Kinetics of DNA binding with chloroquine phosphate using capacitive sensing method

The capacitive sensing method has been applied to study the binding of DNA with chloroquine phosphate. DNA was immobilized on a gold electrode surface, self-assembled with thioglycolic acid. The results of a quartz crystal impedance (QCI) study indicate that the reaction of double-strand DNA (dsDNA) with chloroquine includes a fast electrostatic attraction and a slow intercalation of chloroquine into double-strand helix. The real-time experimental data obtained by capacitive sensing also revealed two distinctive kinetics stages during binding of dsDNA with chloroquine, while only one stage exists during reaction of single-strand DNA (ssDNA) with chloroquine. The kinetic parameters were obtained by fitting the real-time experimental data using a two stage reaction model. The rate constants of electrostatic attraction for dsDNA and ssDNA are estimated as 0.014 and 0.018 s(-1), respectively. The rate constant of the second stage of dsDNA is 0.0011 s(-1).     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

Ig H and L chain contributions to autoimmune specificities

An Ig H chain expression vector has been constructed by using the V region of 3H9, an antibody that binds ssDNA, dsDNA, and cardiolipin. The H chain construct was transfected into six hybridoma cell lines expressing Ig L chains. All resulting H and L chain combinations had at least some affinity for ssDNA, whereas five also bound dsDNA to a similar degree as 3H9. The loss of dsDNA binding was correlated with a single amino acid difference between two V kappa 8 L chains. A further characteristic of 3H9, its immunofluorescent staining pattern, was shared by four of the recombinant antibodies, whereas its specificity for cardiolipin was shared with five. The transfections reported here show that a V kappa 3 L chain confers specificity for an RNA-associated epitope and that a V kappa 21E L chain prevents cardiolipin binding. These experiments suggest that the 3H9 H chain contributes essential determinants required for binding to DNA as well as cardiolipin but that L chains can modulate or prevent this binding. L chains may also expand the specificity of a recombinant antibody.     

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.     

Self-assembly DNA-conjugated polymer for detection of single nucleotide polymorphism

We developed a self-assembly DNA-conjugated polymer based on polyacrylic acid (PAA) for DNA chip fabrication. A 20-mer single-stranded DNA (ssDNA, probe-1), and 3-(2-pyridyldithio)propionyl hydrazide (PDPH), for promoting self-assembled immobilization, were both covalently attached to PAA as sidechains. This DNA-conjugated PAA was then spontaneously immobilized on a gold substrate. Probe-1 on the immobilized polymer was hybridized to a 34-mer ssDNA (probe-2), which had the sequence desired for analyzing the target DNA. The fluorescence intensity after incubating the P-1 DNA-conjugated polymer with probe-2 DNA was much higher than with control sequence in the first hybridization. The interactions between target DNA and the DNA-conjugated PAA were investigated by fluorescence measurement. The interaction of fully matched target DNA with this immobilized DNA conjugated polymer has been studied at different ion strength conditions. SNP sequences as targets showed less than 15% the intensity of fully matched target DNA in the second hybridization, indicating that the gold surfaces coated with the DNA-conjugated PAA was highly specific to fully matched DNA. The DNA-conjugated PAA immobilized on a gold substrate is characterized by reduced nonspecific adsorption, due to less electrostatic repulsion as well as the polymer coating. Therefore, DNA-conjugated PAA can be used for probe DNA immobilization method.     

Osmoelastic coupling in biological structures: a comprehensive thermodynamic analysis of the osmotic response of phospholipid vesicles and a reevaluation of the "dehydration force" theory

A comprehensive thermodynamic analysis of the osmotic response of phospholipid vesicles is presented, using the Gibbs free energy of a vesicle suspension including the elastic contribution of the bilayer membrane. The results indicate that, in addition to the hydrostatic pressure difference across the membrane and the interbilayer pressure due to electrostatic repulsion, the elastic pressure arising from the coupling between the osmotic stress and the elasticity of the membrane (osmoelastic coupling) should participate in the osmotic response of phospholipid vesicles. The data of Cowley et al. [Cowley, A. C., Fuller, N. L., Rand, R. P., & Parsegian, V. A. (1978) Biochemistry 17, 3163-3168] and of Parsegian et al. [Parsegian, V. A., Fuller, N., & Rand, R. P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2750-2754] on the osmotic shrinkage of multilayer vesicles are discussed in terms of the elastic pressure and the interbilayer pressure, and the proposed "dehydration force" theory is reevaluated from the viewpoint of the present analysis.     

