ULTRAVIOLET INDUCED BINDING OF DAUNOMYCIN TO DNA

Up to 10–15% of daunomycin present in solutions of DNA-daunomycin remains irreversibly bound to DNA after 2 h of UV irradiation. Three times as much drug is irreversibly bound to DNA when oxygen is present, compared to deoxygenated solutions. Maximal amounts of the irreversibly bound drug are obtained between pH 5.5 and 7.5 and at an ionic strength of 0.76 M. Although the presence of daunomycin inhibits the extent of UV-induced degradation of the DNA, the DNA is still extensively degraded and denatured in the time required for optimal yields of the DNA-daunomycin photoproduct. This degradation of the DNA may impair its use as a lysosomotropic carrier of daunomycin.     

Estimation of binding sites from the adsorption profile of complexed DNA

The adsorption profiles of free and Daunomycin bound DNA at an alumina–H₂O interface have been studied. Proper experimental conditions were maintained to considerably reduce intercalation and to make electrostatic binding predominant in the DNA–Daunomycin complex. For both free and complexed DNA, Langmuir plots gave straight lines. Adsorption of drug bound DNA was less than that of free DNA. This has been explained from the viewpoint of electrostatic interactions between adsorbate and adsorbent. A Langmuir-type model for adsorption of polymers combined with Scatchard equation have been used to estimate the average value of percent of phosphates in DNA stacked with Daunomycin.     

盐酸表柔比星与核酸相互作用的共振瑞利散射光谱研究及其分析应用

用共振瑞利散射光谱研究了盐酸表柔比星(EPI)与小牛胸腺DNA(ctDNA)、鲑鱼DNA(sDNA)、鲱鱼精DNA(hsDNA)和酵母RNA(yRNA)等核酸之间的相互作用. 实验表明在pH 2.0左右的酸性介质中, 表柔比星及核酸本身的共振瑞利散射(RRS)均十分微弱, 但是当它们相互作用形成结合产物时, 将导致RRS增强并出现新RRS光谱. 不同核酸与表柔比星结合产物的RRS 光谱特征略有差异, 散射增强程度则各不相同, 其相对散射强度的顺序是ctDNA≈sDNA＞hsDNA＞yRNA . 在一定范围内核酸浓度与散射强度成正比, 据此可以建立一种新的用表柔比星测定DNA的 RRS 法, 方法具有高灵敏度, 对于不同DNA 其检出限(3s)在24.0 ng/mL 至28.0 ng/mL 之间, 用于合成样品分析, 结果满意. 文中还研究了适宜的反应条件, 影响因素和结合产物的分析化学性质. 结合吸收光谱和荧光光谱特征对表柔比星与DNA 的反应机理进行了讨论.     

类黄酮化合物对G-四链体及双链DNA选择性的电喷雾质谱研究

利用电喷雾质谱(ESI-MS)研究了4种常见的类黄酮化合物芦丁、 槲皮素、 葛根素和柚皮苷与2种不同形态结构的G-四链体DNA和3种双链DNA的非共价相互作用, 比较了这些小分子化合物与不同形态结构DNA结合的强弱及形成复合物的化学计量. 结果表明, 芦丁和槲皮素对G-四链体DNA具有一定的选择性, 同时它们对双链DNA的选择性也较高; 而葛根素和柚皮苷对G-四链体DNA仅显示了较低的选择性.     

A comparison of the binding of metal complexes to duplex and quadruplex DNA

Electrospray ionisation mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to compare the binding of mononuclear nickel, ruthenium and platinum complexes to double stranded DNA (dsDNA) and quadruplex DNA (qDNA). CD studies provided evidence for the binding of intact complexes of all three metal ions to qDNA. ESI mass spectra of solutions containing platinum or ruthenium complexes and qDNA showed evidence for the formation of non-covalent complexes consisting of intact metal molecules bound to DNA. However, the corresponding spectra of solutions containing nickel complexes principally contained ions consisting of fragments of the initial nickel molecule bound to qDNA. In contrast ESI mass spectra of solutions containing nickel, ruthenium or platinum complexes and dsDNA only showed the presence of ions attributable to intact metal molecules bound to DNA. The fragmentation observed in mass spectral studies of solutions containing nickel complexes and qDNA is attributable to the lower thermodynamic stability of the former metal complexes relative to those containing platinum or ruthenium, as well as the slightly harsher instrumental conditions required to obtain spectra of qDNA. This conclusion is supported by the results of tandem mass spectral studies, which showed that ions consisting of intact nickel complexes bound to qDNA readily undergo fragmentation by loss of one of the ligands initially bound to the metal. The ESI-MS results also demonstrate that the binding affinity of each of the platinum and ruthenium complexes towards qDNA is significantly less than that towards dsDNA.     

