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H H Thorp

Publications and source records attributed to H H Thorp.

16 recordsLinked to original sources
PubMed2001

Modification of indium tin oxide electrodes with repeat polynucleotides: electrochemical detection of trinucleotide repeat expansion.

Genomic expansion of the triplet repeat sequences 5'-(CTG)n and 5'-(CGG)n leads to myotonic dystrophy and fragile X syndrome, respectively. Methods for determining the number of repeats in unprocessed nucleic acids would be useful in diagnosing diseases based on triplet repeat expansion. Electrochemical reactions based on the oxidation of guanine were expected to give larger signals per strand for expansion of repeats containing guanine. A novel PCR reaction was used to generate fragments containing 150, 230, 400, and 830 repeats of (CTG)n, which codes for myotonic dystrophy, and 130 and 600 repeats of (CGG)n, which codes for fragile X syndrome. These PCR fragments were immobilized to indium tin oxide electrodes, and oxidation of guanine in the fragments was realized using electrocatalysis by Ru(bpy)3(2+) (bpy = 2,2'-bipyridine). The catalytic currents due to oxidation of the immobilized guanines by Ru(bpy)3(3+) increased with the number of repeats and were a linear function of the repeat number when normalized to the number of strands immobilized. These results suggest a sensing strategy for repeat length based on the combination of the electrocatalytic strategy for determining the repeat length combined with existing methods for determining the number of strands.

DNA↗
PubMed2001

Oxidation of 7-deazaguanine by one-electron and oxo-transfer oxidants: mismatch-dependent electrochemistry and selective strand scission.

Addition of oligonucleotides containing 7-deazaguanine (Z) to solutions containing Ru(dmb)3(2+) (dmb = 4,4'-dimethyl-2,2'-bipyridine) produces an enhancement in the oxidative current in the cyclic voltammogram of the metal complex that can be used, through digital simulation, to determine the rate of oxidation of 7-deazaguanine by Ru(dmb)3(3+). The measured rate constants are about 10 times higher than those for oxidation of guanine by Ru(bpy)3(3+), even though the redox potential of Ru(dmb)3(3+/2+) is 200 mV lower. A potential of 0.75 V (vs Ag/AgCl) can therefore be estimated for the oxidation of 7-deazaguanine, which can be selectively oxidized over guanine when Ru(dmb)3(3+) is the oxidant. The rate of oxidation was much faster in single-stranded DNA, and the difference between rates of single-stranded and duplex DNA was higher than for guanine. The oxidation rate was also sensitive to the presence of a single-base mismatch at the 7-deazaguanine in the order Z.C < Z.T < Z.G approximately Z.A < single-stranded. The Z.T mismatch was much more readily distinguished than the G.T mismatch, consistent with the overall greater sensitivity to secondary structure for Z. The oxidation reaction was also probed by monitoring piperidine-labile cleavage at the Z nucleotide, which could be generated by treatment with either photogenerated Ru(bpy)3(3+) or the thermal oxidant Ru(tpy)(bpy)O2+ (tpy = 2,2',2' '-terpyridine). These oxidants gave qualitatively similar selectivities to the electron-transfer rates from cyclic voltammetry, although the magnitudes of the selectivities were considerably lower on the sequencing gels.

Base Pair Mismatch↗
PubMed2001

Oxidation kinetics of guanine in DNA molecules adsorbed onto indium tin oxide electrodes.

Oligonucleotides containing the guanine nucleobase were adsorbed onto ITO electrodes from mixtures of DMF and acetate buffer. Chronocoulometry and chronoamperometry were performed on the modified electrodes in both phosphate buffer and buffer containing low concentrations of the inorganic complex Ru(bpy)3(2+) (bpy = 2,2' bipyridine), which catalyzes guanine oxidation. The charge and current evolution with and without the catalyst were compared to the charge and current evolution for electrodes that were treated with identical oligonucleotides that were substituted at every guanine with the electrochemically inert nucleobase hypoxanthine. Chronocoulometry over 2.5 s shows that roughly 2 electrons per guanine were transferred to the electrode in both the presence and absence of Ru(bpy)3(2+), although at a slower rate for the uncatalyzed process. Chronoamperograms measured over 250 ms can be fit to a double exponential decay, with the intensity of the fast component roughly 6-20 times greater than that of the slow component. First- and second-order rate constants for catalytic and direct guanine oxidation were determined from the fast component. The maximum catalytic enhancement for immobilized guanine was found to be i(cat)/i(d) = 4 at 25 microM Ru(bpy)3(2+). The second-order rate constant for the catalyzed reaction was 1.3 x 10(7) M(-1) s(-1), with an apparent dissociation constant of 8.8 microM. When compared to parallel studies in solution, a smaller value of the dissociation constant and a larger value of the second-order rate constant are observed, probably due to distortion of the immobilized DNA, an increase in the local negative charge due to the oxygen sites on the ITO surface, and redox cycling of the catalyst, which maintains the surface concentration of the active form.

