Islet surface modification with urokinase through DNA hybridization

Transplantation of islets of Langerhans (islets) has been proposed as a safe, effective approach to treating patients with insulin-dependent diabetes mellitus (type I diabetes). It has been reported, however, that many islets are lost in the early phase after intraportal transplantation by instant blood coagulation-mediated inflammatory reactions. In this study, DNA hybridization was applied to conjugate the fibrinolytic enzyme urokinase on the islet surface. We synthesized amphiphilic polymers, PEG-lipids carrying oligo(dT)(20) (oligo(dT)(20)-PEG-lipid; PEG MW = 5000) and urokinase (UK) carrying oligo(dA)(20). The oligo(dT)(20)-PEG-lipid was spontaneously incorporated into the cell membrane through interactions between the hydrophobic parts of the PEG-lipids and the lipid bilayer, and UK was conjugated on the cell surface through DNA hybridization between oligo(dT)(20) on the cell and complementary oligo(dA)(20) on the UK. The activity of UK was maintained on the islet surface. The surface modification with UK did not influence islet morphology or islet ability to secrete insulin in response to changes in glucose concentration. No practical volume increase was observed with our method, indicating that islet graft loss could be suppressed at the early stage of intraportal islet transplantation.     

DNA hybridization biosensors using polylysine modified SPCEs

Two electrochemical DNA hybridization biosensors (genosensors) for the detection of a 30-mer sequence unique to severe acute respiratory syndrome (SARS) virus are described in this work. Both genosensors rely on the hybridization of the oligonucleotide target with its complementary probe, which is immobilized on positively charged polylysine modified screen-printed carbon electrodes (SPCEs), through electrostatic interactions. In one design, a biotinylated target is used and the detection of the hybridization reaction is monitored using alkaline phosphatase labeled streptavidin (S-AP). This enzyme catalyzes the hydrolysis of the substrate 3-indoxyl phosphate (3-IP) to indigo, which is then solubilized to indigo carmine and detected by means of cyclic voltammetry (CV). In the other design, the target is labeled using an Au(I) complex, sodium aurothiomalate, and the duplex formation is detected by measuring, for first time, the current generated by the hydrogen evolution catalyzed by the gold label. Using 30 min of hybridization time, a detection limit of 8 pM is calculated for the enzymatic genosensor. Although this good sensitivity cannot be reached with the metal label (0.5 nM), the use of this label allows a considerable decrease of the analysis time. Both genosensors do not require the modification of the oligonucleotide probe and using stringent experimental conditions (60 min of hybridization time and 50% formamide in the hybridization buffer) can discriminate between a complementary oligonucleotide and an oligonucleotide with a three-base mismatch.     

The effect of surface probe density on DNA hybridization

The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.     

Competitive displacement of DNA during surface hybridization

Using real-time dual-color fluorescence detection, we have experimentally tracked individual target species during competitive DNA surface hybridization in a two-component sample. Our experimental results demonstrate displacement of the lower affinity species by the higher affinity species and corroborate recent theoretical models describing competitive DNA surface hybridization. Competition at probe sites complementary to one of the two DNA species was monitored in separate experiments for two different target pairs. Each pair differs in sequence by a single nucleotide polymorphism, and one pair includes a folding target. We propose a mechanistic interpretation of the differences between hybridization curves of targets in multi-component and single-component experiments.     

Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We discuss the implications with respect to DNA microarray detection.     

Electrochemical impedance detection of DNA hybridization based on dendrimer modified electrode

Bioactive ultrathin films with the incorporation of amino-terminated G4 PAMAM dendrimers have been prepared via layer-by-layer self-assembly methods on a gold electrode and used for the DNA hybridization analysis. Surface plasmon resonance (SPR), X-ray photoelectron spectroscopy (XPS), and electrochemical impedance spectroscopy (EIS) are used to characterize the successful construction of the multicomponent film on the gold substrate. The dendrimer-modified surfaces improve the immobilization capacity of the probe DNA greatly, compared to the AET (2-aminoethanethiol) SAM sensor surfaces without dendrimer molecules. DNA hybridization analysis is monitored by EIS. The dendrimer-based electrochemical impedance DNA biosensor shows high sensitivity and selectivity for DNA hybridization assay. The multicomponent films also display a high stability during repeated regeneration and hybridization cycles.     

