Label-free molecular beacon system based on DNAs containing abasic sites and fluorescent ligands that bind abasic sites

A new class of label-free molecular beacon (MB) system based on DNA strands that contain abasic (AP) sites (AP-DNA) and adopt stem-loop structures, in combination with fluorescent ligands that bind these AP sites, has been developed. Unlike a conventional MB, which requires covalent labeling of the MB with a fluorophore and a quencher, the developed system (APMB) does not require covalent attachment of signal transduction units. Detailed sensing functions of a series of APMB systems were examined with the aid of the fluorescent ligand named ATMND to provide insight into the design strategy for APMB systems. The effects of the stem length and the position of the AP site in the stem moiety on the fluorescence response of the APMB system were examined. Genotyping of a G/C SNP of PCR amplification products was successfully demonstrated with the APMB system and blue-fluorescent ATMND as a ligand. The APMB system was further extended to a system that utilized green-fluorescent lumiflavin.     

Detection of labeled abasic sites in damaged DNA by capillary electrophoresis with laser-induced fluorescence

Removal of nucleobases from the DNA backbone leads to the formation of abasic sites. The rate of abasic site formation is significantly increased for chemically damaged nucleobases. Thus, abasic sites serve as general biomarkers for the quantification of DNA damage. Herein, we show that capillary electrophoresis with laser-induced fluorescence (CE-LIF) can be used to detect the amount of abasic sites with very high sensitivity. For proof of concept, DNA was incubated with methylmethane sulfonate (MMS) and the damaged bases were removed by incubation at 80 °C. The resulting abasic sites were then tagged with a fluorescent aldehyde-reactive probe (FARP). The DNA was precipitated with ethanol, and then analyzed by CE-LIF. CE-LIF and HPLC analysis shows that the fluorescently tagged DNA (DNA-FARP) had a peak area directly proportional to the amount of N-7 methyl guanines. The CE-LIF method had a detection limit of 1.2 abasic sites per 1,000,000 bases or ca. 20 attomoles of abasic sites. This provides a general method for detecting DNA damage that is not only faster but also has comparable or better sensitivity than the alternative ELISA-like method.     

Molecular dynamics simulation of clustered DNA damage sites containing 8-oxoguanine and abasic site

Clustered DNA damage sites induced by ionizing radiation have been suggested to have serious consequences to organisms, such as cancer, due to their reduced probability to be repaired by the enzymatic repair machinery of the cell. Although experimental results have revealed that clustered DNA damage sites effectively retard the efficient function of repair enzymes, it remains unclear as to what particular factors influence this retardation. In this study, approaches based on molecular dynamics (MD) simulation have been applied to examine conformational changes and energetic properties of DNA molecules containing clustered damage sites consisting of two lesioned sites, namely 7,8-dihydro-8-oxoguanine (8-oxoG) and apurinic/apyrimidinic (AP) site, located within a few base pairs of each other. After 1 ns of MD simulation, one of the six DNA molecules containing a clustered damage site develops specific characteristic features: sharp bending at the lesioned site and weakening or complete loss of electrostatic interaction energy between 8-oxoG and bases located on the complementary strand. From these results it is suggested that these changes would make it difficult for the repair enzyme to bind to the lesions within the clustered damage site and thereby result in a reduction of its repair capacity.     

Molecular modeling on garuganin-I

Summary The conformation of garuganin-I was analyzed in terms of the mobility of the cyclic structure. Molecular mechanics calculations were applied to show that the interconversion of the optical isomers is not possible at room temperature due to steric interactions. Different possibilities of the distortion of the molecular conformations were calculated and compared with molecular dynamics simulations.

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Molekulares Modelling von Garuganin-I

Zusammenfassung Die Konformation von Garuganin-I wurde bezüglich der Mobilität der cyclischen Struktur analysiert. Es wurden molekularmechanische Berechnungen herangezogen, die zeigten, daß die Interkonversion der optischen Isomeren bei Raumtemperatur wegen sterischer Wechselwirkungen nicht möglich ist. Es wurden verschiedene Möglichkeiten von Konformationsänderungen berechnet und mit einer Simulation der Molekulardynamik verglichen.

