Electrochemical detection of in situ adriamycin oxidative damage to DNA

Adriamycin intercalation and in situ interaction with double helix DNA was investigated using a voltammetric DNA-biosensor. Oxidation and reduction of adriamycin molecules intercalated in double helix DNA were investigated in order to understand the in vivo mechanism of action with this anti-neoplasic drug. The results showed that the interaction of adriamycin with DNA is potential-dependent causing contact between DNA guanine and adenine bases and the electrode surface such that their oxidation is easily detected. A mechanism for adriamycin reduction and oxidation in situ when intercalated in double helix DNA immobilised onto the glassy carbon electrode surface is presented and the formation of the mutagenic 8-oxoguanine explained.     

Anticancer Drug Modified GCFE,for the Study of Its In‐Vivo Interaction Mechanism

An anticancer drug (Adriamycin) modified Glassy Carbon Fiber Electrode (GCFE) has been prepared to study its interaction with ds‐DNA. The redox reaction of Adriamycin molecules at the chemically modified GCFE helps in understanding the in‐vivo mechanism of action of this anti cancer drug. The modified electrode has been fabricated by the adsorption of Adriamycin on GCFE surface. The results of Differential Pulse Voltammetric (DPV) analysis in acetate buffer of pH 4.5 ± 0.1, showed that the interaction between DNA guanine and adenine bases and electrode surface, is easily detected. A suitable mechanism for the oxidation and reduction of Adriamycin in‐situ intercalated in ds‐DNA immobilized on to the GCFE surface has been explained. The drug‐DNA complex formation at GCFE surface has also been studied. The prepared modified electrode is of utmost relevance because the mechanism of interaction of DNA‐Adriamycin at charged interfaces is parallel to the in‐vivo DNA‐Adriamycin complex reaction, where the nucleic acid is in close contact with charged phospholipid membranes and proteins. The interaction studies of Adriamycin at modified GCFE using DPV method help in understanding the DNA‐Adriamycin reaction mechanism.     

The Synthesis of 1-Aminoxanthones

The importance of the anthracycline antibiotics as anticancer agents, particularly Adriamycin, is well established. It is generally accepted that Adriamycin has the broadest spectrum of activity among all the anticancer agents in clinical use¹.     

Reversed-phase liquid chromatographic determination of plasma levels of adriamycin and adriamycinol

A method is given for the determination of adriamycin and its main metabolite, adriamycinol in plasma from cancer patients after administration of adriamycin as the free drug or as a complex with DNA. Adriamycin and adriamycinol are extracted in a column from 1 ml of plasma (pH 8.6) using a mixture of chloroform--1-heptanol (8:2). After re-extraction into phosphate buffer pH 2.2, the separation is performed as reversed-phase liquid chromatography on a LiChrosorb RP-2 (5 micron) column with a mobile phase of acetonitrile-water, acidified with phosphoric acid. The precision by quantitation with photometric detection was better than 5% within the range 50-300 ng/ml. Plasma levels of adriamycin and adriamycinol in a cancer patient are presented in this paper.     

DNA double helices recognize mutual sequence homology in a protein free environment

The structure and biological function of the DNA double helix are based on interactions recognizing sequence complementarity between two single strands of DNA. A single DNA strand can also recognize the double helix sequence by binding in its groove and forming a triplex. We now find that sequence recognition occurs between intact DNA duplexes without any single-stranded elements as well. We have imaged a mixture of two fluorescently tagged, double helical DNA molecules that have identical nucleotide composition and length (50% GC; 294 base pairs) but different sequences. In electrolytic solution at minor osmotic stress, these DNAs form discrete liquid-crystalline aggregates (spherulites). We have observed spontaneous segregation of the two kinds of DNA within each spherulite, which reveals that nucleotide sequence recognition occurs between double helices separated by water in the absence of proteins, consistent with our earlier theoretical hypothesis. We thus report experimental evidence and discuss possible mechanisms for the recognition of homologous DNAs from a distance.     

