Reversible photopadlocking on double-stranded DNA

We describe a highly efficient method for reversible photocircularization of oligonucleotide (ODN) on a double-stranded DNA template: 5-carboxyvinyl-2'-deoxyuridine-containing ODN was reversibly circularized around the target sequence of the double-stranded plasmid DNA resulting in formation of a catenated plasmid.     

Capillary electrophoresis of double-stranded DNA in an untreated capillary.

Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.     

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.     

An ion conductor that recognizes osmotically-stressed phospholipid bilayers

A synthetic ion conductor (1), derived from cholic acid and spermine, has been found capable of recognizing osmotic stress in liposomes made from 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine [(C16:1)PC]. Thus, when large unilamellar vesicles of (C16:1)PC are placed under hypotonic conditions, the Na+/Li+ transport activity of 1 increases by as much as 1 order of magnitude, relative to isotonic conditions     

Microdetermination of double-stranded DNA by linear sweep voltammetry with phenosafranine.

A novel voltammetric method for the determination of microamounts of fish sperm double-stranded (ds) DNA based on its interaction with phenosafranine (PSF) is proposed in this paper. In a pH 3.5 Britton-Robinson (B-R) buffer solution, PSF had a well-defined second-order derivative linear-sweep voltammetric reductive peak at -0.32 V (vs. SCE) on a mercury electrode. After the addition of dsDNA into the PSF solution, the reductive peak current decreased significantly without a shift of the peak potential, and no new peak appeared. The experiment results showed that a new supramolecular complex was formed after the interaction of dsDNA with PSF, which resulted in a decrease of the diffusion coefficient, and then a decrease of the reductive peak current. The interaction conditions and the electrochemical detection conditions were carefully investigated. Under the optimal conditions, the decrease of the peak current was proportional to the dsDNA concentration in the range 1.0 - 40.0 microg/mL with the linear regression equation DeltaI(p)'(nA) = 32.59C(microg/mL) - 4.03 (n = 13, gamma = 0.998) and a detection limit of 0.25 microg/mL (3 sigma). The interaction mechanism was considered based on the aggregation of the dsDNA-PSF supramolecular complex; the stoichiometry of this supramolecular complex was calculated based on voltammetric data with a binding number of 3 and a binding constant of 2.76 x 10(12). This method was successfully applied to the determination of synthetic samples and the polymerase chain reaction (PCR) product of the nopaline synthase gene (NOS) DNA from genetically modified organisms (GMOs) with satisfactory results.     

Short lexitropsin that recognizes the DNA minor groove at 5'-ACTAGT-3': understanding the role of isopropyl-thiazole

Isopropyl-thiazole ((iPr)Th) represents a new addition to the building blocks of nucleic acid minor groove-binding molecules. The DNA decamer duplex d(CGACTAGTCG)(2) is bound by a short lexitropsin of sequence formyl-PyPy(iPr)Th-Dp (where Py represents N-methyl pyrrole, (iPr)Th represents thiazole with an isopropyl group attached, and Dp represents dimethylaminopropyl). NMR data indicate ligand binding in the minor groove of DNA to the sequence 5'-ACT(5)AG(7)T-3' at a 2:1 ratio of ligand to DNA duplex. Ligand binding, assisted by the enhanced hydrophobicity of the (iPr)Th group, occurs in a head-to-tail fashion, the formyl headgroups being located toward the 5'-ends of the DNA sequence. Sequence reading is augmented through hydrogen bond formation between the exocyclic amine protons of G(7) and the (iPr)Th nitrogen, which lies on the minor groove floor. The B(I)/B(II) DNA backbone equilibrium is altered at the T(5) 3'-phosphate position to accommodate a B(II) configuration. The ligands bind in a staggered mode with respect to one another creating a six base pair DNA reading frame. The introduction of a new DNA sequence-reading element into the recognition jigsaw, combined with an extended reading frame for a small lexitropsin with enhanced hydrophobicity, holds great promise in the development of new, potentially commercially viable drug lead candidates for gene targeting.     

Sieving of double-stranded DNA during agarose gel electrophoresis

Agarose gel electrophoresis is used to fractionate linear, double-stranded DNA by its length. Sieving of the gel is the cause of this fractionation and has been investigated by developing theoretical models and by quantifying sieving observed during electrophoresis. Here are reviewed the following aspects of the fractionation of linear, double-stranded DNA by agarose gel electrophoresis: (1) the basic observations that qualitatively characterize these fractionations, (2) evidence for the deformation of DNA's random coil, (3) quantitative analysis of the relationship of observed electrophoretic mobility to the DNA's length, (4) theoretical models that have been developed to explain data presented in Sections 1-3, (5) observations not yet quantitatively explained by models, and (6) some aspects of the use of a variable electrical field (pulsed-field gel electrophoresis) to improve separations.     

Electrophoretic chip for high-fidelity fractionation of double-stranded DNA

We report the high fidelity, on-chip fractionation of selected segments from an electrophoretic flow of separated fragments. dsDNA fragments (10-330 base pairs (bp)) were initially separated using a 6.5 cm long channel with an electric field strength of 150 V/cm. As an example of the fractionation process, a target fragment of 20 bp was selected and extracted from the separation channel. The extraction was confirmed and evaluated by fluorescence imaging. High resolution and extraction fidelity were achieved by introducing new procedures for (i) extraction channel-blocking and (ii) segment transfer with cleaning. These procedures are necessary for the development of a practical, fully automated multitarget fractionation electrophoretic chip. A kind of CCD image processing method was introduced to monitor, control, and evaluate the procedure of fractionation. The resolution limits of the separation and extraction are discussed.     