A modified hybridization protocol for low-density diagnostic DNA microarrays

To promote the application of DNa microarrays for clinical diagnosis, the problems of cross-hybridization and low signal intensity in the hybridization processes has been addressed. We tested a new hybridization protocol for low-density diagnostic DNA microarrays, by skipping the purification step during sample labeling, while elevating the hybridization temperature from 42°C to 52°C, adding a step of distilled water rinsing immediately after hybridization and before the low stringency washing steps. It was found that the modified hybridization protocol works well in our study, which increased detection sensitivity and eliminated nonspecific signals.     

Use of surface plasmon-coupled emission to measure DNA hybridization

The authors describe a new approach to measuring DNA hybridization based on surface plasmon-coupled emission (SPCE). SPCE is the resonance coupling of excited fluorophores with electron motions in thin metal films, resulting in efficient transfer of energy through the film and radiation into the glass substrate. The authors evaluated the use of SPCE for detection of DNA hybridization. An unlabeled capture biotinylated oligonucleotide was attached near the surface of a thin (50 nm) silver film using streptavidin. The authors then measured the emission intensity of single-stranded Cy5-labeled DNA upon binding to a complementary oligomer attached to a silver film. Hybridization could be detected by an increase in SPCE, which appeared as light radiated into the substrate at a sharply defined angle near 73 degrees from the normal. The largest signals were observed when the excitation angle of incidence equaled the surface plasmon wavelength, but directional emission was also observed without excitation by the surface plasmon evanescent field. The increased intensity is due to proximity to the metal surface, so that hybridization can be detected without a change in the quantum yield of the fluorophore. These results indicate that SPCE can provide highly sensitive real-time measurement of DNA hybridization.     

Enhancement of DNA immobilization and hybridization on gold electrode modified by nanogold aggregates

Gold electrodes modified by nanogold aggregates (nanogold electrode) were obtained by the electrodeposition of gold nanoparticles onto planar gold electrode. The Electrochemical response of single-stranded DNA (ssDNA) probe immobilization and hybridization with target DNA was measured by cyclic voltammograms (CV) using methylene blue (MB) as an electroactive indicator. An improving method using long sequence target DNA, which greatly enhanced the response signal during hybridization, was studied. Nanogold electrodes could largely increase the immobilization amount of ssDNA probe. The hybridization amount of target DNA could be increased several times for the manifold nanogold electrodes. The detection limit of nanogold electrode for the complementary 16-mer oligonucleotide (target DNA1) and long sequence 55-mer oligonucleotide (target DNA2) could reach the concentration of 10(-9) mol/L and 10(-11) mol/L, respectively, which are far more sensitive than that of the planar electrode.     

Accurate measurement of psoralen-crosslinked DNA: direct biochemical measurements and indirect measurement by hybridization

This paper evaluates methods to measure crosslinkage due to psoralen plus light in total DNA and in specific sequences. DNA exposed in cells or in vitro to a bifunctional psoralen and near ultraviolet light accumulates interstrand crosslinks. Crosslinkage is the DNA mass fraction that is attached in both strands to a crosslink. We show here biochemical methods to measure psoralen photocrosslinkage accurately in total DNA. We also describe methods to measure photocrosslinkage indirectly, in specific sequences, by nucleic acid hybridization. We show that a single 4,5',8-trimethylpsoralen (TMP) crosslink causes at least 50 kbp of alkali-denatured DNA contiguous in both strands with it to snap back into the duplex form when the denatured preparation is returned to neutral pH. This process was so efficient that the DNA was not nicked by the single-strand nuclease S1 at 100-fold excess after snapping back. Uncrosslinked DNA was digested to acid-soluble material by the enzyme. Crosslinkage therefore equals the fraction of S1-resistant nucleotide in this kind of experiment. We alkali-denatured DNA samples crosslinked to varying degrees by varying TMP concentration at constant light exposure. We then measured crosslinkage by ethidium bromide (EtBr) fluorometry at pH 11.8; by EtBr fluorometry at neutral pH of S1 digests of the DNA; and by the fraction of radioactivity remaining acid insoluble in S1-digests of DNA labeled uniformly with [3H]deoxythymidine. These assays measure distinct physical properties of crosslinked DNA. Numerical agreement is expected only when all three measurements are accurate. Under optimum conditions, the three methods yielded identical results over the range of measurement. Using alkaline EtBr fluorescence in crude cell lysates, we detected crosslinks at frequencies in the range of 1.6 X 10(-7) per base pair. These levels were compatible with cell survival, attesting to the sensitivity of the measurement system. Crosslinkage affected hybridization as well. One crosslink prevented all alkali-denatured DNA contiguous in both strands with it from hybridizing to complementary DNA either on solid supports or in solution. Strand-length effects on crosslinkage and on reassociation caused solution hybridization levels to exceed those predicted by simple theory. In a quantitative, dot-blotting assay hybridization was linear up to membrane saturation by denatured, uncrosslinked DNA of any strand length.(ABSTRACT TRUNCATED AT 400 WORDS)     

