Acceleration of nucleic acid hybridization on DNA microarrays driven by pH tunable modifications.

A series of peptides containing histidine residues were designed as potential hybridization rate enhancers within a polymeric matrix of DNA microarrays. The polymeric matrix modified with these peptides showed strong attraction to DNA molecules under conditions of induction. DNA probes on the peptide-modified sites rapidly hybridized to their complementary targets with single base pair mismatch discrimination.

Oligonucleotides form a duplex with non-helical properties on a positively charged surface.

The double helix is known to form as a result of hybridization of complementary nucleic acid strands in aqueous solution. In the helix the negatively charged phosphate groups of each nucleic acid strand are distributed helically on the outside of the duplex and are available for interaction with cationic groups. Cation-coated glass surfaces are now widely used in biotechnology, especially for covalent attachment of cDNAs and oligonucleotides as surface-bound probes on microarrays. These cationic surfaces can bind the nucleic acid backbone electrostatically through the phosphate moiety. Here we describe a simple method to fabricate DNA microarrays based upon adsorptive rather than covalent attachment of oligonucleotides to a positively charged surface. We show that such adsorbed oligonucleotide probes form a densely packed monolayer, which retains capacity for base pair-specific hybridization with a solution state DNA target strand to form the duplex. However, both strand dissociation kinetics and the rate of DNase digestion suggest, on symmetry grounds, that the target DNA binds to such adsorbed oligonucleotides to form a highly asymmetrical and unwound duplex. Thus, it is suggested that, at least on a charged surface, a non-helical DNA duplex can be the preferred structural isomer under standard biochemical conditions.

Simultaneous multianalyte ELISA performed on a microarray platform.

BACKGROUND: A logical progression of the widely used microtiter plate ELISA is toward a protein array format that allows simultaneous detection of multiple analytes at multiple array addresses within a single well. Here we describe the construction and use of such a multiplex ELISA to measure prostate-specific antigen (PSA), alpha1-antichymotrypsin-bound PSA (PSA-ACT), and interleukin-6 (IL-6). METHODS: We silanized glass plates and printed the appropriate capture antibodies to allow for the construction of "sandwich" ELISA quantification assays. We examined specificity of the assay for appropriate antigen, assembled calibration curves, and obtained PSA concentrations for 14 human serum samples. We compared the serum PSA concentrations derived through the use of our array with values obtained independently using a standard ELISA method. RESULTS: R2 values generated by our microarray for the PSA and PSA-ACT calibration curves were 0.989 and 0.979, respectively. Analyte concentrations used for the construction of these curves were 0.31-20 microg of protein/L of diluent. IL-6 calibration curve concentrations were 4.9-300 ng of IL-6/L of diluent. The R2 value for the IL-6 calibration curve was 0.983. The 14 human serum samples screened by this micro-ELISA technique for PSA concentrations generated a regression equation (linear) with a slope of 0.83 +/- 0.10 and intercept of 0.74 +/- 0.70 (R2 = 0.88). CONCLUSIONS: Multiplexed ELISA arrays are a feasible option for analyte quantification in complex biologic samples.

DNA microarrays based on noncovalent oligonucleotide attachment and hybridization in two dimensions.

Short oligonucleotide probes have been linked to a solid support by simple electrostatic adsorption onto a positively charged surface film. Attachment was obtained by microfluidic application of unmodified oligonucleotides in distilled water onto amino-silanized glass. It has been demonstrated that an extremely stable monolayer of oligonucleotide is obtained by this method, at a density of about 10(11) molecules/mm(2), which approaches the limit expected for a two-dimensional closest-packed array. Application of oligonucleotide by adsorption is followed by capping with acetic anhydride in the vapor phase, and then capping with succinic anhydride in solution to form a surface with weak negative charge. The capping method has been successfully employed for microarray fabrication and for the analysis of single nucleotide polymorphisms in the k-ras gene. The data reveal that, subsequent to capping, the adsorptive association of oligonucleotide to the surface yields a probe layer which is capable of single nucleotide base mismatch discrimination and high apparent binding affinity.

Nearly instantaneous, cation-independent, high selectivity nucleic acid hybridization to DNA microarrays.

Hybridization rate enhancement has been demonstrated for high molecular weight DNA target binding to a microarray. Microarrays were fabricated using biotin-modified oligonucleotides complexed with streptavidin (SA), which serves as an attachment to the underlying surface. It is shown that at low salt and pH 5, where SA develops a positive charge, duplex formation becomes at least 80-fold faster than seen under standard conditions, where SA is neutral or anionic. Duplex formation becomes independent of solution state cation concentration in the low pH state, under conditions where specificity remains high. The utility of such applied surface science is discussed.