Adsorption↗
PubMed2001

An ionic liquid form of DNA: redox-active molten salts of nucleic acids.

Ionic liquids are described that contain duplex DNA as the anion and polyether-decorated transition metal complexes based on M(MePEG-bpy)(3)(2+) as the cation (M = Fe, Co; MePEG-bpy = 4,4'-(CH(3)(OCH(2)CH(2))(7)OCO)(2)-2,2'-bipyridine). When the undiluted liquid DNA-or molten salt-is interrogated electrochemically by a microelectrode, the molten salts exhibit cyclic voltammograms due to the physical diffusion (D(PHYS)) of the polyether-transition metal complex. When M = Co(II), the cyclic voltammogram of the melt shows an oxidative wave due to the Co(III/II) couple at E(1/2) = 0.40 V (versus Ag/AgCl) and a D(PHYS) of 6 x 10(-12) cm(2)/s, which is significantly lower than that for Co(MePEG-bpy)(3)(ClO(4))(2) (D(PHYS) = 2.6 x 10(-10) cm(2)/s) due to greater viscosity provoked by the DNA polymer. When a 1:1 mixture is made of the Co(MePEG-bpy)(3).DNA and Fe(MePEG-bpy)(3)(ClO(4))(2) melts, two redox waves are observed. The first is due to the Co(III/II) couple, and the second is a catalytic wave due to oxidation of guanine in DNA by electrogenerated Fe(III) in the undiluted melt. Independent experiments show that the Fe(III) form of the complex selectively oxidizes guanine in duplex DNA. These DNA molten salts constitute a new class of materials whose properties can be controlled by nucleic acid sequence and that can be interrogated in undiluted form on microelectrode arrays.

DNA↗
PubMed2000

Ethanol oxidation by imidorhenium(V) complexes: formation of amidorhenium(III) complexes.

The reaction of Re(NC6H4R)Cl3(PPh3)2 (R = H, 4-Cl, 4-OMe) with 1,2-bis(diphenylphosphino)ethane (dppe) is investigated in refluxing ethanol. The reaction produces two major products, Re(NC6H4R)Cl(dppe)(2)2+ (R = H, 1-H; R = Cl, 1-Cl; R = OMe, 1-OMe) and the rhenium(III) species Re(NHC6H4R)Cl(dppe)2+ (R = H, 2-H; R = Cl, 2-Cl). Complexes 1-H (orthorhombic, Pcab, a = 22.3075(10) A, b = 23.1271(10) A, c = 23.3584(10) A, Z = 8), 1-Cl (triclinic, P1, a = 11.9403(6) A, b = 14.6673(8) A, c = 17.2664(9) A, alpha = 92.019(1) degrees, beta = 97.379(1) degrees, gamma = 90.134(1) degrees, Z = 2), and 1-OMe (triclinic, P1, a = 11.340(3) A, b = 13.134(4) A, c = 13.3796(25) A, alpha = 102.370(20) degrees, beta = 107.688(17) degrees, gamma = 114.408(20) degrees, Z = 1) are crystallographically characterized and show an average Re-N bond length (1.71 A) typical of imidorhenium(V) complexes. There is a small systematic decrease in the Re-N bond length on going from Cl to H to OMe. Complex 2-Cl (monoclinic, Cc, a = 24.2381(11) A, b = 13.4504(6) A, c = 17.466(8) A, beta = 97.06900(0) degrees, Z = 4) is also crystallographically characterized and shows a Re-N bond length (1.98 A) suggestive of amidorhenium(III). The rhenium(III) complexes exhibit unusual proton NMR spectra where all of the resonances are found at expected locations except those for the amido protons, which are at 37.8 ppm for 2-Cl and 37.3 ppm for 1-H. The phosphorus resonances are also unremarkable, but the 13C spectrum of 2-Cl shows a significantly shifted resonance at 177.3 ppm, which is assigned to the ipso carbon of the phenylamido ligand. The extraordinary shifts of the amido hydrogen and ipso carbon are attributed to second-order magnetism that is strongly focused along the axially compressed amido axis. The reducing equivalents for the formation of the Re(III) product are provided by oxidation of the ethanol solvent, which produces acetal and acetaldehyde in amounts as much as 30 equiv based on the quantity of rhenium starting material. Equal amounts of hydrogen gas are produced, suggesting that the catalyzed reaction is the dehydrogenation of ethanol to produce acetaldehyde and hydrogen gas. Metal hydrides are detected in the reaction solution, suggesting a mechanism involving beta-elimination of ethanol at the metal center. Formation of the amidorhenium(III) product possibly arises from migration of a metal hydride in the imidorhenium(V) complex.