Direct observation of essential DNA dynamics: melting and reformation of the DNA minor groove

The dynamics of bound water and ions present in the minor groove of a dodecamer DNA has been decoupled from that of the long-range twisting/bending of the DNA backbone, using the minor groove binder Hoechst 33258 as a fluorescence reporter in the picosecond-resolved time window. The bound water and ions are essential structural components of the minor groove and are destroyed with the destruction of the minor groove when the dodecamer melts at high temperatures and reforms on subsequent cooling of the melted DNA. The melting and rehybridization of the DNA has been monitored by the changes in secondary structure using circular dichroism (CD) spectroscopy. The change in the relaxation dynamics of the DNA has been studied with picosecond resolution at different temperatures, following the temperature-dependent melting and rehybridization profile of the dodecamer, using time-resolved emission spectra (TRES). At room temperature, the relaxation dynamics of DNA is governed by a 40 ps (30%) and a 12.3 ns (70%) component. The dynamics of bound water and ions present in the minor groove is characterized by the 40 ps component in the relaxation dynamics of the probe bound in the minor groove of the dodecamer DNA. Analyses of the TRES taken at different temperatures show that the contribution of this component decreases and ultimately vanishes with the destruction of the minor groove and reappears again with the reformation of the groove. The dynamical behavior of bound water molecules and ions of a genomic DNA (from salmon testes) at different temperatures is also found to be consistent with that of the dodecamer. The longer component of approximately 10 ns in the DNA dynamics is found to be associated with the long-range bending/twisting of the DNA backbone and the associated counterions. The transition from bound water to free water at the DNA surface, indicative of the change in the hydration number associated with each base pair, has also been ascertained in the case of the genomic DNA at different temperatures by employing densimetric and acoustic techniques.     

NETROPSIN: INTERACTION WITH ULTRAVIOLET IRRADIATED DNA

Abstract—Ultraviolet irradiation of double-stranded DNA reduces the circular dichroism (i < 300 nm) induced when the basic peptide antibiotic netropsin (Nt) is added to DNA subsequent to thc irradiation compared to the CD induced by the same concentrations of Nt added to unirradiated DNA. Nt is known to bind to A T base pairs in duplex DNA but will not bind to single-stranded DNA. The reduction in the maximum induced CD observed with saturating concentrations of Nt is a linear function of the concentration of pyrimidine dimers which. along with other dinucleotide photoproducts. form short disrupted regions in duplex DNA. The decrease in the CD of Nt bound to irradiated DNA could be due to elimination of potential Nt sites in the vicinity of a dimer. reduction in the average magnitude of the CD of Nt bound near a dimer or various combinations of these effects. In addition there is a reduction in the average binding constant for Nt bound to irradiated DNA compared to unirradiated DNA suggesting that formation of dinucleotide photoproducts either tends to preferentially eliminate the tighter binding sites or that tighter sites are converted to weaker ones. A simple model suggests that no more than one-third to one-half of the pyrimidine dimcrs formed in DNA completely eliminate a Nt site.     

Fluorescence quenching of ethidium ion by porphyrin cations and quaternary ammonium surfactants in the presence of DNA

Fluorescence quenching of free and DNA-bound ethidium bromide (EB) by a number of quaternary ammonium and other compounds was studied. For free EB or bound EB at lower DNA concentration the fluorescence quenching follows the Stern–Volmer equation and at higher DNA concentration follows an exponential model. At least at low quencher concentrations the quenching efficiency varies with DNA or NaCl concentrations and is about 100 times greater for bound than free EB. The quenching pathways may involve energy transfer and conformational loosening or distortion of the DNA helix in addition to possible electron transfer.     

Assessment of aptamer-steroid binding using stacking-enhanced capillary electrophoresis

The binding affinity of 17β-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17β-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 ± 10 μM. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.     

DNA-Directed Three-Dimensional Protein Organization

All bound together: Self-assembled symmetric DNA polyhedra were used to organize proteins in 3D space. Biotin moieties were incorporated into the self-assembled symmetric DNA polyhedra. Upon incubation with streptavidin (STV) protein, an STV protein became bound to each polyhedral face, thus resulting in well-structured DNA polyhedra/STV complexes (see picture, TET=tetrahedron). This strategy was also applied to different 3D DNA nanostructures and different proteins.     