Loading/release behavior of (chitosan/DNA)n layer-by-layer films toward negatively charged anthraquinone and its application in electrochemical detection of natural DNA damage

In the present work, positively charged chitosan (CS) and negatively charged DNA were alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes, forming (CS/DNA)(n) layer-by-layer films. Cyclic voltammetry (CV) results showed that negatively charged electroactive probe, 9,10-anthraquinone-2,6-disulfonate (AQDS), could be loaded into the (CS/DNA)(n) films from its solution (1 mM at pH 7.0, containing 0.1 M NaCl), designated as (CS/DNA)(n)-AQDS, and then released from the films in blank buffers. The loading/release behavior of (CS/DNA)(n) films toward AQDS was found to be obviously different between double-stranded (dsDNA) and single-stranded DNA (ssDNA). The release rate of AQDS from (CS/dsDNA)(n) films was much slower than that from the ssDNA counterparts mainly because AQDS could be intercalated into the double helix structure of dsDNA despite the repulsion between likely charged AQDS and DNA. The loading/release behavior of (CS/DNA)(n) films toward AQDS in recognition of dsDNA and ssDNA was then successfully applied to electrochemically detect the damage of natural DNA caused by Fenton reaction. To further understand the essence of the interactions involved in the AQDS loading/release process for (CS/DNA)(n) films, comparison experiments were performed, in which either positively charged intercalator brilliant cresyl blue (BCB) was used to replace AQDS as the redox probe, or poly(diallyldimethylammonium) (PDDA) with relatively high positive charge density was used to replace CS as the constituent of layer-by-layer films with DNA. The loading/release behavior of DNA films toward electroactive intercalator may open new possibilities for dsDNA/ssDNA recognition and of DNA damage detection by electrochemistry.     

Replication protein A interactions with DNA. 2. Characterization of double-stranded DNA-binding/helix-destabilization activities and the role of the zinc-finger domain in DNA interactions

Human replication protein A (RPA) is a heterotrimeric single-stranded DNA-binding protein that is composed of subunits of 70, 32, and 14 kDa. RPA is required for multiple processes in cellular DNA metabolism. RPA has been reported to (1) bind with high affinity to single-stranded DNA (ssDNA), (2) bind specifically to certain double-stranded DNA (dsDNA) sequences, and (3) have DNA helix-destabilizing ("unwinding") activity. We have characterized both dsDNA binding and helix destabilization. The affinity of RPA for dsDNA was lower than that of ssDNA and precisely correlated with the melting temperature of the DNA fragment. The rates of helix destabilization and dsDNA binding were similar, and both were slow relative to the rate of binding ssDNA. We have previously mapped the regions required for ssDNA binding [Walther et al. (1999) Biochemistry 38, 3963-3973]. Here, we show that both helix-destabilization and dsDNA-binding activities map to the central DNA-binding domain of the 70-kDa subunit and that other domains of RPA are needed for optimal activity. We conclude that all types of RPA binding are manifestations of RPA ssDNA-binding activity and that dsDNA binding occurs when RPA destabilizes a region of dsDNA and binds to the resulting ssDNA. The 70-kDa subunit of all RPA homologues contains a highly conserved putative (C-X2-C-X13-C-X2-C) zinc finger. This motif directly interacts with DNA and contributes to dsDNA-binding/unwinding activity. Evidence is presented that a metal ion is required for the function of the zinc-finger motif.     