16(R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes.

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16(R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108+/-26 pg/10(8) cells) and 20-HETE (341+/-69 pg/10(8) cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16(R)-HETE via a cytochrome P450 omega- and omega-4 oxygenase(s).

MerMade: an oligodeoxyribonucleotide synthesizer for high throughput oligonucleotide production in dual 96-well plates.

We have designed and constructed a machine that synthesizes two standard 96-well plates of oligonucleotides in a single run using standard phosphoramidite chemistry. The machine is capable of making a combination of standard, degenerate, or modified oligos in a single plate. The run time is typically 17 hr for two plates of 20-mers and a reaction scale of 40 nM. The reaction vessel is a standard polypropylene 96-well plate with a hole drilled in the bottom of each well. The two plates are placed in separate vacuum chucks and mounted on an xy table. Each well in turn is positioned under the appropriate reagent injection line and the reagent is injected by switching a dedicated valve. All aspects of machine operation are controlled by a Macintosh computer, which also guides the user through the startup and shutdown procedures, provides a continuous update on the status of the run, and facilitates a number of service procedures that need to be carried out periodically. Over 25,000 oligos have been synthesized for use in dye terminator sequencing reactions, polymerase chain reactions (PCRs), hybridization, and RT-PCR. Oligos up to 100 bases in length have been made with a coupling efficiency in excess of 99%. These machines, working in conjunction with our oligo prediction code are particularly well suited to application in automated high throughput genomic sequencing.

Post-receptor signal transduction and regulation of 14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET) binding in U-937 cells.

14(R),15(S)-epoxyeicosatrienoic acid (14,15-EET), a cytochrome P-450 monooxygenase (epoxygenase) metabolite of arachidonic acid has been reported to induce adhesion of a monocyte cell line (U-937) to cultured endothelial cells. In this study, we identified a population of specific, high affinity binding sites for 14(R),15(S)-EET in U-937 cell surface with Kd of 13.84 +/- 2.58 nM and Bmax of 3.54 +/- 0.28 pmol/10(6) cells. The specific binding of [3H]-14,15-EET on U-937 cells is more effectively displaced by 14(R),15(S)-EET than the 14(S),15(R)-isomer thus indicating stereospecificity. The binding was sensitive to various protease treatments suggesting the binding site is protein in nature. 14,15-EET binding in U937 cells is attenuated by cholera toxin (CT) and dibutyryl cAMP. Mean binding site density (Bmax) decreased 31.61% and 34.8% by the pretreatment with cholera toxin (200 micrograms/ml) and dibutyryl cAMP (300 nM), respectively, without affecting the dissociation constant. Under similar conditions, pertussis toxin (20-200 ng/ml) was less effective as compared to CT and dibutyryl cAMP. The down regulation of 14,15-EET binding caused by dibutyryl cAMP in U-937 cell was reversed by a specific protein kinase A (PKA) inhibitor, H-89, but not by the PKC inhibitor K252a. Thus, the results suggest that the specific binding site of 14,15-EET in U-937 cells is associated with a receptor that could be down regulated through an increase in intracellular cAMP and activation of a PKA signal transduction mechanism. We propose that the signal transduction mechanism of 14,15-EET begins with the binding of the receptor, which leads to the increase of intracellular cAMP levels and the activation of PKA, and finally with the down regulation of 14,15-EET receptor binding.

The highly stereoselective oxidation of polyunsaturated fatty acids by cytochrome P450BM-3.

Cytochrome P450BM-3 catalyzes NADPH-dependent metabolism of arachidonic acid to nearly enantiomerically pure 18(R)-hydroxyeicosatetraenoic acid and 14(S), 15(R)-epoxyeicosatrienoic acid (80 and 20% of total products, respectively). P450BM-3 oxidizes arachidonic acid with a rate of 3.2 +/- 0.4 micromol/min/nmol at 30 degrees C, the fastest ever reported for an NADPH-dependent, P450-catalyzed reaction. Fatty acid, oxygen, and NADPH are utilized in an approximately 1:1:1 molar ratio, demonstrating efficient coupling of electron transport to monooxygenation. Eicosapentaenoic and eicosatrienoic acids, two arachidonic acid analogs that differ in the properties of the C-15-C-18 carbons, are also actively metabolized by P450BM-3 (1.4 +/- 0.2 and 2.9 +/- 0.1 micromol/min/nmol at 30 degrees C, respectively). While the 17,18-olefinic bond of eicosapentaenoic acid is epoxidized with nearly absolute regio- and stereochemical selectivity to 17(S),18(R)-epoxyeicosatetraenoic acid (>/=99% of total products, 97% optical purity), P450BM-3 is only moderately regioselective during hydroxylation of the eicosatrienoic acid omega-1, omega-2, and omega-3 sp3 carbons, with 17-, 18-, and 19-hydroxyeicosatrienoic acid formed in a ratio of 2.4:2.2:1, respectively. Based on the above and on a model of arachidonic acid-bound P450BM-3, we propose: 1) the formation by P450BM-3 of a single oxidant species capable of olefinic bond epoxidation and sp3 carbon hydroxylation and 2) that product chemistry and, thus, catalytic outcome are critically dependent on active site spatial coordinates responsible for substrate binding and productive orientation between heme-bound active oxygen and acceptor carbon bond(s).