Enzymatic Fabrication of High‐Density RNA Arrays

A powerful new strategy for the fabrication of high‐density RNA arrays is described. A high‐density DNA array is fabricated by standard photolithographic methods, the surface‐bound DNA molecules are enzymatically copied into their RNA complements from a surface‐bound RNA primer, and the DNA templates are enzymatically destroyed, leaving behind the desired RNA array. The strategy is compatible with 2′‐fluoro‐modified (2′F) ribonucleoside triphosphates (rNTPs), which may be included in the polymerase extension reaction to impart nuclease resistance and other desirable characteristics to the synthesized RNAs. The use and fidelity of the arrays are explored with DNA hybridization, DNAzyme cleavage, and nuclease digestion experiments.     

Endonuclease-based Method for Detecting the Sequence Specific DNA Binding Protein on Double-stranded DNA Microarray

The double-stranded DNA (dsDNA) probe contains two different protein binding sites. One is for DNA- binding proteins to be detected and the other is for a DNA restriction enzyme. The two sites were arranged together with no base interval. The working principle of the capturing dsDNA probe is described as follows: the capturing probe can be cut with the DNA restriction enzyme (such as EcoR I) to cause a sticky terminal, if the probe is not bound with a target protein, and the sticky terminal can be extended and labeled with Cy3-dUTP by DNA polymerase. When the probe is bound with a target protein, the probe is not capable to be cut by the restriction enzyme because of space obstruction. The amount of the target DNA binding proteins can be measured according to the variations of fluorescent signals of the corresponding probes.     

Electrochemical DNA Sensor Using Ferrocenyl Naphthalene Diimide Ligand

Searching for the method for gene detection is vitally important with the development of genetic diagnosis. Generally gene detection is carried out by the judgment of existence of signal of DNA probe (labeled complementary sequence) bound to target DNA sequence. Since sample DNA is immobilized on the solid support, non-bound DNA probe is washed out from the support.     

FLUORESCENCE STUDY OF THE PHYSICO-CHEMICAL PROPERTIES OF A BENZO(A)PYRENE7,8-DIHYDRODIOL9,10-OXIDE DERIVATIVE BOUND COVALENTLY TO DNA

Abstract— Covalent complexes between 7 ,8-dihydrodiol 9.10-oxide benzo(a)pyrene (BPDE) and DNA with a modification of one BPDE molecule per 1000 DNA bases were prepared in vitro . The same stereoselective and chemically homogeneous binding of BPDE to native DNA was observed, as reported earlier for human and bovine bronchial explants. The fluorescence of the pyrene-like aromatic moiety of BPDE bound to DNA in vitro was used as a probe of the microenvironment of the BPDE molecule in order to obtain information about the structure of the BPDE-DNA complex dissolved in aqueous solution. Fluorescence techniques, based on the quenching of the singlet excited states by metal ions such as Ag⁺, by iodide ions, and by molecular oxygen are described, which provide a method for differentiating between external and internal (intercalation) binding of polycyclic aromatic molecules to DNA. Silver ions, which bind specifically to DNA bases, exhibit a strong quenching effect on noncovalently bound, intercalated benzo(a)pyrene; on the other hand, there is no quenching effect on the fluorescence of BPDE in the covalent DNA adduct. Quenchers such as O₂ and iodide ions, which do not specifically bind to DNA and are dissolved in the solution external to the DNA molecule, exhibit a quenching effect on the BPDE chromophore. Furthermore, the fluorescence yield of the BPDE-DNA complex decreases with increasing DNA concentration, an effect which is not observed with non-covalently bound intercalated benzo(a)pyrene-DNA complexes, and which is attributed to intermolecular DNA-DNA interactions. The results of these studies indicate that the pyrene-like chromophore in the covalent BPDE-DNA complex is not intercalated between the base pairs, and that it is located in an accessible region external to the DNA helix. Possible structures are discussed.     

The dynamics of water at DNA interfaces: computational studies of Hoechst 33258 bound to DNA

Together, spectroscopy combined with computational studies that relate directly to the experimental measurements have the potential to provide unprecedented insight into the dynamics of important biological processes. Recent time-resolved fluorescence experiments have shown that the time scales for collective reorganization at the interface of proteins and DNA with water are more than an order of magnitude slower than in bulk aqueous solution. The molecular interpretation of this change in the collective response is somewhat controversial some attribute the slower reorganization to dramatically retarded water motion, while others describe rapid water dynamics combined with a slower biomolecular response. To connect directly to solvation dynamics experiments of the fluorescent probe Hoechst 33258 (H33258) bound to DNA, we have generated 770 ns of molecular dynamics (MD) simulations and calculated the equilibrium and nonequilibrium solvation response to excitation of the probe. The calculated time scales for the solvation response of H33258 free in solution (0.17 and 1.4 ps) and bound to DNA (1.5 and 20 ps) are highly consistent with experiment (0.2 and 1.2 ps, 1.4 and 19 ps, respectively). Decomposition of the calculated response revealed that water solvating the probe bound to DNA was still relatively mobile, only slowing by a factor of 2-3, while DNA motion was responsible for the long-time component (approximately 20 ps).     