DNA-cationic surfactant interactions are different for double- and single-stranded DNA

The stability of DNA in solution and the phase behavior in mixtures with dodecyltrimethylammonium bromide (DTAB) were investigated. By means of circular dichroism, UV absorption, and differential scanning calorimetry, we found that for dilute solutions of DNA with no addition of salt the DNA molecules are in the single-stranded conformation, whereas the addition of a small amount of NaBr, 1 mM, is sufficient to stabilize the DNA double-helix. Furthermore, at higher DNA concentrations, native DNA becomes the most stable structure, which is due to a self-screening effect. By phase diagram determinations of the DNA-surfactant system, we found that the effect of salt on phase behavior mainly relates to a difference in interaction of the amphiphile between single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). The difference in association between ss and dsDNA with surfactants of different chain lengths can be interpreted in terms of an interplay between hydrophobic and electrostatic interactions, the latter being influenced by polymer flexibility. In this way, a nonmonotonic variation can be rationalized. A crossing of the phase separation lines with DNA concentration can be rationalized in terms of a change in relative stability of ss and dsDNA. The fact that ssDNA phase separates earlier than dsDNA in association with DTAB, may serve as a basis for a method of easily separating dsDNA from ssDNA by the addition of surfactant; this is verified as monitored by circular dichroism measurements.     

DNA hybridization detection with organic thin film transistors: toward fast and disposable DNA microarray chips

We demonstrate a novel DNA hybridization detection method with organic thin film transistors. DNA molecules are immobilized directly on the surface of organic semiconductors, producing an unambiguous doping-induced threshold voltage shift upon hybridization. With these shifts, single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) are differentiated successfully. This method is expected to result in higher sensitivity than the main competitive technology, ISFET-based sensors because of the direct exposure of DNA molecules to sensitive layers. Factors that influence sensor sensitivity have been analyzed and optimum conditions have been determined using statistically designed experiments. Under the optimum conditions, the maximum difference between saturation current ratios caused by ssDNA and dsDNA reaches as high as 70%. In order to make DNA detection fast, we also demonstrate rapid on-chip electrically enhanced hybridization using the TFTs. These technologies together will enable the realization of disposable, rapid-turnaround tools for field-deployable genomic diagnosis.     

The TREX1 double-stranded DNA degradation activity is defective in dominant mutations associated with autoimmune disease

Mutations in TREX1 have been linked to a spectrum of human autoimmune diseases including Aicardi-Goutières syndrome (AGS), familial chilblain lupus (FCL), systemic lupus erythematosus, and retinal vasculopathy and cerebral leukodystrophy. A common feature in these conditions is the frequent detection of antibodies to double-stranded DNA (dsDNA). TREX1 participates in a cell death process implicating this major 3' --> 5' exonuclease in genomic DNA degradation to minimize potential immune activation by persistent self DNA. The TREX1 D200N and D18N dominant heterozygous mutations were identified in AGS and FCL, respectively. TREX1 enzymes containing the D200N and D18N mutations were compared using nicked dsDNA and single-stranded DNA (ssDNA) degradation assays. The TREX1WT/D200N and TREX1WT/D18N heterodimers are completely deficient at degrading dsDNA and degrade ssDNA at an expected approximately 2-fold lower rate than TREX1WT enzyme. Further, the D200N- and D18N-containing TREX1 homo- and heterodimers inhibit the dsDNA degradation activity of TREX1WT enzyme, providing a likely explanation for the dominant phenotype of these TREX1 mutant alleles in AGS and FCL. By comparison, the TREX1 R114H homozygous mutation causes AGS and is found as a heterozygous mutation in systemic lupus erythematosus. The TREX1R114H/R114H homodimer has dysfunctional dsDNA and ssDNA degradation activities and does not detectibly inhibit the TREX1WT enzyme, whereas the TREX1WT/R114H heterodimer has a functional dsDNA degradation activity, supporting the recessive genetics of TREX1 R114H in AGS. The dysfunctional dsDNA degradation activities of these disease-related TREX1 mutants could account for persistent dsDNA from dying cells leading to an aberrant immune response in these clinically related disorders.     