Covalently coupling the antibody on an amine-self-assembled gold surface to probe hyaluronan-binding protein with capacitance measurement

Hyaluronan-binding proteins (HABPs), the important structural components of extracellular matrices, served important structural and regulatory functions during development and in maintaining adult tissue homestats. A sensitive, specific and rapid-responsing immunosensor to probe hyaluronan-binding cartilage protein was presented in this work. The novel immunosensor supplied a label-free detection method for HABP, which was based on measuring the capacitance change in-between the unlabeled HABP (antigen) and rabbit-anti-HABP (Ra-HABP, antibody). The HABP immunosensor was prepared by covalently coupling Ra-HABP on an amine-self-assembled gold surface with glutaraldehyde. The capacitance change corresponding to the concentration of HABP, the target antigen, was evaluated by an electrochemical approach called potentiostatic-step in microseconds. The immunosensor showed a specific response to HABP in the range 10-1000 ng/ml. The presented work supplied a promising clinical screening method.     

Nanogold hollow balls with dendritic surface for hybridization of DNA

Au hollow balls are fabricated by adsorption of gold 3.5 nm in diameter onto a mixed vesicle composed of mixed polymerized diacetylene which made of negative charged 10,12-pentacosadiynoic acid (PCDA) and positive charged 10,12-pentacosadiynoic acid 2'-aminoethylamide (PCDA-NH(2)). The morphology of these hollow spheres could be controlled by changing the ratio of PCDA and PCDA-NH(2) and the immobilization and hybridization ability of the gold hollow ball have been investigated using a quartz crystal microbalance (QCM). It was found that a dendritic surface in an appropriate ratio existed. The hybridization amount of target DNA is about three to five times for the Au-mixed hollow ball at an optimal ratio (PCDA/PCDA-NH(2)=1/3) as compared with that for pure Au-PCDA-NH(2), though the immobilization amount of ssDNA on these two samples are almost the same, and the detected limitation of target DNA is extend from 10(-9) to 10(-12) M. The stability against breakage by transportation, combined with the simplicity and efficiency of detection, would offer an important advantage over unpolymerized one. This result shows the possibility to control the morphology and surface of nanogold hollow spheres by changing the ratio of PCDA and PCDA-NH(2) for the develop of a better DNA detection assay, further proving the idea that low surface coverage and higher DNA probe to target DNA ratios lead to optimal hybridization.     

The isolation and measurement of tRNAmeti using RNA/DNA hybridization

A pBR322 plasmid containing the initiator tRNAmet gene of Xenopus (pt145 - donated by Stuart Clarkson) will specifically bind to mouse initiator tRNAmet (tRNAmeti) when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band (95%) and a minor band. The hybridizing tRNA has been identified as initiator tRNAmet by RNA sequencing. Hybridization of tRNAtotal to another plasmid containing the Xenopus gene for tRNAasn results in two bound species with different electrophoretic mobilities than the tRNA bound to the initiator tRNAmet gene. pt145 has been used to measure the steady state concentration of initiator tRNAmet in the uninduced and erythroid Friend cell, and in the unfertilized egg and 21 h blastula of the sea urchin. Initiator tRNAmet represents 0.91% and 0.52% of the tRNA populations extracted from uninduced and erythroid Friend cells, respectively. Based upon the total tRNA content per cell, there is a 3.8 fold decrease in initiator tRNAmet per cell during erythroid differentiation. tRNA extracted from unfertilized eggs and 21 h blastula of the sea urchin both have 0.5% of total tRNA as initiator tRNAmet (approximately 1.5 pg).     

Frequency dependent and surface characterization of DNA immobilization and hybridization

The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued.     

Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison

The hybridization kinetics for a series of designed 25mer probe–target pairs having varying degrees of secondary structure have been measured by UV absorbance and surface plasmon resonance (SPR) spectroscopy in solution and on the surface, respectively. Kinetic rate constants derived from the resultant data decrease with increasing probe and target secondary structure similarly in both solution and surface environments. Specifically, addition of three intramolecular base pairs in the probe and target structure slow hybridization by a factor of two. For individual strands containing four or more intramolecular base pairs, hybridization cannot be described by a traditional two-state model in solution-phase nor on the surface. Surface hybridization rates are also 20- to 40-fold slower than solution-phase rates for identical sequences and conditions. These quantitative findings may have implications for the design of better biosensors, particularly those using probes with deliberate secondary structure.     