The microenvironment of DNA switches the activity of singlet oxygen generation photosensitized by berberine and palmatine

The effect of the interaction between DNA and the photosensitizer on photosensitized singlet oxygen (1O2) generation was investigated using DNA-binding alkaloids, berberine and palmatine. These photosensitizers were bound to DNA by electrostatic force. Near-infrared luminescence measurement demonstrated that the photoexcited alkaloids can generate 1O2 only when the photosensitizers are bound to DNA. A fluorescence decay study showed significant enhancement of the lifetime of their photoexcited state with the DNA binding. A calculation study suggested that the electrostatic interaction with DNA inhibits the quenching of the photoexcited state of these alkaloids via intramolecular electron transfer, leading to the prolongation of the lifetime of their excited state. This effect should enhance their intersystem crossing and the yield of energy transfer to molecular oxygen. The results show that the electrostatic interaction with DNA significantly affects the 1O2 generation activity of a photosensitizer. In addition, this interaction may be applied to the control and the design of photosensitizers for medical applications such as photodynamic therapy.     

Functional insights gained from structural analyses of DNA duplexes that contain UV-damaged photoproducts

Ultraviolet photolesions endow DNA with distinct structural and dynamic properties. Biophysical studies of photoproduct-containing DNA have shown that these lesions affect the mutagenic properties of DNA and damage recognition by DNA repair systems. Recently obtained high-resolution cocrystal structures of damaged DNA bound to either DNA polymerase or DNA repair enzymes have enriched our understanding of the mechanisms by which DNA lesions are bypassed or recognized by DNA metabolizing proteins. Here, we summarize the results of these structural studies and discuss their implications for DNA metabolism.     

Adiabatically bound valence anions of guanine

It is believed that guanine, a basic component of DNA and RNA, has the smallest affinity to an excess electron among all nucleic acid bases. Our experimental and computational findings indicate, however, that many so far neglected tautomers of guanine support adiabatically bound anionic states in the gas phase. The computed values of electron vertical detachment energy for the most stable anionic tautomers are within a broad range of the dominant feature of the photoelectron spectrum. We suggest that guanine might be the strongest excess electron acceptor among nucleic acid bases. Thus it might be critical to radiobiological damage of DNA and it might contribute to those chemical transformations of DNA that proceed through bound anionic states.     

Bioconjugated nanoparticle for DNA protection from ultrasound damage.

To research whether poly-L-lysine-starch nanoparticle (PLL-StNP) could protect DNA from ultrasound damage or not, a series experiments were carried out: plasmid DNA-PLL-StNP complexes were treated with ultrasound for diverse times; the electrophoresis result proved that DNA bound to the complexes all the same. To investigate whether the conjugated DNA was completely protected or not, cDNA fragments bound to PLL-StNP were treated with ultrasound, and matched molecular beacons (MBs) were added. The cDNA-MB-PLL-StNP complexes exhibited dramatically increasing fluorescence, and had the same intensity as that of those MBs that were hybridized with free cDNA fragments. After being treated by ultrasound, the pIRGFP plasmid DNA-PLL-StNP complexes were transferred into COS-7 cells mediated by ultrasound. Green fluorescence protein expressed in most of the cells. Those results indicated that PLL-StNP could completely protect DNA from ultrasound damage. Furthermore, the DNA kept the same function as untreated one.     

Nature of minor-groove binders-DNA complexes in the gas phase

The structure of noncovalent complexes of DNA duplex with minor groove binders (mG-binders) has been analyzed by state of the art molecular dynamics (MD) simulations. More than 3.3 micros of MD trajectories (including 4 x 0.5 micros trajectories) were collected for the Dickerson's dodecamer bound to DAPI, Hoechst 33258, and Netropsin. Comparison of these trajectories with control simulations in water allowed us to determine that the extreme dehydration and partial neutralization occurring during electrospray experiments does not produce the disruption of the DNA:mG-binder complexes or the dissociation of the two strands of the duplex. Irrespective of the drug and the simulation conditions the mG-binders remains bound to the DNA near the preferential binding position in aqueous conditions. Large distortions appear in the two DNA strands, which maintain however a memory of the original DNA duplex structure in water, and a general helical-like conformation.