Journal Article↗
PubMed2000

Kinetics of metal-mediated one-electron oxidation of guanine in polymeric DNA and in oligonucleotides containing trinucleotide repeat sequences.

The oxidation of guanines in DNA by Ru(III) is investigated by catalytic electrochemistry and stopped-flow spectrophotometry. The reactions of calf thymus DNA (20% guanine) and herring testes DNA (25% guanine) with Ru(bpy)3(3+) (bpy = 2,2'-bipyridine) show biexponential decays in stopped-flow spectrophotometric experiments with the fast and slow components in 2:1 ratios and average rate constants in 880 mM NaCl of = 18,700 M-1 s-1 for calf thymus DNA and = 24,600 M-1 s-1 for herring testes DNA. The higher rate constant for the more guanine-rich DNA is possibly due to a higher density of electron-rich guanine multiplets. The observation of a biexponential decay is incorporated into digital simulations of the catalytic voltammograms observed for Ru(bpy)3(2+) in the presence of DNA. For both DNAs, the rates observed by voltammetry are somewhat slower than those observed by stopped-flow spectrophotometry and the dependence of the rate constants on scan rate using the biexponential model is less pronounced than when only one decay is treated, supporting the notion that the scan rate dependence arises from the multiphasic decay. At low salt concentrations, where binding of the metal complex to DNA increases the effective catalytic rate constant, rates can be measured by stopped-flow spectrophotometry only with a less oxidizing complex, Fe(bpy)3(3+/2+), which yields trends in the rate constants similar to those observed for the case of Ru(bpy)3(3+/2+) at high ionic strength. Oligonucleotides based on the trinucleotide repeat sequences (AGT)n and (GAA)n produce significant catalytic currents, which are readily interpreted in terms of the guanine concentration and the secondary structure discerned from gel electrophoresis experiments. These experiments may provide a basis for sensing secondary structures and repeat numbers in biologically relevant DNAs.

Animals↗
PubMed2000

Redox pathways in DNA oxidation: kinetic studies of guanine and sugar oxidation by para-substituted derivatives of oxoruthenium(IV).

The oxidation of nucleotides and DNA by a series of complexes based on Ru(tpy)(bpy)O2+ (1) was investigated (tpy = 2,2':6',2"-terpyridine; bpy = 2,2'-bipyridine). These complexes were substituted with electron-donating or-withdrawing substituents in the para positions of the polypyridyl ligands so that the oxidation potentials of the complexes were affected but the reaction trajectory of the oxo ligand with DNA was the same throughout the series. The prepared complexes were (with E1/2(III/II) and E1/2(IV/III) values in volts versus Ag/AgCl) Ru(4'-EtO-tpy)(bpy)O2+ (2; 0.47, 0.60), Ru(4'-Cl-tpy)(bpy)O2+ (3; 0.55, 0.63), Ru(tpy)(4,4'-Me2-bpy)O2+ (4; 0.48, 0.62), and Ru(tpy)(4,4'-Cl2-bpy)O2+ (5; 0.58, 0.63). The complexes oxidized deoxycytosine 5'-monophosphate at the sugar moiety (k = 0.24-0.47 M-1 s-1) and guanosine 5'-monophosphate at the base moiety (k = 6.1-15 M-1 s-1). The rate constants increase across these ranges in the order 3 > 1 > 4 > 2, which is the same order as the redox potentials of the complexes. The effect of the base on these reactions was also studied, and xanthine was found to react with 1 much faster than guanine while hypoxanthine was less reactive than the sugar moiety. The complexes all oxidized oligonucleotides to generate base-labile lesions at guanine and a combination of spontaneous and base-labile scission at the sugar functionalities. The selectivity of cleavage in duplex and single-stranded DNA was not a strong function of the substituents on the metal complex.

