Molecular recognition of a RNA:DNA hybrid structure.
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DNA↗
Biomedical subjects
N Dattagupta
Publications and source records attributed to N Dattagupta.
A transcriptionally amplified DNA probe assay with ligatable probes and immunochemical detection.
Transcriptionally amplified DNA probes are valuable tools in the development of sensitive nucleic acid-based diagnostic assays. Here we describe a model assay using a novel oligonucleotide hairpin probe that encodes a T7 RNA polymerase promoter. The hairpin probe and an adjacently hybridizing biotinylated capture probe were hybridized to target DNA and the duplex was captured onto streptavidin-coated magnetic particles. After ligation of the immobilized probes, which served to maintain specificity, the hairpin probe was transcribed by T7 RNA polymerase. The amplified RNA product was hybridized to the capture probe and bound to the streptavidin-coated magnetic particles. The immobilized heteroduplex was detected with an antibody-alkaline phosphatase conjugate specific for DNA:RNA hybrids, and the chemiluminescent substrate adamantyl-1,2-dioxetane phenyl phosphate. Ten attomoles of target DNA could be detected in a background of 5 micrograms of unrelated DNA. The chemiluminescent immunoassay was as sensitive as radioactive detection of specific product after gel electrophoresis.
Base Sequence↗
Monoadduct forming photochemical reagents for labeling nucleic acids for hybridization.
We describe the synthesis of three angelicin derivatives which can be used for labeling nucleic acids with biotin. These compounds were used to label nucleic acids in the presence of lysed cell constituents. The resulting labelled nucleic acids show hybridization to a genus specific probe for E. coli. The relative comparison of sensitivity indicates that a polyamine linker is better than a polyethylene oxide linker between the biotin and angelicin moieties.
Affinity Labels↗
Rapid identification of microorganisms by nucleic acid hybridization after labeling the test sample.
A novel method for rapidly identifying microorganisms has been developed. This method employs a monoadduct-forming furocoumarin derivative, which can photochemically label nucleic acids. The labeled nucleic acid can, in turn, be hybridized simultaneously to a panel of immobilized probe DNAs arrayed as dots on a solid support such as nitrocellulose. This procedure offers several advantages over more conventional hybridization techniques in that sample nucleic acids can be photolabeled without substantial sample preparation and that identification can be achieved by a single, rapid hybridization reaction.
Bacterial Typing Techniques↗
Detection of biotinylated nucleic acid hybrids by antibody-coated gold colloid.
A simple and sensitive nonradioactive method for detecting biotinylated nucleic acid hybrids on nitrocellulose paper is described. The method employs anti-biotin antibody-coated gold colloid and silver enhancement. Comparative results indicate sensitivity similar to that of a commonly used enzymatic colorimetric detection method.
Affinity Labels↗
Anthracycline antibiotics. Interaction with DNA and nucleosomes and inhibition of DNA synthesis.
We report studies of the interaction of four anthracycline antibiotics, iremycin (IM), daunomycin (DM), aclacinomycin A (AM), and violamycin B1 (VM), with naked DNA, nucleosomal core particles, and 175 base pair (bp) nucleosomes lacking histone H1. In all cases the binding strength increases in the order IM less than DM approximately AM less than VM. The binding substrates increased in affinity for the drugs in the following order: core particles less than 175-bp nucleosomes less than DNA. The apparent DNA length increment per drug bound decreases in the progression IM greater than DM greater than AM greater than VM, the same serial order as is characterized by increasing binding affinity. Dichroism amplitude measurements show that for all drugs the long-wavelength absorbance transition moment is tilted by 26-29 degrees relative to the plane perpendicular to the helix axis; this angle probably corresponds to the long axis tilt of the intercalated chromophore. Finally, it was found that the ability of the drugs to inhibit DNA synthesis by Escherichia coli DNA polymerase I increases in the same order as their binding affinity.
Aclarubicin↗
A simple DNA diagnostic method for human genetic disorders.
A fast, reliable, and simple technique for detecting point mutations in unfractionated human DNA has been developed. Oligonucleotide probes complementary to either sickle- or normal beta-globin DNA are labeled by primer extension and hybridized to DNA applied to nitrocellulose paper in a dot-blot format. A short hybridization time (about 1 h) and low probe concentration (about 1 nM) yield low background and high specificity. Double-blind trials show 100% agreement with restriction fragment length polymorphism (RFLP) analysis of DNA from normal, sickle, and heterozygous subjects.