20-Hydroxyeicosatetraenoic acid is formed in response to EGF and is a mitogen in rat proximal tubule.

Hydroxyeicosatetraenoic acid (20-HETE) is a major cytochrome P-450-arachidonic acid metabolite in the rat kidney, and its synthesis along the nephron is specifically localized to the proximal tubule, where receptor density for epidermal growth factor (EGF) is the highest. EGF stimulated endogenous 20-HETE formation in a concentration and time-dependent manner, i.e., from 1.6 to 2.6 +/- 0.3 and 3.0 +/- 0.6 pmol 20-HETE.mg-1.min-1 at 10(-8) and 10(-7) M EGF, respectively. The effect of 20-HETE on proximal tubular cell proliferation was examined using primary cultures of rat proximal tubular cells and proximal tubular-derived cell lines, LLC-PK1 and opossum kidney OK. In both cell lines, 20-HETE increased thymidine incorporation into DNA with maximal effect at 10(-9) M. Addition of 20-HETE to serum-deprived LLC-PK1 or OK cells for 48 h caused a concentration-dependent increase in cell number with maximal effect at 10(-9) M. This effect was specific, as structurally similar eicosanoids such as 20-COOH-arachidonic acid, 19(R)-HETE, and 19(S)-HETE did not increase cell number. In 4-day primary cultures of proximal tubular cells, EGF (10(-9) M) and 20-HETE (10(-9) M) increased bromodeoxyuridine (BrdU) incorporation by 40 and 28%, respectively. Addition of both resulted in a twofold increase in BrdU incorporation. Although 20-HETE synthesis in cultured cells is greatly diminished with time, significant picomolar concentrations can be obtained in 4-day cultures. Addition of 17-octadecynoic acid (17-ODYA), an inhibitor of 20-HETE synthesis, significantly inhibited EGF-stimulated BrdU incorporation. The demonstrations that EGF stimulates proximal tubular 20-HETE production and that the latter is a potent mitogen to these cells suggests that 20-HETE may act as a mediator of the EGF effect on cellular growth in the proximal tubule.

The catalytic outcomes of the constitutive and the mitogen inducible isoforms of prostaglandin H2 synthase are markedly affected by glutathione and glutathione peroxidase(s).

Reduced glutathione (GSH), at physiological concentrations, was found to markedly alter the profile of arachidonate metabolism by prostaglandin H2 synthase. In 1 mM GSH, the constitutive (COX-1) and the mitogen inducible (COX-2) isoforms metabolized arachidonate to 12-hydroxyheptadecatrienoic acid (12-HHT) (88% and 78% of total products, respectively). Prostanoid formation was consequently reduced to only 12% (COX-1) and 19% (COX-2) of the total metabolites. The GSH-dependent production of 12-HHT was regio- and enantioselective for the 12(S)-isomer. We propose that 12(S)-HHT is formed by a GSH-dependent enzymatic cleavage of the PGH2 8,9 and 11,12 carbon-carbon bonds based on the following: (a) nonsignificant GSH-dependent formation of 12(S)-HHT during chemical decomposition of synthetic PGH2, (b) the structural similarities between the asymmetric carbons at C(12) in 12-HHT and C(15) in PGH2, (c) the GSH concentration-dependent product/precursor relationship between 12-HHT and prostanoid production, and (d) aspirin inhibition of 12-HHT formation by both enzymes. Arachidonic acid oxidation by COX-1, and not by COX-2, was inhibited by the combined presence of GSH and liver cytosol. In contrast, metabolism by neither isoform was inhibited when the cytosol was obtained from selenium-depleted animals. This is consistent with a unique, selenium dependent, cytosolic GSH peroxidase that inhibits specifically prostanoid and 12(S)-HHT formation by COX-1. These results suggest an additional role for GSH and GSH peroxidase(s) in regulating prostanoid biosynthesis. Differences between the isoforms in their sensitivities to GSH peroxidase may reflect differences in their requirements for an "initiator hydroperoxide".