DNA-bound lipids: computer modeling of DNA interaction with stearic acid and unsaturated fatty acids

It was shown for the first time by computer experiments that fatty acids are strongly bound to DNA. This is consistent with the presence of free fatty acids in the specimens of DNA-bound lipids isolated from various cells. Binding of all fatty acids to the DNA minor groove is stronger than to the major groove, which is correlated with the presence of two pools of free fatty acids isolated from DNA specimens by biochemical methods. Since DNA polymerase is also bound to the DNA minor groove, fatty acids can play an important role in the regulation mechanism of DNA replication and signal transmission. The energy of interaction of fatty acids with DNA depends on both the number of double bonds and the geometric configuration of the fatty acid and the nucleotide composition of DNA. Dependence on the bond energy in the DNA—fatty acid complex on the nucleotide composition attests to the possibility of site-specific binding of lipids to DNA. On passing from a saturated fatty acid to unsaturated acids containing one, two, or three double trans-bonds, the bond energy of DNA with the fatty acid gradually decreases. The presence of one or three double cis-bonds results in weakening of the strength of the DNA—fatty acid complexes compared to those with the saturated acid. The strongest binding between DNA and fatty acid was found for the unsaturated acid with two double cis-bonds (linoleic). This can be explained by the fact that the bent (boomerang) shape of the molecule of this acid follows the curve of the DNA helix. The pattern of variation of the energy of DNA complexes with stearic, linoleic, oleic, and linolenic acids correlates with experimental data on the melting points of these complexes: the more stable the DNA—fatty acid complex, the lower the melting point of DNA.     

双链DNA分子内电荷转移超交换机理

设计并合成了一系列寡聚核苷酸组成的双链DNA分子,通过检测样品中二氨基嘌呤(Ap)荧光峰强度和相对荧光量子产率来研究DNA分子内电荷转移.实验中直接分辨和观测到双链DNA分子内电荷转移超交换机理,超交换机理在近距离起作用；而电荷转移跳跃机理,可能是通过极子运动形式体现.     

Exonucleolytic degradation of high-density labeled DNA studied by fluorescence correlation spectroscopy

The exonucleolytic degradation of high-density labeled DNA by exonuclease III was monitored using two-color fluorescence correlation spectroscopy (FCS). One strand of the double stranded template DNA was labeled on either one or two base types and additionally at one end via a 5' Cy5 tagged primer. Exonucleolytic degradation was followed via the diffusion time, the brightness of the remaining DNA as well as the concentration of released labeled bases. We found a hydrolyzation rate of about 11 to 17 nucleotides per minute per enzyme (nt/min/enzyme) for high-density labeled DNA, which is by a factor of about 4 slower than for unlabeled DNA. The exonucleolytic degradation of a 488 base pair long double stranded DNA resulted in a short double stranded DNA segment of 112 ± 40 base pairs (bp) length with two single-stranded tails.     

Changes in hydration of lanthanide ions on binding to DNA in aqueous solution

The interaction of the trivalent lanthanides Ce(III), Eu(III), and Tb(III) with sodium deoxyribonucleic acid (DNA) in aqueous solution has been studied using their luminescence spectra and decays. Complexation with DNA is indicated by changes in luminescence intensity. In the system terbium(III)-DNA, changes in luminescence with pH are suggested to be due to the protonation of phosphate groups. The degree of hydration of Tb(III) on binding to DNA is followed by luminescence lifetime measurements in water and deuterium oxide solutions, and it is found that the lanthanide ion loses at least one hydration water on binding to long double stranded DNA at pH 4.7 and pH 7. Rather different behavior is observed on binding to long or short single stranded DNA, where six water molecules are lost, independent of pH. It is suggested that in this case the lanthanide probably binds to the bases of the DNA backbone. The DNA conformation seems to be an important factor in the binding. In addition, the isotopic effect on terbium luminescence lifetime may provide a useful method to distinguish between single and double stranded DNA. DSC results are consistent with cleavage of the double helix of DNA at pH 9 in the presence of terbium.     

Poly-intercalators Carrying Threading Intercalator Moieties as Novel DNA Targeting Ligands

Threading intercalators are a novel class of intercalators that carry two substituents along the diagonal positions of an aromatic ring. These substituents are projecting out in DNA grooves when bound to DNA. Poly-intercalators carrying threading intercalating parts are quite novel and were recently found to show a unique DNA binding behavior. We review herein two types of poly-intercalators. First, tris-intercalators carrying a threading intercalator part in the middle of the molecule are described. These intercalators appear to intercalate into double stranded DNA in a special binding manner, which we call the penetrating mode, in which all the three intercalating units are arranged linearly with one of them penetrating into the DNA ladder. We synthesized two tris-intercalators ( 3 and 4) of this type and studied their binding behavior for double stranded DNA. All the experimental results were consistent with the proposed penetrating mode. Another type of threading poly-intercalators is a macrocyclic bis-threading intercalator ( 5). We found that this compound can bis-intercalate to double stranded DNA when the base pairing is disrupted temporarily to form a complex with a unique structure like a catenane. On the basis of a study of the interaction of such intercalators we envisage that DNA is a flexible and dynamic entity. These novel families of poly-intercalators will expand the scope of DNA poly-intercalation chemistry with possible medicinal applications.     