Carbohydrates↗
PubMed2000

The importance of being r: greater oxidative stability of RNA compared with DNA.

The 2'-hydroxyl group of ribose imparts hydrolytic lability on RNA, which provides a mechanism for numerous biological functions. Recent evidence from chemical cleavage studies shows that this hydroxyl group also stabilizes the sugar moiety in RNA towards oxidation relative to DNA. Is this just because RNA needs to be distinguishable from DNA or does it have other evolutionary significance?

Animals↗
PubMed1999

Site-selective electron transfer from purines to electrocatalysts: voltammetric detection of a biologically relevant deletion in hybridized DNA duplexes.

BACKGROUND: The one-electron oxidation of guanine nucleobases is of interest for understanding the mechanisms of mutagenesis, probing electron-transfer reactions in DNA, and developing sensing schemes for nucleic acids. The electron-transfer rates for oxidation of guanine by exogenous redox catalysts depend on the base paired to the guanine. An important goal in developing the mismatch sensitivity is to identify a means for monitoring the current resulting from electron transfer at a single base in the presence of native oligonucleotides that contain all four bases. RESULTS: The nucleobase 8-oxo-guanine (8G) is selectively oxidized by the redox catalyst Os(bpy)(3)(3+/2+) (bpy = 2,2'-bipyridine) in the presence of native guanine. Cyclic voltammograms of Os(bpy)(3)(2+) show current enhancements indicative of nucleobase oxidation upon addition of oligonucleotides that contain 8G, but not in the presence of native guanine. As expected, similar experiments with Ru(bpy)(3)(2+) show enhancement with both guanine and 8G. The current enhancements for the 8G/Os(III) reaction increase in the order 8G-C approximately 8G.T < 8G.G < 8G.A < 8G, the same order as that observed for guanine/Ru(III). This site-selective mismatch sensitivity can be applied to detection of a TTT deletion, which is important in cystic fibrosis. CONCLUSIONS: The base 8G can be effectively used in conjunction with a low-potential redox catalyst as a probe for selective electron transfer at a single site. Because of the high selectivity for 8G, rate constants can be obtained that reflect the oxidation of only one base. The mismatch sensitivity can be used to detect biologically relevant abnormalities in DNA.

Base Pair Mismatch↗
PubMed1998

Effects of secondary structure on DNA and RNA cleavage by diplatinum(II).

The photochemistry of Pt2(pop)44- with nucleic acids has been studied using radiolabeled oligomers of DNA and RNA and high-resolution electrophoresis (pop is P2O5H22-). Photolysis of Pt2(pop)44- with duplex DNA produces an even cleavage ladder and relatively little enhancement of cleavage upon treatment with piperidine. In contrast, the cleavage pattern is far less regular with single-stranded DNA, and there is a large enhancement in cleavage upon treatment with piperidine. Accordingly, photolysis of Pt2(pop)44- with the DNA hairpin 5'-d[ATCCTATTTATAGGAT] produces a much larger piperidine enhancement at the loop and end nucleotides than in the stem. There is an additional piperidine enhancement that occurs selectively at guanine residues either in RNA or in DNA at low Mg2+ concentrations that is attributed to outer-sphere electron transfer on the basis of the known excited-state redox potentials of Pt2(pop)44- and the expected oxidative chemistry of guanine. The extent of guanine oxidation is higher compared to the extent of sugar oxidation at low Mg2+ concentrations, which can be attributed to a shallower distance dependence for electron transfer compared to that for atom transfer. The effects of Mg2+ and piperidine or aniline treatment were examined on stem-loop structures of DNA and RNA and gave partial images of the expected secondary structures.

Bacterial Proteins↗
PubMed1998

Bioinorganic chemistry and drug design: here comes zinc again.

The structures and reactions of metal ions in proteins are of tremendous interest in bioinorganic chemistry, as is the potential for metals in creating novel medicines. New results combine these aspects in describing an unexpected mode for metal-mediated drug efficacy that relies on well-established principles of metalloprotein structure.

Animals↗
PubMed1998

Oxidation of guanines in the iron-responsive element RNA: similar structures from chemical modification and recent NMR studies.