Anemia, Sickle Cell↗
Kinetics of the daunomycin--DNA interaction.
The kinetics of the interaction of daunomycin with calf thymus DNA are described. Stopped-flow and temperature-jump relaxation methods, using absorption detection, were used to study the binding reaction. Three relaxation times were observed, all of which are concentration dependent, although the two slower relaxations approach constant values at high reactant concentrations. Relaxation times over a wide range of concentrations were gathered, and the data were fit by a minimal mechanism in which a rapid bimolecular association step is followed by two sequential isomerization steps. The six rate constants for this mechanism were extracted from our data by relaxation analysis. The values determined for the six rate constants may be combined to calculate an overall equilibrium constant that is in excellent agreement with that obtained by independent equilibrium measurements. Additional stopped-flow experiments, using first sodium dodecyl sulfate to dissociate bound drug and second pseudo-first-order conditions to study the fast bimolecular step, provide independent verification of three of the six rate constants. The temperature dependence of four of the six rate constants was measured, allowing estimates of the activation energy of some of the steps to be made. We speculate that the three steps in the proposed mechanism may correspond to a rapid "outside" binding of daunomycin to DNA, followed by intercalation of the drug, followed by either conformational adjustment of the drug or DNA binding site or redistribution of bound drug to preferred sites.
DNA↗
Binding of daunomycin to calf thymus nucleosomes.
We report equilibrium, hydrodynamic, and electric dichroism studies of the complex of daunomycin with H1-depleted 175 base pair nucleosomes, along with some comparative data for ethidium. In contrast to ethidium, daunomycin binding to nucleosomes is strongly reduced relative to the affinity for free DNA. The salt concentration dependence of the binding constant indicates that approximately one Na+ ion is released from both nucleosomes and free DNA upon daunomycin binding. The early melting transition of nucleosomes is preferentially stabilized by low levels of both drugs, but more markedly by ethidium. Ethidium also stabilizes the second nucleosome melting transition, but daunomycin barely does so. Dichroism and rotational relaxation time measurements indicate that daunomycin unfolds nucleosomes in a manner analogous to the influence of ethidium, although about twice as much daunomycin as ethidium is required complete the unfolding process. The data support an unfold structure in which the nucleosome elongates along the DNA superhelical axis. Levels of daunomycin greater than about 0.15 per DNA base pair promote nucleosome aggregation. To relate our results to the activity of daunomycin as an antitumor agent, we propose that the drug, because of its special intercalation geometry, strongly prefers free DNA regions over the bent helices found in nucleosomes and chromatin. The result of this preference should be an increased local concentration of the drug in the genetically active regions of nuclear DNA in which nucleosomal structure is less prevalent. Presumably the abundance of such regions in tumor cells makes them especially sensitive to daunomycin.
Animals↗
Orientation of nucleosomes in the thirty-nanometer chromatin fiber.
We have measured the linear dichroism of Mg2+-stabilized and dimethylsuberimidate cross-linked 30-nm chromatin fibers, using electric fields to produce orientation. The limiting dichroism of Mg2+-stabilized fibers at low monovalent ion concentration is -0.09, with indistinguishable results for avian erythrocyte and calf thymus chromatin. Correction of the apparent dichroism signal for an absorbance change that becomes significant at high electric field yields the dichroism of -0.09 and reveals that the fibers reach saturation of their orientation by about 15 kV/cm. Calf thymus chromatin fibers cross-linked at 100 mM NaCl concentration have a dichroism of +0.05, with a dependence of orientation on field similar to that observed for Mg2+-stabilized fibers. Comparative sedimentation studies of Mg2+-stabilized fibers and fibers in 100 mM cross-linking buffer revealed a 25% sedimentation coefficient increase accompanying the dichroism increase from -0.09 to +0.05. We interpret the results to mean that the nucleosomal disk diameters form an angle of about 30 degrees to the chromatin fiber axis in Mg2+-stabilized fibers at low monovalent ion concentration. When 100 mM NaCl is added, the fiber becomes more compact, and the disk diameter angles increase by 8 degrees, to about 38 degrees. Cross-linking itself may also contribute slightly to fiber compaction. The results are consistent with a large longitudinal compressibility of the 30-nm fiber, a feature required for the bendability necessary when the fiber is further coiled to form structures such as chromosomes. Our results indicate that compression is accommodated by small changes in the angular orientation of the nucleosomal disks.
Animals↗
Self-association of daunomycin.