Direct measurement of the force between two lipid bilayers and observation of their fusion

The force between two phosphatidylcholine bilayers is measured as a function of their separation, showing a strong hydration repulsion at short range, as previously reported by LeNeveu et al. (LeNeveu, D.M., Rand, R.P., Parsegian, V.A. and Gingell, D. (1977) (Biophys. J. 18, 209–230). The experimental technique also allows direct observation of the molecular process by which two bilayers fuse into one.     

电场驱动微悬臂生物传感器及对DNA 的快速、高灵敏度检测

微悬臂列阵传感器在生物检测方面具有快速、痕量和非标记的特性. 我们以镀金并在其上固定了 DNA 探针的微悬臂为正极,在靶杂交液槽内引入另一电极作为负极,构成电场驱动微悬臂 DNA 生物传感器. 对该传感器系统施加静电场,驱动 DNA 分子朝正极迁移,使溶液中的 DNA 分子富集在微悬臂上,促进 DNA 分子的杂交. 结果表明： a. DNA 在微悬臂上的杂交时间仅需 3 min,加快了微悬臂生物传感器对 DNA 分子的检测速度; b. 提高了微悬臂生物传感器的灵敏度,可以检测到皮克级的 DNA 分子.     

Antibody-nucleic acid complexes. Conformational and base specificities associated with spontaneously occurring poly- and monoclonal anti-DNA antibodies from autoimmune mice

An enzyme-linked immunosorbent assay (ELISA) was developed to characterize spontaneously occurring, mono-and polyclonal anti-DNA antibodies. The assay consists of adsorbing single- (ss) and double-stranded (ds) DNA and various nucleoside-bovine serum albumin conjugates (e.g., A-, G-BSA, etc.) to microtiter wells and assesses the ability of various antibodies to bind to these immobilized antigens. The conformational and base specificity of two monoclonal antibodies (designated MRss-1 BWds-3) was examined in this manner. The exclusive binding of MRss-1 to ssDNA and guanosine-BSA (G-BSA) confirms our previous findings [Munns, T.W., Liszewski, M.K., Tellam, J.T., Ebling, F. M., & Hahn, B.H. (1982) Biochemistry 21,2929-2936] that this antibody recognizes single-stranded nucleic acids by virtue of their guanine content. The extensive binding of BWds-3 to dsDNA, its limited binding to ssDNA, and complete absence of binding to nucleoside-BSA antigens implied a double-stranded conformational specificity. Further, competitive studies with naturally occurring and synthetic alternating copolymers indicated that BWds-3 preferentially recognized the native dsDNA antigens. ELISA analysis of the spontaneously occurring, polyclonal anti-DNA antibodies from MRL/lpr and NZB/NZW-F1 mice revealed that the majority of anti-ssDNA antibodies bound to nucleoside-BSA conjugates. Anti-G antibodies were most prominent in both strains of mice, yet lesser and more variable quantities of anti-A, -C, -U, and -T antibodies were also detected. Preadsorption of serum with G-BSA/Sepharose resulted in the complete removal of anti-G antibodies and a 60% reduction in anti-ssDNA antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermine-induced aggregation of DNA, nucleosome, and chromatin.

We have analyzed the conditions of aggregation or precipitation of DNA in four different states: double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), mononucleosome core particles (NCP), and H1-depleted chromatin fragments (ChF) in the presence of the multivalent cation spermine (4+). In an intermediate regime of DNA concentration, these conditions are identical for the four states. This result demonstrates that the mechanism involved is general from flexible chains to rigid rods and quasi-colloidal states. It is dominated by local electrostatic attractions that are considered, for instance, by the "ion-bridging" model. The onset of precipitation does not require the electroneutrality of the DNA chains. Above a given spermine concentration dsDNA aggregates remain neutral, whereas NCP aggregates turn positively charged. The difference is thought to originate from the extension of the positively charged proteic tails of the NCP. This suggests that local fluctuations of polyamine concentrations can induce either positively or negatively charged chromatin domains.     