     

Molecular modeling and dynamics of neuropeptide Y

Summary A combination of molecular modeling and molecular dynamics (MD) is used to determine a theoretical structure for neuropeptide Y (NPY). Starting with the X-ray structure for avian pancreatic polypeptide (APP), the substituted amino acids were mutated, the side chains oriented to local potential energy minima, and the entire structure minimized and subjected to an MD simulation. Comparison of the resulting NPY structure with APP X-ray and MD results showed secondary structural elements to be maintained and RMS fluctuations to be similar, although differences in both were observed. The approach presented offers a means to study the structure-function relationships of NPY and other similar polypeptides when combined with pharmacological measurements.Abbreviations NPY Neuropeptide Y - APP Avian pancreatic polypeptide - ABNR Adopted-basis Newton Raphson - MD Molecular dynamics     

A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site

Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.     

Impedance‐Based DNA Biosensor Employing Molecular Beacon DNA as Probe and Thionine as Charge Neutralizer

In this paper, we present an impedance‐based DNA biosensor using thionine intercalation to amplify DNA hybridization signal. Beacon single‐stranded DNA (ssDNA) probe and mercaptoacetic acid were self‐assembled onto a Au electrode by forming Au? S bonds. These beacon ssDNAs were hybridized with the complementary sequences around the loop structure. Then thionine was intercalated into the double‐stranded DNA (dsDNA) immobilized on the Au electrode surface. Due to the neutralization of the negative charges of dsDNA by the intercalated thionine, the electronic transfer resistance (R_et) of the DNA modified Au electrode was significantly diminished. Herein, the decreased value of R_et resulted from the thionine intercalating into dsDNA was employed as the hybridization signal. SDS was used to reduce the unspecific adsorption between ssDNA and thionine. Several experimental conditions, including the surface coverage of ssDNA probe on Au electrode, the hybridization temperature and time were all optimized. Moreover, the hybridization reactions of the unstructured linear ssDNA probe and the structured beacon ssDNA probe with their complementary sequences were compared in this work. The sensitivity of the presented DNA biosensor highlighted that the intercalation of thionine into dsDNA was an efficient approach to amplify the hybridization signal using impedance detection technique. Additionally, in this DNA biosensing protocol, beacon ssDNA has a good ability to distinguish target DNA sequences. This results in a higher specificity than using traditional unstructured DNA probe.     

Simple fluorescent pyrimidine analogues detect the presence of DNA abasic sites

A family of simple pyrimidine analogues has been synthesized, and their photophysical properties have been investigated. The most responsive of the family was incorporated in DNA. This isosteric fluorescent DNA analogue monitors denaturation of a DNA duplex via fluorescence and positively detects the presence of abasic sites in DNA duplexes.     

New potential DNA intercalators of the carbazole series from indole-2,3-quinodimethanes: Synthesis,crystal structure,and molecular modeling with a watson-crick mini-helix

Summary 1-Alkylpyrano[3,4-b]indol-3-ones3 react via a Diels-Alder step with an aryne or N-phenylmaleimide to furnish the new [b]annellated carbazoles4–10 in a one-pot process. In an analogous procedure, the in situ generated N-benzoylindole-2,3-quinodimethane (13) reacted with quinones to furnish the dioxocarbazoles14–16. Compounds4–8 and14–16 with a coplanar skeleton are members of a class of potential DNA intercalators, as has been shown for5 and8 by X-ray structural analysis. On the basis of the geometries determined by X-ray crystallography, the intercalative binding of these molecules with a Watson-Crick mini-helix was predicted by molecular modeling methods.