The detection of intact double-stranded DNA by MALDI

DNA fragments have been analyzed by matrix-assisted laser desorption ionization (MALDI) and electrospray mass spectrometry. In many cases, only the single-stranded oligonucleotides have been detected. Recently, spectra of intact double-stranded DNA have been obtained in both electrospray and massive cluster impact ionization. We show here the first MALDI spectra of intact double-stranded DNA (EcoR1 adaptor 12/16) that is clearly not due to nonspecific dimer formation. 6-Aza-2-thiothymine was used as the matrix in the presence of ammonium citrate. Via the same procedure but with other matrices commonly employed for oligonucleotide analysis, the intact DNA duplex was not detected. No sign of the homodimer of either of the single strands is observed. Although the spectrum also shows peaks attributable to each of the single strands, these are demonstrated to arise from the DNA solution and not the sample preparation or desorption process.     

Interaction of double-stranded DNA inside single-walled carbon nanotubes

Deoxyribonucleic acid (DNA) is the genetic material for all living organisms, and as a nanostructure offers the means to create novel nanoscale devices. In this paper, we investigate the interaction of deoxyribonucleic acid inside single-walled carbon nanotubes. Using classical applied mathematical modeling, we derive explicit analytical expressions for the encapsulation of DNA inside single-walled carbon nanotubes. We adopt the 6–12 Lennard–Jones potential function together with the continuous approach to determine the preferred minimum energy position of the dsDNA molecule inside a single-walled carbon nanotube, so as to predict its location with reference to the cross-section of the carbon nanotube. An analytical expression is obtained in terms of hypergeometric functions which provides a computationally rapid procedure to determine critical numerical values. We observe that the double-strand DNA can be encapsulated inside a single-walled carbon nanotube with a radius larger than 12.30 ?, and we show that the optimal single-walled carbon nanotube to enclose a double-stranded DNA has radius 12.8 ?.     

Electrocatalytic intercalator-induced winding of double-stranded DNA with polyaniline

The intercalation of doxorubicin into double-stranded DNA stimulates the electocatalyzed oxidation of aniline to polyaniline and its winding on the DNA template.     

Metal-containing trifurcate receptor that recognizes and senses citrate in water

[reaction: see text] The binding tendencies toward carboxylates of a trifurcated receptor containing three copper(II)-cyclam subunits have been investigated in pure water, through the displacement of a fluorescent indicator. The receptor is tailor-made for the recognition of tricarboxylates, e.g., citrate, whose three negatively charged oxygen atoms interact with the three coordinatively unsaturated Cu(II) centers.     

Positive ion electrospray ionization mass spectrometry of double-stranded DNA/drug complexes.

Positive ion electrospray ionization mass spectra of 16 base-pair double-stranded (ds)DNA have been obtained with essentially no ions from single-stranded DNA present. Single-stranded DNA was minimized by: (1) careful choice of DNA sequences; (2) the use of a relatively high salt concentration (0.1 M ammonium acetate, pH 8.5), and, (3) a low desolvation temperature (40 degrees C). Similarly, ESI-MS complexes of dsDNA with cisplatin, daunomycin and distamycin were obtained that contained only negligible amounts of single-stranded DNA. The complexes with daunomycin and distamycin were more stable to strand separation in the gas phase than dsDNA alone. This is in agreement with solution studies and with other recent gas phase results. These data contrast with many earlier ESI-MS studies of dsDNA and DNA/drug complexes in which ions from ssDNA are also normally observed.     

Trastuzumab quantification in serum: a new,rapid, robust ELISA assay based on a mimetic peptide that specifically recognizes trastuzumab

Trastuzumab, a humanized monoclonal antibody directed against the epidermal growth factor receptor 2 (HER2), is a milestone in the treatment of HER2-overexpressing breast cancer patients. An enzyme-linked immunosorbent assay (ELISA) for trastuzumab has been developed for routine use in the laboratory to support clinical and pharmacokinetic studies to optimize therapy. The method relies on an antigen peptide linked to a 96-well plate via the streptavidin/biotin system. The peptide sequence mimics the extracellular portion of the HER2 receptor that is recognized by trastuzumab. The calibration range of the assay is 10 to 360 ng/mL per well, corresponding to a trastuzumab serum concentration from 5 to 180 μg/mL with a lower limit of quantification of 10 μg/mL. Validation results demonstrate that trastuzumab can be accurately and precisely quantified in human serum using this assay. The procedure was also tested in sera obtained from breast cancer patients to evaluate trastuzumab serum levels, confirming the applicability of method that could be a valid assay to use in daily laboratory practice.     

Optimization of on-chip elongation for fabricating double-stranded DNA microarrays

The sequence-specific recognitions between DNA and proteins are playing important roles in many biological functions. The double-stranded DNA microarrays (dsDNA microarrays) can be used to study the sequence-specific recognitions between DNAs and proteins in highly parallel way. In this paper, two different elongation processes in forming dsDNA from the immobilized oligonucleotides have been compared in order to optimize the fabrication of dsDNA microarrays: (1) elongation from the hairpins formed by the self-hybridized oligonucleatides spotted on a glass; (2) elongation from the complementary primers hybridized on the spotted oligonucleatides. The results suggested that the dsDNA probes density produced by the hybridized-primer extension was about four times lower than those by the self-hybridized hairpins. Meanwhile, in order to reduce the cost of dsDNA microarrays, we have replaced the Klenow DNA polymerase with Taq DNA polymerase, and optimized the reaction conditions of on-chip elongation. Our experiements showed that the elongation temperature of 50 °C and the Mg²⁺ concentration of 2.5 mM are the optimized conditions in elongation with Taq DNA polymerase. A dsDNA microarray has been successfully constructed with the above method to detect NF-kB protein.     

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.