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T J Meade

Publications and source records attributed to T J Meade.

14 recordsLinked to original sources
PubMed2001

Electronic detection of single-base mismatches in DNA with ferrocene-modified probes.

Genotyping and gene-expression monitoring is critical to the study of the association between genetics and drug response (pharmacogenomics) and the association of sequence variation with heritable phenotypes. Recently, we developed an entirely electronic method for the detection of DNA hybridization events by the site-specific incorporation of ferrocenyl derivatives into DNA oligonucleotides. To perform rapid and accurate point mutation detection employing this methodology, two types of metal-containing signaling probes with varying redox potentials are required. In this report we describe a new ferrocene-containing phosphoramidite 9 that provides a range of detectable redox potentials. Using automated DNA/RNA synthesis techniques the two ferrocenyl complexes were inserted at various positions along oligonucleotide probes. Thermal stability analysis of these metal-containing DNA oligonucleotides indicates that incorporation of 9 resulted in no destabilization of the duplex. A mixture of oligonucleotides containing compounds 9 and I was analyzed by alternating current voltammetry (ACV) monitored at the 1st harmonic. The data demonstrate that the two ferrocenyl oligonucleotide derivatives can be distinguished electrochemically. A CMS-DNA array was prepared on an array of gold electrodes on a printed circuit board substrate with a self-assembled mixed monolayer, coupled to an electronic detection system. Experiments for the detection of a single-base match utilizing two signaling probes were carried out. The results demonstrate that rapid and accurate detection of a single-base mismatch can be achieved by using these dual-signaling probes on CMS-DNA chips.

Base Pair Mismatch↗
PubMed2001

Automated synthesis of 3'-metalated oligonucleotides.

We report the first synthesis of a metallonucleoside bound to a solid support and subsequent oligonucleotide synthesis with this precursor. Large-scale syntheses of metal-containing oligonucleotides are achieved using a solid support modified with [Ru(bpy)(2)(impy')](2+) (bpy is 2,2'-bipyridine; impy' is 2'-iminomethylpyridyl-2'-deoxyuridine). A duplex formed with the metal-containing oligonucleotide exhibits superior thermal stability when compared to the corresponding unmetalated duplex (T(m) = 50 degrees C vs T(m) = 48 degrees C). Electrochemical (E(1/2) = 1.3 V vs NHE), absorption (lambda(max) = 480 nm), and emission (lambda(max) = 720 nm, tau = 44 ns, Phi = 0.11 x 10(-)(3)) data for the ruthenium-modified oligonucleotides indicate that the presence of the oligonucleotide does not perturb the electronic properties of the ruthenium complex. The absence of any change in the emission properties upon duplex formation suggests that the [Ru(bpy)(2)(impy)](2+) chromophore will be a valuable probe for DNA-mediated electron-transfer studies. Despite the relatively high Ru(III/II) reduction potential, oxidative quenching of photoexcited [Ru(bpy)(2)(impy)](2+) does not lead to oxidative damage of guanine or other DNA bases.

Base Sequence↗
PubMed2000

In vivo visualization of gene expression using magnetic resonance imaging.

High-resolution in vivo imaging of gene expression is not possible in opaque animals by existing techniques. Here we present a new approach for obtaining such images by magnetic resonance imaging (MRI) using an MRI contrast agent that can indicate reporter gene expression in living animals. We have prepared MRI contrast agents in which the access of water to the first coordination sphere of a chelated paramagnetic ion is blocked with a substrate that can be removed by enzymatic cleavage. Following cleavage, the paramagnetic ion can interact directly with water protons to increase the MR signal. Here, we report an agent where galactopyranose is the blocking group. This group renders the MRI contrast agent sensitive to expression of the commonly used marker gene, beta-galactosidase. To cellular resolution, regions of higher intensity in the MR image correlate with regions expressing marker enzyme. These results offer the promise of in vivo mapping of gene expression in transgenic animals and validate a general approach for constructing a family of MRI contrast agents that respond to biological activity.

Animals↗
PubMed1999

Looking deeper into vertebrate development.

Magnetic resonance imaging (MRI) is a non-invasive imaging method that provides three-dimensional (3-D) images of the internal structure of opaque objects, such as humans and mice. In optimal situations, spatial resolution can approach the micron level. Arbitrarily oriented single-slice images can be obtained in seconds, with full 3-D volume images taking tens of minutes to collect. The exquisite sensitivity of MRI to the local physical and chemical environment provides a wide range of mechanisms giving rise to intrinsic contrast in the MR experiment, thus providing images with dramatic differences between different tissue types (e.g. white vs grey matter, myelinated vs unmyelinated fibres, and brain parenchyma vs ventricles). The recent advent of physiologically sensitive MRI contrast agents opens up a wealth of new avenues of study, even including the in vivo imaging of gene expression.