Dominant mutation of the TREX1 exonuclease gene in lupus and Aicardi-Goutieres syndrome

TREX1 is a potent 3' → 5' exonuclease that degrades single- and double-stranded DNA (ssDNA and dsDNA). TREX1 mutations at amino acid positions Asp-18 and Asp-200 in familial chilblain lupus and Aicardi-Goutières syndrome elicit dominant immune dysfunction phenotypes. Failure to appropriately disassemble genomic DNA during normal cell death processes could lead to persistent DNA signals that trigger the innate immune response and autoimmunity. We tested this concept using dsDNA plasmid and chromatin and show that the TREX1 exonuclease locates 3' termini generated by endonucleases and degrades the nicked DNA polynucleotide. A competition assay was designed using TREX1 dominant mutants and variants to demonstrate that an intact DNA binding process, coupled with dysfunctional chemistry in the active sites, explains the dominant phenotypes in TREX1 D18N, D200N, and D200H alleles. The TREX1 residues Arg-174 and Lys-175 positioned adjacent to the active sites act with the Arg-128 residues positioned in the catalytic cores to facilitate melting of dsDNA and generate ssDNA for entry into the active sites. Metal-dependent ssDNA binding in the active sites of the catalytically inactive dominant TREX1 mutants contributes to DNA retention and precludes access to DNA 3' termini by active TREX1 enzyme. Thus, the dominant disease genetics exhibited by the TREX1 D18N, D200N, and D200H alleles parallel precisely the biochemical properties of these TREX1 dimers during dsDNA degradation of plasmid and chromatin DNA in vitro. These results support the concept that failure to degrade genomic dsDNA is a principal pathway of immune activation in TREX1-mediated autoimmune disease.     

Optimization of DNA-directed immobilization on mixed oligo(ethylene glycol) monolayers for immunodetection

The development of protein chips has suffered from problems regarding long-term protein stability and activity. We present a protein sensor surface for immunodetection that is prepared by a DNA-directed protein immobilization method on a mixed self-assembled monolayer (SAM). By this approach, an immobilized single-stranded DNA (ssDNA) surface can be transferred/modified into a protein chip by flowing in ssDNA-conjugated protein when the protein chip measurement is needed. Therefore, the long-term stability of the protein chip will not be a problem for various applications. We tried various compositions for the SAM layer, the length of the ssDNA spacer, the end-point nucleotide composition, and the processes of ssDNA immobilization of the SAM for an optimized condition for shifting the DNA chip to a protein chip. The evaluations were made by using surface plasmon resonance. Our results indicated that a 50:1 ratio of oligo(ethylene glycol) (OEG)/COOH-terminated OEG and DNA sequences with 20mer are the best conditions found here for making a protein chip via a DNA-directed immobilization (DDI) method. The designed end-point nucleotide composition contains a few guanines or cytosines, and ssDNA immobilization of the SAM by dehybridizing immobilized double-stranded DNA (dsDNA) can improve the hybridization efficiency.     

Electrostatic-assembly-driven formation of supramolecular rhombus microparticles and their application for fluorescent nucleic acid detection

In this paper, we report on the large-scale formation of supramolecular rhombus microparticles (SRMs) driven by electrostatic assembly, carried out by direct mixing of an aqueous HAuCl(4) solution and an ethanol solution of 4,4'-bipyridine at room temperature. We further demonstrate their use as an effective fluorescent sensing platform for nucleic acid detection with a high selectivity down to single-base mismatch. The general concept used in this approach is based on adsorption of the fluorescently labeled single-stranded DNA (ssDNA) probe by SRM, which is accompanied by substantial fluorescence quenching. In the following assay, specific hybridization with its target to form double-stranded DNA (dsDNA) results in desorption of ssDNA from SRM surface and subsequent fluorescence recovery.