Daunomycin, a potent anthracycline antibiotic, self-associates in aqueous solution at concentrations greater than 10 microM. We report here visible absorbance, sedimentation equilibrium, and proton nuclear magnetic resonance (NMR) experiments that characterize this self-association. In contrast to earlier reports that the process is a simple dimerization, we find that an indefinite association model best fits our data, with an intrinsic association constant, Ki, equal to 1500 M-1. From the temperature dependence of the observed NMR spectra, an enthalpy of approximately -8.0 kcal/mol is calculated. The NMR data show that the aromatic protons of the anthracycline portion of the drug are most affected by aggregation, probably due to stacking of the anthracycline rings. Knowledge of the applicable model for the self-association process, and the equilibrium constant that describes the process, enables us to assess quantitatively the possible effects of drug aggregation on the interpretation of drug-DNA binding data. For the ionic conditions most commonly used in such studies, the amount of aggregated daunomycin will be slight and may safely be ignored.
Chemical Phenomena↗
Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: equilibrium binding studies on interaction of daunomycin with deoxyribonucleic acid.
We have used equilibrium dialysis and fluorescence and absorbance titration to study the interaction of daunomycin with DNA. Our data at 200 mM Na+ are best fit by the neighbor exclusion model, with K = 7.0 x 10(5) M-1 and an exclusion parameter of three to four base pairs. The binding is dependent on ionic strength, with d log K/d log [Na+] = -0.84, from which we may estimate quantitatively ion release and the binding free energy corrected for the free energy of counterion release. From the temperature dependence of the binding constant, we find the binding to be exothermic, with a van't Hoff enthalpy of -12.8 kcal/mol. Competition dialysis experiments show that G+C base pairs are slightly preferred as binding sites for the drug and suggest that daunomycin binds preferentially to G+C pairs at low r. Cesium chloride density gradient sedimentation experiments provide an experimental demonstration of this preference. Daunomycin increases the Tm for DNA melting by some 30 degrees C as binding approaches saturation, with biphasic melting at low drug/base pair ratios. The data from these equilibrium studies are consistent with intercalative binding of daunomycin and provide a solid foundation for further structural and kinetic studies.
Base Composition↗
Studies on interaction of anthracycline antibiotics and deoxyribonucleic acid: geometry of intercalation of iremycin and daunomycin.
The structure of iremycin [10-(alpha-L-rhodosaminyl)-gamma-rhodomycinone] hydrochloride has been confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. We studied the interaction of iremycin and the related compound daunomycin with DNA by transient electric dichroism and by sedimentation analysis of supercoiled closed duplex DNA. The apparent length increase of sonicated calf thymus DNA (150 +/- 20 base pairs) in 2.5 mM sodium cacodylate buffer (pH 7) at 12 degrees C was determined to be 0.40 +/- 0.02 nm/bound drug). The Cu(II) complex of iremycin with a metal/drug ratio of 0.7 induces a length increase of DNA of 0.44 +/- 0.02 nm/added drug. The alignment of the iremycin chromophore with respect to the DNA helix axis was determined from the electric dichroism of the complex. The tilt (long axis) and twist (short axis) of the chromophore are both 28 +/- 4 degrees, whereas for daunomycin the long axis is perpendicular to the helix axis and the short axis is twisted by about 25 degrees. Intercalation of iremycin between DNA base pairs is supported by unwinding of the supercoiled closed duplex form of pBR 322 plasmid DNA from Escherichia coli. In 2.5 mM sodium cacodylate buffer at pH 7 and at 25 degrees C, the unwinding induced by iremycin is 15.0 +/- 1.5 degrees/bound drug. Under identical conditions daunomycin shows on unwinding angle of 15.4 +/- 1.5 degrees. The superhelical density of pBR 322 DNA (sigma 0) was determined to be -0.087 +/- 0.002 at standard conditions (0.2 M NaCl, 37 degrees C).
Anthracyclines↗
Unfolding of 175-base-pair nucleosomes.
Calf thymus nucleosomes containing 175 base pairs of DNA unfold in two steps as the salt concentration is lowered, as detected by electric dichroism measurements. The transition midpoints are at 2.9 and 1.1 mM ionic strength at 7 degrees C with at most a small dependence on temperature. We identify the product of the 2.9 mM transition as an expanded disklike structure similar to the product of the 1.0--1.3 mM unfolding transition of 146-base-pair nucleosomes. The product of the 1.1 mM transition in 175-base-pair nucleosomes is elongated into a more asymmetric particle.
Animals↗
Neocarzinostatin chromophore binds to deoxyribonucleic acid by intercalation.