Molecular evidence on maize specific DNA fragment transferred into wheat through sexual hybridization

Ｗｈｅａｔｈａｐｌｏｉｄｃａｎｂｅｐｒｏｄｕｃｅｄｂｙｅｍｂｒｙｏｒｅｓｃｕｅａｆｔｅｒｗｈｅａｔａｎｄｍａｉｚｅｃｒｏｓｓｓｉｎｃｅｔｈｅｍａｉｚｅｃｈｒｏｍｏｓｏｍｅｓａｒｅｒａｐｉｄｌｙｅｌｉｍｉｎａｔｅｄｆｒｏｍｔｈｅｅａｒｌｙｚｙｇｏｔｉｃｅｍｂｒｙｏｓ[1,2].Ｕｓｕａｌｌｙ2%—5%ｏｆｗｈｅａｔｄｏｕｂｌｅｄｈａｐｌｏｉｄｓｈｏｗｓａｇｒｏｎｏｍｉｃｖａｒｉａｔｉｏｎｓ,ｂｕｔｎｏｏｂｖｉｏｕｓｍｏｒｐｈｏｌｏｇｉｃａｌｔｒａｉｔｆｒｏｍｍａｉｚｅｉｓｏｂｓｅｒｖｅｄ.Ｔｈｅｙｗｅｒｅｃｏ…     

Monitoring human exposure to carcinogens by DNA adduct measurement

Sensitive immunologic techniques are now available for the detection and quantitation of carcinogen-DNA adducts. We have developed a number of specific monoclonal antibodies which recognize DNA modified by particular carcinogens, including benzo[a]-py-rene, aminopyrene, 8-methoxypsoralen plus UVA light and vinyl chloride. These antibodies can be used in competitive enzyme-linked immunosorbent assays to quantitate adducts in DNA isolated from biological samples. Samples from treated animals as well as from humans with occupational or environmental exposure to carcinogens have been studied. In addition, antibodies can be used in indirect immunohistochemical studies to localize adduct formation in various tissues or cell types.Abbreviations BPDE-1 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - CTCL Cutaneous T cell lymphoma - ELISA enzyme linked immunosorbent assay - 8-MOP 8-methoxypsoralen - PUVA 8-MOP plus UVA irradiation     

Grating-based surface plasmon resonance detection of core-shell nanoparticle mediated DNA hybridization

In this report, we have investigated enhanced surface plasmon resonance (SPR) detection of DNA hybridization using gold core - silica shell nanoparticles in localized plasmonic fields. The plasmonic fields were localized by periodic linear gratings. Experimental results measured for hybridization of 24-mer single-stranded DNA oligomers suggest that core-shell nanoparticles (CSNPs) on gratings of 400 nm period provide enhanced optical signatures by 36 times over conventional thin film-based SPR detection. CSNP-mediated DNA hybridization produced 3 times larger angular shift compared to gold nanoparticles of the same core size. We have also analyzed the effect of structural variation. The enhancement using CSNPs was associated with increased surface area and index contrast that is combined by improved plasmon coupling with localized fields on gratings. The combined approach for conjugated measurement of a biomolecular interaction on grating structures is expected to lower the limit of detection to the order of a few tens of fg/mm(2).     

Introduction of antioxidant-loaded liposomes into endothelial cell surfaces through DNA hybridization

Ischemia–reperfusion damage is a problem in organ transplantation. Reactive oxygen species are produced in cells by blood-mediated reactions at the time of blood reperfusion. In this study, we developed a method to immobilize and internalize antioxidants in endothelial cells, using vitamin E-loaded liposomes. The liposomes loaded with vitamin E and human umbilical vein endothelial cells (HUVECs) were modified with poly(ethylene glycol)–phospholipid conjugates carrying 20-mer of deoxyadenylic acid (oligo(dA)₂₀) and 20-mer of complementary deoxythymidylic acid (oligo(dT)₂₀), respectively. The liposomes were effectively immobilized on HUVECs through DNA hybridization between oligo(dA)₂₀ and oligo(dT)₂₀. The liposomes loaded with vitamin E were gradually internalized into HUVECs. Then, the cells were treated with antimycin A to induce oxidative stress. We found the amount of reactive oxygen species was greatly reduced in HUVECs carrying vitamin E-loaded liposomes.