阿霉素的光谱电化学研究

利用循环伏安、紫外可见光谱电化学、荧光光谱电化学、圆二色光谱电化学等方法研究了阿霉素(ADM)在石墨电极上的电化学行为.结果发现,阿霉素在+0.2～+0.7V和-0.2～-0.7V范围内分别出现一对氧化还原峰.正电位下,蒽环上的酚羟基发生单电子氧化,并伴随后续化学反应.负电位范围内,阿霉素经历ECE电极反应过程,即蒽醌经单电子还原生成半醌自由基,半醌可发生不可逆的化学反应,脱去配氧糖基,转变为7-去氧柔毛霉醌(7-deoxyadriamycinone),后者在更负的电位下形成一对可逆的新的氧化还原峰.     

Single,double, and multiple double strand breaks induced in DNA by 3-100 eV electrons

Nonthermal secondary electrons with initial kinetic energies below 100 eV are an abundant transient species created in irradiated cells and thermalize within picoseconds through successive multiple energy loss events. Here we show that below 15 eV such low-energy electrons induce single (SSB) and double (DSB) strand breaks in plasmid DNA exclusively via formation and decay of molecular resonances involving DNA components (base, sugar, hydration water, etc.). Furthermore, the strand break quantum yields (per incident electron) due to resonances occur with intensities similar to those that appear between 25 and 100 eV electron energy, where nonresonant mechanisms related to excitation/ionizations/dissociations are shown to dominate the yields, although with some contribution from multiple scattering electron energy loss events. We also present the first measurements of the electron energy dependence of multiple double strand breaks (MDSB) induced in DNA by electrons with energies below 100 eV. Unlike the SSB and DSB yields, which remain relatively constant above 25 eV, the MDSB yields show a strong monotonic increase above 30 eV, however with intensities at least 1 order of magnitude smaller than the combined SSB and DSB yields. The observation of MDSB above 30 eV is attributed to strand break clusters (nano-tracks) involving multiple successive interactions of one single electron at sites that are distant in primary sequence along the DNA double strand, but are in close contact; such regions exist in supercoiled DNA (as well as cellular DNA) where the double helix crosses itself or is in close proximity to another part of the same DNA molecule.     

A temperature-dependent interaction of neutral red with calf thymus DNA

Neutral red (NR) is used as a probe to study the temperature and concentration dependent interaction of a cationic dye with nucleic acid. A temperature-dependent interaction of NR with calf thymus DNA (CT DNA) has been studied by differential pulse voltammetry (DPV), UV-Visible absorption, circular dichroism (CD) and fluorescence spectroscopy. The experimental results of increasing peak current, changes in the UV-Visible absorption and fluorescence spectra of NR and decreasing the induced circular dichroism (ICD) intensity show that (i) the binding mode of NR molecules is changed from intercalating into DNA base pairs to aggregating along the DNA double helix and (ii) the orientation of NR chromophore in DNA double helix is also changed with the temperature.     

中性介质中中性红与双链DNA作用的光谱

通过分子吸收、荧光发射和共振光散射测定,表征了在水溶液介质中中性红(NR)与双 螺旋DNA的作用.在pH 7.63和离子强度低于0.01的水溶液介质中,随着NR与DNA的摩尔比(R)变 化,存在有两种结合方式.第一种结合方式发生在R > 2.22,此时获得共振光散射光谱增强信 号,表明NR在DNA分子表面发生聚集,集聚特性可使用RLS测定数据进行Scatchard分析;第二种 结合方式发生在R < 2.22,此时NR内嵌到DNA分子的双链碱基对之间,具有特征波长红移和分 子吸收增色效应,发生了从DNA到NR的分子能量转移,能观察到荧光增强.     