The nonprotein chromophore of neocarzinostatin was found to share many of the characteristics of classical intercalators in its interaction with DNA. Viscosity studies with PM2 DNA indicated that the DNA helix unwinding induced by the chromophore was 0.82 times that of ethidium or 21 degrees. Electric dichroism of the chromophore--DNA complex showed that each bound chromophore molecule lengthened DNA by 3.3 A and that absorbance transitions of the chromophore at 315--385 nm were oriented approximately parallel to DNA bases, as expected for an intercalated aromatic ring. Binding to DNA induced strong hypochromicity and a pronounced red shift in the absorbance spectrum of the chromophore. Spectrophotometric titrations suggested at least two types of chromophore binding sites on DNA; one type of site was saturated at rb = 0.125 chromophore molecule/nucleotide, but binding to additional sites continued to at least rb = 0.3. These physical--chemical studies were performed at pH 4--5 in order to keep the chromophore stable, but chromophore bound to an excess of DNA at pH 7 showed a stable absorbance spectrum identical with that seen at pH 4--5, suggesting that a similar type of binding occurs at neutral pH. Chromophore which had spontaneously degraded in pH 8 buffer did not bind to DNA at all, as judged by absorbance spectroscopy. The degree of protection afforded by DNA against spontaneous chromophore degradation implied a dissociation constant of approximately 5 microM for the DNA--chromophore complex at neutral pH and physiological ionic strength. Supercoiled DNA was nearly twice as effective as relaxed DNA in protecting chromophore from degradation, providing additional evidence for intercalation at neutral pH. Comparison of absorbance, fluorescence, and dichroism spectra suggests that the naphthalene ring system is the intercalating moiety.
Antibiotics, Antineoplastic↗
Solution structural studies of the Ag(I)-DNA complex.
We report equilibrium dialysis and electric dichroism studies of the two strong complexes (I and II) of silver ion with DNA. Cooperative conversion of DNA to the stronger type I complex results in a 9% length decrease, and a structure in which intercalated ethidium is perpendicular to the helix axis. Upon addition of more Ag+ to form the type II complex, the DNA length reverts to its original value and bound ethidium once again becomes tilted from the plane perpendicular to the helix axis. In both type I and type II Ag (I) - DNA complexes, ethidium binding is mildly cooperative. We interpret the results in terms of a sequence of silver-induced cooperative switches of DNA from its B-form structure with propeller twisted base pairs to a structure with flat base pairs in the type I complex, and back again to propellered base pairs in the type II complex.
Animals↗
Synthesis and properties of acrylamide-substituted base pair specific dyes for deoxyribonucleic acid template mediated synthesis of dye polymers.
We have tried to construct synthetic polymers for sequence-specific recognition and complexation of longer deoxynucleotide sequences. For this purpose, we developed a method of template-directed polymerization of basic pair specific DNA ligands such as basic dyes. The template-directed polymerization consists in a copolymerization of various dyes of different specificities staying simultaneously in a binding equilibrium with DNA. In the present paper, we describe the synthesis and the properties of base pair specific monomers especially designed for performance of radical chain polymerization reactions in aqueous medium at room temperature. Different acrylamide derivatives of well-known dyes, such as AT-specific malachite green and GC-specific phenyl neutral red, were synthesized and studied for their ability of base pair specific complex formation with DNA of different base composition. Partition equilibrium dialysis and dye titration agarose gel electrophoresis were used to ensure for several dyes that they may be incorporated into different polymers via copolymerization of their acrylamido derivatives with various small base-unspecific monomers without substantial change of their binding parameters. Furthermore, we demonstrate that acrylamide and other small acrylamide derivatives can be used as building blocks for the synthesis of polymeric links between base pair specific monomers. The results and their consequences for template polymerization reactions are discussed.
Acrylamides↗
Carcinogen-induced alteration of DNA structure.
We have prepared covalent complexes between defined length DNA fragments and a diol epoxide derivative of the carcinogenic polycyclic aromatic hydrocarbon, benzo(a)pyrene. We have studied the structure of these complexes, using transient electric dichroism, circular dichroism, fluorescence quenching, thermal denaturation, polyacrylamide gel electrophoresis, and nuclease digestion. Our observations suggest that th covalently bound carcinogen is intercalated within the helix, forming a wedge-shaped complex. Binding of the carcinogen distorts the structure of the DNA over a region extending beyond the immediate binding site. The most striking aspect of this distortion is that it produces a bend in the helix.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