Animals↗
PubMed1999

Inhibition of thermolysin and human alpha-thrombin by cobalt(III) Schiff base complexes.

Cobalt(III) Schiff base complexes have been shown to inhibit the replication of the ocular herpes virus. It is well known that these complexes have a high affinity for nitrogenous donors such as histidine residues, and it is possible that they bind to (and inhibit) an enzyme that is crucial to viral replication. In model studies, we have found that [Co(acacen)(NH3)2]+ is an effective irreversible inhibitor of thermolysin at millimolar concentrations; it also inhibits human alpha-thrombin. Axial ligand exchange with an active-site histidine is the proposed mechanism of inhibition. The activity of thermolysin and thrombin can be protected by binding a reversible inhibitor to the active site before addition of the cobalt(III) complex.

Binding Sites↗
PubMed1998

Genetic determinants of host-specificity in bipartite geminivirus DNA A components.

Geminiviruses are small, ssDNA-containing plant viruses. Bean golden mosaic virus (BGMV) and tomato golden mosaic virus (TGMV) have bipartite genomes, the components of which are designated A and B. Although they are closely related, BGMV and TGMV nevertheless exhibit distinct host-specific phenotypes, with BGMV being well adapted to beans and TGMV being well adapted to Nicotiana benthamiana. A previous study showed that the two open reading frames (ORFs) of DNA B only partially determine the host-adapted phenotypes of BGMV and TGMV. We have now investigated the contributions of A component ORFs to host adaptation. Co-inoculated TGMV DNA A enhances the accumulation of BGMV in N. benthamiana. Using mutant and hybrid TGMV A components, the determinant of this phenotype was mapped to a region encompassing the overlapping AL2 and AL3 ORFs (AL23). BGMV- and TGMV-based hybrid A components containing the heterologous AL23 region each displayed host-specific gain-of-function phenotypes, which indicates that these sequences contribute to host adaptation in both viruses. In N. benthamiana, al2 and al3 mutants of either virus can be complemented in trans by the heterologous A component, so adaptation of the AL23 region to this host is likely mediated through a virus nonspecific, trans-acting factor. In beans, however, co-inoculated BGMV A does not affect the accumulation of TGMV, and TGMV did not complement BGMV al2 or al3 mutants. Thus host-adaptation of the AL23 region may have a different mechanistic basis in beans than it does in N. benthamiana. Although our experiments did not reveal significant host adaptation of the coat protein, which is encoded by the AR1 ORF, a virus-specific effect on viral ssDNA accumulation was observed.

Capsid↗
PubMed1998

Isolation of a myoglobin molten globule by selective cobalt(III)-induced unfolding.

Reaction of the Schiff-base complex [Co(acetylacetonate-ethylenediimine)(NH3)2]+ with metmyoglobin at pH 6.5 yields a partially folded protein containing six Co(III) complexes. Although half of its alpha-helical secondary structure is retained, absorption and CD spectra indicate that the tertiary structure in both B-F and AGH domains is disrupted in the partially folded protein. In analogy to proton-induced unfolding, it is likely that the loss of tertiary structure is triggered by metal-ion binding to histidines. Cobalt(III)-induced unfolding of myoglobin is unique in its selectivity (other proteins are unaffected) and in allowing the isolation of the partially folded macromolecule (the protein does not refold or aggregate upon removal of free denaturant).

Animals↗
PubMed1998

A cobalt complex that selectively disrupts the structure and function of zinc fingers.

Zinc finger domains are structures that mediate sequence recognition for a large number of DNA-binding proteins. These domains consist of sequences of amino acids containing cysteine and histidine residues tetrahedrally coordinated to a zinc ion. In this report, we present a means to selectively inhibit a zinc finger transcription factor with cobalt(III) Schiff-base complexes. 1H NMR spectroscopy confirmed that the structure of a zinc finger peptide is disrupted by axial ligation of the cobalt(III) complex to the nitrogen of the imidazole ring of a histidine residue. Fluorescence studies reveal that the zinc ion is displaced from the model zinc finger peptide in the presence of the cobalt complex. In addition, gel-shift and filter-binding assays reveal that cobalt complexes inhibit binding of a complete zinc finger protein, human transcription factor Sp1, to its consensus sequence. Finally, a DNA-coupled conjugate of the cobalt complexes selectively inhibited Sp1 in the presence of several other transcription factors.

Amino Acid Sequence↗
PubMed1997

Stretching and breaking duplex DNA by chemical force microscopy.