The Detection and Stability of DNA Duplexes Probed by MALDI Mass Spectrometry

Matrix‐assisted laser desorption/ionization (MALDI) with nonacidic matrices is shown to generate intact gas‐phase ions of double‐stranded DNA. Control experiments show that specific DNA duplexes are detected by this method, while nonspecific adducts form only when high monomer concentrations are present in the MALDI sample. The relative intensity of the duplex‐ion signal is found to reflect the solution‐phase stability of the double‐stranded DNA.     

Pseudohexagonal 2D DNA crystals from double crossover cohesion

Two-dimensional pseudohexagonal trigonal arrays have been constructed by self-assembly from DNA. The motif used is a bulged-junction DNA triangle whose edges and extensions are DNA double crossover (DX) molecules, rather than conventional DNA double helices. Experiments were performed to establish whether the success of this system results from the added stiffness of DX molecules or the presence of two sticky ends at the terminus of each edge. Removal of one sticky end precludes lattice formation, suggesting that it is the double sticky end that is the primary factor enabling lattice formation.     

Naphthalene Diimide Carrying Two Cysteine Termini at Both Imide Linkers as a Molecular Staple

Naphthalene diimide ( 1 ) carrying cysteines at the termini of amide substituents were synthesized to act as a molecular staple of double stranded DNA. Since 1 is able to bind to double stranded DNA with threading intercalation, the complex of 1 with double stranded DNA can be topologically immobilized on a gold surface through the S? Au linkage as confirmed by cyclic voltammetric experiment. Ferrocenyl‐double stranded 23‐mertic oligonucleotide, dsFcODN, was immobilized on gold electrode with 1.0×10¹² molecules cm^?2 when electrode was treated with 2.0 µM dsFcODN and 4.0 µM 1 for 1 h at room temperature. The coverage density was similar to that obtained for the terminal thiol‐modified oligonucleotide. Compound 1 was applied to detect the 321‐meric PCR product of P. gingivalis, which is important in the diagnosis of periodontal disease. This experiment, coupled with the use of ferrocenylnaphthalene diimide, FND as electrochemical indicator for double stranded DNA, resulted in quantitative detection of PCR product within the range of 10 pg µL^?1–10 ng µL^?1 (15 nM–15 µM). The 1 and FND established a simple and rapid detection method of double stranded PCR product with a detection limit of 10 pg µL^?1 (15 nM).     

Topological solitons in an inhomogeneous DNA molecule

The dynamics of topological solitons describing the opening of the double helix of a DNA molecule is studied. The estimated actual values of the rigidity of the polynucleotide chains made it possible to develop a more precise DNA model and to show that four types of topological solitons can appear in the DNA double helix. Interactions between solitons are studied, as well as their interaction with the chain inhomogeneities and the stability of solitons with respect to thermal fluctuations. Thermal fluctuations promote propagation of solitons along an inhomogeneous base sequence.     

Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity

The present work demonstrates a novel signal-off electrochemical method for the determination of DNA methylation and the assay of methyltransferase activity using the electroactive complex [Ru(NH₃)₆]³⁺ (RuHex) as a signal transducer. The assay exploits the electrostatic interactions between RuHex and DNA strands. Thiolated single strand DNA1 was firstly self-assembled on a gold electrode via Au–S bonding, followed by hybridization with single strand DNA2 to form double strand DNA containing specific recognition sequence of DNA adenine methylation MTase and methylation-responsive restriction endonuclease Dpn I. The double strand DNA may adsorb lots of electrochemical species ([Ru(NH₃)₆]³⁺) via the electrostatic interaction, thus resulting in a high electrochemical signal. In the presence of DNA adenine methylation methyltransferase and S-adenosyl-l-methionine, the formed double strand DNA was methylated by DNA adenine methylation methyltransferase, then the double strand DNA can be cleaved by methylation-responsive restriction endonuclease Dpn I, leading to the dissociation of a large amount of signaling probes from the electrode. As a result, the adsorption amount of RuHex reduced, resulting in a decrease in electrochemical signal. Thus, a sensitive electrochemical method for detection of DNA methylation is proposed. The proposed method yielded a linear response to concentration of Dam MTase ranging from 0.25 to 10 U mL⁻¹ with a detection limit of 0.18 U mL⁻¹ (S/N = 3), which might promise this method as a good candidate for monitoring DNA methylation in the future.