo-ss DNA)were studied by synchrotron small-angle X-ray scattering.It was found that the DNA length and flexibility,together with the positive/negative charge ratio,determined the final structure.At higher charge ratios,the DNA length exhibited negligible effect.Both oligo-ds DNA and salmon DNA formed inverted hexagonal packing of cylinders with CTAB,as well as bilayered lamella with DDAB.However,at lower charge ratios,oligo-ds DNA formed a distorted hexagonal phase with CTAB and a new structure with DDAB,which was different from the behaviors of salmon DNA.The flexible oligo-ss DNA formed rich structures that were subject to environmental disturbance.Kinetic study also indicated that the structures of the complex formed by oligo-ss DNA took much longer to mature than the structures formed by oligo-ds DNA.We attributed this result to the conformational adjustment of oligo-ss DNA in the complex.     

High resolution mass spectrometry based profiling of diet-related deoxyribonucleic acid adducts

Exposure of DNA to endo- and exogenous DNA binding chemicals can result in the formation of DNA adducts and is believed to be the first step in chemically induced carcinogenesis. DNA adductomics is a relatively new field of research which studies the formation of known and unknown DNA adducts in DNA due to exposure to genotoxic chemicals. In this study, a new UHPLC-HRMS(/MS)-based DNA adduct detection method was developed and validated. Four targeted DNA adducts, which all have been linked to dietary genotoxicity, were included in the described method; O⁶-methylguanine (O⁶-MeG), O⁶-carboxymethylguanine (O⁶-CMG), pyrimidopurinone (M1G) and methylhydroxypropanoguanine (CroG). As a supplementary tool for DNA adductomics, a DNA adduct database, which currently contains 123 different diet-related DNA adducts, was constructed. By means of the newly developed method and database, all 4 targeted DNA adducts and 32 untargeted DNA adducts could be detected in different DNA samples. The obtained results clearly demonstrate the merit of the described method for both targeted and untargeted DNA adduct detection in vitro and in vivo, whilst the diet-related DNA adduct database can distinctly facilitate data interpretation.     

Magnetically trigged direct electrochemical detection of DNA hybridization using Au67 quantum dot as electrical tracer

A novel gold nanoparticle-based protocol for detection of DNA hybridization based on a magnetically trigged direct electrochemical detection of gold quantum dot tracers is described. It relies on binding target DNA (here called DNA1) with Au(67) quantum dot in a ratio 1:1, followed by a genomagnetic hybridization assay between Au(67)-DNA1 and complementary probe DNA (here called DNA2) marked paramagnetic beads. Differential pulse voltammetry is used for a direct voltammetric detection of resulting Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate on magnetic graphite-epoxy composite electrode. The characterization, optimization, and advantages of the direct electrochemical detection assay for target DNA are demonstrated. The two main highlights of presented assay are (1) the direct voltammetric detection of metal quantum dots obviates their chemical dissolution and (2) the Au(67) quantum dot-DNA1/DNA2-paramagnetic bead conjugate does not create the interconnected three-dimensional network of Au-DNA duplex-paramagnetic beads as previously developed nanoparticle DNA assays, pushing down the achievable detection limits.     

Pressure-induced transport of DNA confined in narrow capillary channels

Pressure-induced transport of double-stranded DNA (dsDNA) from 10 base pairs (bp) to 1.9 mega base pairs (Mbp) confined in a 750-nm-radius capillary was studied using a hydrodynamic chromatographic technique and four distinct length regions (rod-like, free-coiled, constant mobility, and transition regions) were observed. The transport behavior varied closely with region changes. The rod-like region consisted of DNA shorter than the persistence length (～150 bp) of dsDNA, and these molecules behaved like polymer rods. Free-coiled region consisted of DNA from ～150 bp to ～2 kilo base pairs (kbp), and the effective hydrodynamic radius R(HD) of these DNA scaled to L(0.5) (L is the DNA length in kbp), a characteristic property of freely coiled polymers. Constant mobility region consisted of DNA longer than ～100 kbp, and these DNA had a constant hydrodynamic mobility and could not be resolved. Transition region existed between free-coiled and constant mobility regions. The transport mechanism of DNA in this region was complicated, and a general empirical equation was established to relate the mobility with DNA length. Understanding of the fundamental principles of DNA transport in narrow capillary channels will be of great interest in the development of "lab-on-chip" technologies and nongel DNA separations.     