BACKGROUND: Specific interactions between complementary strands of DNA and other molecules are central to the storage, retrieval and modification of information in biological systems. Although in many cases the basic structures of duplex DNA and the binding energetics have been well characterized, little information is available about the forces in these systems. These forces are of critical importance because they must be overcome, for example, by protein machines during transcription and repair. Recent developments in atomic force microscopy make possible direct measurements of such forces between the individual oligonucleotide strands that form DNA duplexes. RESULTS: We used the chemical force microscopy technique, in which oligonucleotides are covalently linked to the force microscope probe tip and the sample surface, to measure the elongation and binding forces of individual DNA duplexes. The separation forces between complementary oligonucleotide strands were found to be significantly larger than the forces measured between noncomplementary strands, and to be consistent with the unbinding of a single DNA duplex. With increasing applied force, the separation of complementary strands proceeded in a stepwise manner: B-form DNA was stretched, then structurally transformed to a stable form of DNA approximately twice the length of the B form, and finally separated into single-stranded oligonucleotides. These data provide a direct measurement of the forces required to elastically deform and separate double-stranded DNA into single strands. CONCLUSIONS: Force microscopy provides a direct and quantitative measurement of the forces and energetics required to stretch and unbind DNA duplexes. Because the measurements can be carried out readily on synthetic oligonucleotides and in the presence of exogenous molecules, this method affords an opportunity for directly assessing the energetics of distorting and unbinding specific DNA sequences and DNA complexes. Such data could provide unique insights into the mechanistic steps following sequence-specific recognition by, for example, DNA repair and transcription factors.

DNA↗
PubMed1995

Receptor-targeted co-transport of DNA and magnetic resonance contrast agents.

BACKGROUND: Ligand molecules conjugated to polylysine can be electrostatically bound to DNA and can bind receptors or antigens on the surface of cells, delivering the DNA into specific cells and tissues. Several researchers have used this approach to generate non-viral vehicles for the efficient delivery of DNA to specific cells. We have attempted to adopt this general approach to the cell-specific delivery of magnetic contrast agents for use in magnetic resonance imaging (MRI). RESULTS: We have synthesized a new class of agents capable of both transfecting genes into cells and enhancing the contrast of the targeted cells for MRI. DNA is used both to encode a marker gene and as a molecular scaffold, which electrostatically binds polylysine conjugated to transferrin, an iron uptake protein, and polylysine modified with gadolinium chelated to diethylenetriaminepetaacetic acid. When cells displaying the transferrin receptor are treated with these particles, high levels of gene expression are observed, higher than with control particles composed only of transferrin, polylysine and DNA. The treated cells show specific MRI contrast enhancement, which did not require expression of the marker gene. CONCLUSIONS: The development of this class of particles permits the use of novel protocols by which genes for genetic therapy and agents for MRI contrast are co-transported. These protocols may allow non-invasive MRI monitoring of DNA delivery for gene therapy in real time.

Cells, Cultured↗
PubMedDate not supplied

Transfection of folate-polylysine DNA complexes: evidence for lysosomal delivery.

We are utilizing the folate receptor for the intracellular delivery of DNA. In this study, a folate-poly-L-lysine (FPLL) conjugate was synthesized and equilibrated with plasmid DNA encoding the firefly luciferase gene. The FPLL-DNA complexes were added to KB cells treated with chloroquine. Luciferase activity of cells incubated with FPLL-DNA was 6-fold higher than of cells exposed to poly-L-lysine (PLL)-DNA. The addition of free folic acid competitively inhibited the enhancement of gene expression. Removal of chloroquine from the media significantly inhibited transfection efficiency of FPLL-DNA complexes. We conclude that FPLL-DNA complexes are delivered into KB cells via folate receptor-mediated endocytosis and likely follow a lysosomal pathway into the cytoplasm.

Animals↗
PubMedDate not supplied

Fluorescently detectable magnetic resonance imaging agents.

This report describes the synthesis, characterization, and in vivo testing of several bifunctional contrast-enhancing agents for optical and magnetic resonance imaging (MRI) of experimental animals. These new agents integrate the advantages of both techniques since they can be visualized simultaneously by light and MRI microscopy. Employing this strategy allows the same biological structures of a specimen to be studied at dramatically different resolutions and depths. The complexes possess a metal chelator for binding a paramagnetic ion, gadolinium (Gd3+), and a covalently attached fluorescent dye. The first class of complexes are low-molecular weight species that are composed of the macrocyclic tetraamine 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) as the metal-chelating ligand coupled to tetramethylrhodamine. The second class of MRI-enhancing agents are composed of high-molecular weight polymers that are membrane impermeable and once injected into a cell or cells are trapped inside. These complexes possess multiple copies of both the metal-chelator-diethylenetriaminepentaacetic acid (DTPA) and the tetramethylrhodamine attached to a macromolecular framework of either poly(D-lysine) (pdl) or dextran. Images acquired of single cells after injection with these bifunctional agents enabled us to follow the relative motions and reorganizations of different cell layers during amphibian gastrulation and neurulation in Xenopus laevis embryos.

Animals↗