BACKGROUND: The translation or stability of the mRNAs from ferritin, maconitase, erythroid aminoevulinate synthase and the transferrin receptor is controlled by the binding of two iron regulatory proteins to a family of hairpin-forming RNA sequences called iron-responsive elements (IREs). The determination of high-resolution nuclear magnetic resonance (NMR) structures of IRE variants suggests an unusual hexaloop structure, leading to an intra-loop G-C base pair and a highly exposed loop guanine, and a special internal loop/bulge in the ferritin IRE involving a shift in base pairing not predicted with standard algorithms. RESULTS: Cleavage of synthetic 55- and 30-mer RNA oligonucleotides corresponding to the ferritin IRE with complexes based on oxoruthenium(IV) shows enhanced reactivity at a hexaloop guanine and at a guanine adjacent to the internal loop/bulge with strong protection at a guanine in the internal loop/bulge. These results are consistent with the recent NMR structures. The synthetic 55-mer RNA binds the iron-regulatory protein from rabbit reticulocyte lysates. The DNA analogs of the 55- and 30-mers do not show the same reactivity pattern. CONCLUSIONS: The chemical reactivity of the guanines in the ferritin IRE towards oxoruthenium(IV) supports the published NMR structures and the known oxidation chemistry of the metal complexes. The results constitute progress towards developing stand-alone chemical nucleases that reveal significant structural properties and provide results that can ultimately be used to constrain molecular modeling.

Animals↗
PubMed1996

Iron chelates bind nitric oxide and decrease mortality in an experimental model of septic shock.

The hydroxamic acid siderophore ferrioxamine B [FeIII(HDFB)+] and the iron complex of diethylenetri-aminepentaacetic acid [FeIII(DTPA)2-] protected mice against death by septic shock induced by Corynebacterium parvum + lipopolysaccharide. Although FeIII(DTPA)2- was somewhat more effective than FeIII(HDFB)+, the iron-free ligand H4DFB+ was significantly more effective than DTPA. The hydroxamic acid chelator has a much higher iron affinity than the amine carboxylate, allowing for more efficient formation of the FeIII(HDFB)+ complex upon administration of the iron-free ligand. Electrochemical studies show that FeIII(DTPA)2- binds NO stoichiometrically upon reduction to iron(II) at biologically relevant potentials to form a stable NO adduct. In contrast, FeIII(HDFB)+ is a stable and efficient electrocatalyst for the reduction of NO to N2O at biologically relevant potentials. These results suggest that the mechanism of protection against death by septic shock involves NO scavenging and that particularly effective drugs that operate a low dosages may be designed based on the principle of redox catalysis. These complexes constitute a new family of drugs that rely on the special ability of transition metals to activate small molecules. In addition, the wealth of information available on siderophore chemistry and biology provides an intellectual platform for further development.

Animals↗
PubMedDate not supplied

Probing biomolecule recognition with electron transfer: electrochemical sensors for DNA hybridization.

Identifying infectious organisms, quantitating gene expression, and sequencing genomic DNA on chips all rely on the detection of nucleic acid hybridization. Described here is a novel assay for detection of the hybridization of products of the polymerase chain reaction using electron transfer from guanine to a transition-metal complex. The hybridization assay was modeled in solution by monitoring the cyclic voltammetry of Ru(bpy)3(2+) (bpy = 2,2'-bipyridine) in the presence of a probe strand containing only A, T, and C prior to and after hybridization to a complement that contained seven guanines, which led to high catalytic current due to the oxidation of guanine by Ru(bpy)3(3+). To allow recognition of all four bases in the target sequence, it was shown that inosine 5'-monophosphate was 3 orders of magnitude less reactive than guanosine 5'-monophosphate, suggesting that effective hybridization sensors could be realized by immobilization of probe strands in which inosine was substituted for guanosine; hybridization to guanosine-containing target strands would then provide high catalytic currents. A sensor design was tested in a model system for the detection of a synthetic 21-mer oligonucleotide patterned on the sequence of the ras oncogene, which gave an increase in charge collected of 35 +/- 5 microC after hybridization and of only 8 +/- 5 microC after exposure to noncomplementary DNA. Independent quantitation of probe and target by radiolabeling showed that the hybridized electrode contained 3.0 +/- 0.3 ng of target. New sensor electrodes were then prepared for the detection of PCR-amplified genomic DNA from herpes simplex virus type II, genomic DNA from Clostridium perfringens, and genomic RNA from human immunodeficiency virus and gave an additional charge of 35-65 microC for hybridization to complementary amplicon and of only 2-10 microC after exposure to noncomplementary DNA.

Biosensing Techniques↗