Chemiluminescent detection of DNA hybridization and single-nucleotide polymorphisms on a solid surface using target-primed rolling circle amplification

A new chemiluminescent (CL) method has been developed for the sensitive detection of DNA hybridization and single-nucleotide polymorphisms (SNPs) with target-primed rolling circle amplification (RCA). The capture oligonucleotide probe is firstly immobilized on a polystyrene well plate and then hybridized with the wild DNA target. A designed padlock probe is circularized after perfect hybridization to the DNA target. Then the RCA reaction can be initiated from the DNA target that acts as a primer and generates a long tandem single-strand of DNA with repeat sequences. In contrast, the mutant DNA target, which contains a mismatched base with the padlock probe, cannot initiate the RCA reaction and primes only a limited extension with the unligated padlock probe. Afterwards, a biotinylated oligonucleotide is used to hybridize with the RCA product in each repeat sequence and streptavidin-alkaline phosphatase (STV-AP) is employed to combine the anchored biotin. The DNA target is detected with the CL reaction of STV-AP and 3-(2'-spiroadamantane)-4-methoxy-4-(3'-phosphoryloxy)phenyl-1,2-dioxetane (AMPPD). With the RCA-based method, the sensitivity of DNA detection can be increased by about two orders of magnitude compared with that of direct DNA hybridization. A DNA target as low as 3.6 pM can be detected. Wild-type DNA and the one-base mutant DNA can be differentiated with high selectivity through this RCA reaction.     

Properties of hybridized DNA arrays on single-crystalline undoped and boron-doped (100) diamonds studied by atomic force microscopy in electrolytes

Properties of hybridized deoxyribonucleic acid (DNA) arrays on single-crystalline undoped and boron-doped diamonds are studied at the microscopic level by atomic force microscopy (AFM) in buffered electrolytic solutions. DNA is linked to diamond via aminodecene molecules (TFAAD) that are attached to undoped diamonds by a photochemical reaction and via nitrophenyl-diazonium molecules attached electrochemically to boron-doped diamonds. Both H-terminated and oxidized diamond surfaces are used in this process. On H-terminated surfaces, AFM measurements detect compact DNA layers. By analyzing phase and height contrast in AFM, a DNA layer height of 76 A is determined on the photochemically functionalized diamonds and a DNA layer height of up to 92 A is determined on the electrochemically functionalized diamonds. Based on the layer thickness, the DNA chains are tilted under the angle of 31 degrees . The morphology of the DNA layers exhibits long-range (30-50 nm) undulations of 20 A in height and a nanoroughness of 8 A. Using Hertz's model for calculating the contact area of the AFM tip on a DNA layer and a geometrical model of DNA arrangement on diamond yields the DNA density on diamonds of 6 x 10(12) cm(-2) on both photochemically and electrochemically functionalized diamonds. The structure of these dense DNA layers is not significantly influenced by variations in buffer salinity of 1-300 mM NaCl. DNA molecules can be removed from the diamond surface by contact-mode AFM with forces >or= 45 nN and >or= 76 nN on photochemically and electrochemically functionalized diamonds, respectively, indicating that DNA is bonded covalently and stronger on diamond than on gold substrates. The DNA arrangement and bonding strength are similar on oxidized diamond surfaces when using an electrochemical process. On oxidized surfaces after photochemical processing, DNA is bonded noncovalently as deduced from the removal force < 6 nN. The presence of hybridized DNA as well as the selective removal of DNA by AFM scanning are corroborated by fluorescence microscopy.     

Sequence-specific detection of femtomolar DNA via a chronocoulometric DNA sensor (CDS): effects of nanoparticle-mediated amplification and nanoscale control of DNA assembly at electrodes

We herein report a novel nanoparticle-based electrochemical DNA detection approach. This DNA sensor is based on a "sandwich" detection strategy, which involves capture probe DNA immobilized on gold electrodes and reporter probe DNA labeled with gold nanoparticles that flank the target DNA sequence. Electrochemical signals are generated by chronocoulometric interrogation of [Ru(NH(3))(6)](3+) that quantitatively binds to surface-confined capture probe DNA via electrostatic interactions. We demonstrated that the incorporation of a gold nanoparticle in this sensor design significantly enhanced the sensitivity and the selectivity. Nanoscale control of the self-assembly process of DNA probes at gold electrodes further increased the sensor performance. As a result of these two combined effects, this DNA sensor could detect as low as femtomolar (zeptomoles) DNA targets and exhibited excellent selectivity against even a single-base mismatch. In addition, this novel DNA sensor showed fairly good reproducibility, stability, and reusability.     

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by ¶1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.     

Separation of single-stranded DNA fragments at a 10-nucleotide resolution by stretching in microfluidic channels

We report a novel DNA separation method by tethering DNA chains to a solid surface and then stretching the DNA chains with an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of its size. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA strands from the surface and thus DNA separation. Here we show that this method, originally proposed for separation of long double-stranded DNA chains (>10,000 base pairs), is also applicable to single-stranded (ss) DNA fragments with less than 100 nucleotides (nt). Theoretical analysis indicates that the separation resolution is limited by the fluctuation forces on tethered DNA chains. By employing a microfluidic platform with narrow channels filled with a buffer of low ionic conductivity, we are able to apply a strong electric field to the DNA fragments with negligible Joule heating. Upon stepwise increments of the electric field, we demonstrate efficient separation of short ssDNA fragments at a 10-nt resolution.     

On 3-D graphical representation of DNA primary sequences and their numerical characterization

In this article we (1) outline the construction of a 3-D "graphical" representation of DNA primary sequences, illustrated on a portion of the human beta globin gene; (2) describe a particular scheme that transforms the above 3-D spatial representation of DNA into a numerical matrix representation; (3) illustrate construction of matrix invariants for DNA sequences; and (4) suggest a data reduction based on statistical analysis of matrix invariants generated for DNA. Each of the four contributions represents a novel development that we hope will facilitate comparative studies of DNA and open new directions for representation and characterization of DNA primary sequences.     

Solution structure of a DNA duplex containing the potent anti-poxvirus agent cidofovir

Cidofovir (1(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine, CDV) is a potent inhibitor of orthopoxvirus DNA replication. Prior studies have shown that, when CDV is incorporated into a growing primer strand, it can inhibit both the 3'-to-5' exonuclease and the 5'-to-3' chain extension activities of vaccinia virus DNA polymerase. This drug can also be incorporated into DNA, creating a significant impediment to trans-lesion DNA synthesis in a manner resembling DNA damage. CDV and deoxycytidine share a common nucleobase, but CDV lacks the deoxyribose sugar. The acyclic phosphonate bears a hydroxyl moiety that is equivalent to the 3'-hydroxyl of dCMP and permits CDV incorporation into duplex DNA. To study the structural consequences of inserting CDV into DNA, we have used (1)H NMR to solve the solution structures of a dodecamer DNA duplex containing a CDV molecule at position 7 and of a control DNA duplex. The overall structures of both DNA duplexes were found to be very similar. We observed a decrease of intensity (>50%) for the imino protons neighboring the CDV (G6, T8) and the cognate base G18 and a large chemical shift change for G18. This indicates higher proton exchange rates for this region, which were confirmed using NMR-monitored melting experiments. DNA duplex melting experiments monitored by circular dichroism revealed a lower T(m) for the CDV DNA duplex (46 °C) compared to the control (58 °C) in 0.2 M salt. Our results suggest that the CDV drug is well accommodated and stable within the dodecamer DNA duplex, but the stability of the complex is less than that of the control, suggesting increased dynamics around the CDV.     

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.     

Gaussian fluctuations in tethered DNA chains

In a recent work [Gao et al., Appl. Phys. Lett. 134, 113902 (2007)], we reported a novel DNA separation method by tethering DNA chains to a solid surface and then stretching the DNA chains with an electric field. The anchor is such designed that the critical force to detach a DNA chain is independent of its length. Because the stretching force is proportional to the DNA net charge, a gradual increase of the electric field leads to size-based removal of the DNA strands from the surface and thus DNA separation. Originally proposed for separation of long double-stranded DNA chains (>10 000 bps), this method has been proven useful also for short single-stranded DNA fragments (<100 bases) for which the fluctuation force induced by the solvent becomes significant. Here we show that the fluctuation force can be approximately represented by a gaussian model for tethered DNA chains. Analytical expressions have been derived to account for the dependence of the fluctuation force on the surface confinement, the polymer chain length, and the DNA tethering potential. The theoretical predictions are found to coincide with experiment.     

Mechanistic insight into the structure and dynamics of entangled and hydrated λ-phage DNA

Intrinsic dynamics of DNA plays a crucial role in DNA-protein interactions and has been emphasized as a possible key component for in vivo chromatin organization. We have prepared an entangled DNA microtube above the overlap concentration by exploiting the complementary cohesive ends of λ-phage DNA, which is confirmed by atomic force microscopy and agarose gel electrophoresis. Photon correlation spectroscopy further confirmed that the entangled solutions are found to exhibit the classical hydrodynamics of a single chain segment on length scales smaller than the hydrodynamic length scale of single λ-phage DNA molecule. We also observed that in 41.6% (gm water/gm DNA) hydrated state, λ-phage DNA exhibits a dynamic transition temperature (T(dt)) at 187 K and a crossover temperature (T(c)) at 246 K. Computational insight reveals that the observed structure and dynamics of entangled λ-phage DNA are distinctively different from the behavior of the corresponding unentangled DNA with open cohesive ends, which is reminiscent with our experimental observation.