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Neue potentielle DNA-Interkalatoren der Carbazol-Reihe aus Indol-2,3-chinodimethanen: Synthese, Kristallstruktur und Molecular Modeling mit einer Watson-Crick Minihelix

Zusammenfassung 1-Alkylpyrano[3,4-b]indol-3-one3 reagieren über einen Diels-Alder-Schritt mit Arin oder N-Phenylmaleinimid zu [b]annellierten Carbazolen4–10 in einer Einstufenreaktion. In analoger Weise reagiert ein in situ erzeugtes N-Benzoylindol-2,3-chinodimethan13 mit Chinonen zu den Dioxocarbazolen14–16. Die Verbindungen4–8 und14–16 gehören infolge ihrer coplanaren Struktur zur Klasse potentieller DNA-Interkalatoren. Auf der Basis von Röntgenstrukturanalysen von5 und8 wird die interkalative Bindung mit einer Watson-Crick Minihelix durch Molecular Modeling vorhergesagt.

     

Λ-及Δ-[Ru(phen)2dppz]n+对错配DNA的识别作用的分子模拟

本文通过在ESFF(Extensible Systematic Force Field)力场下对其作用中的体系势能进行分子力学计算，分析了手性金属配合物Λ－及Δ-[Ru(phen)2dppz]^n 对错配DNA d(CCGAATGAGG)2的识别机理，并在分子水平上对其做了详细解释。     

Molecular modeling on the recognition of DNA sequence and conformational repair of sheared DNA by novel chiral metal complex D, L-[Co(phen)_2hpip]~(3 )

Recent studies revealed that DNA, once considered as a very stable macromolecular, is rather unstable. Familiar factors, like heavy metal, microbe, high fre-quency electromagnetic radiation and so on, could easily damage the structure of DNA in different …     

Interstrand cross-link of DNA by covalently linking a pair of abasic sites

A pair of apurinic/apyrimidinic sites formed in DNA has been covalently connected with bis(aminooxy) derivatives. The efficacy of the interstrand cross-link is associated with the structural tethering of two aminooxy groups. The interstrand cross-link constructed stable DNA scaffolds for enzyme alignment.     

Computer simulation of the binding of amonafide and azonafide to DNA

Summary Intercalative binding of the antitumor drugs amonafide and azonafide to the oligonucleotide duplex d(GGCCGGCCGG)·d(CCGGCCGGCC) was compared using molecular dynamics in vacuum with the AMBER force field. A number of reasonable possible binding conformations were obtained, with the azonafide complexes favored over the amonafide complexes in net binding enthalpy. In comparison with amonafide, the larger chromophore of azonafide permits greater DNA distortion and wider side-chain swings, without falling out of the intercalation site. The best model obtained was used for further dynamics on amonafide and azonafide with solvent and counterions present, and again the azonafide complex had a more favorable enthalpy. Furthermore, the enthalpy change on going from solvent into the intercalation site was less unfavorable for azonafide. These results are consistent with the stronger DNA binding of azonafide compared to amonafide, as observed in relative melting transition temperature increases and tumor inhibition in cell cultures.     

Determination of a set of parameters for the molecular modelling of phosphorothioate DNA

Phosphorothioate DNAs, have emerged as a new class of potent drugs. They are obtained by the replacement of the anionic oxygens of the phophodiester backbone by sulphur. A set of parameters has been developed for the FLEX force field implemented in JUMNA 10.0 to take into account the influence of sulphur on the structure of the DNA double helix. The consistency of our parameters was tested by modelling a phosphorothioate oligomer namely d(GC)8. d(GC)8. Results, obtained on both R-p_S and S-p_S diastereoisomers, were compared to the phosphodiester counterpart and are in agreement with available experimental data. Thus, our set of parameters seems suitable for further molecular modelling of other phosphorothioate oligomers. Received: 15 July 1998 / Accepted: 8 September 1998 / Published online: 10 December 1998     

分子力场发展的新趋势

分子模拟中的力场方法是用来精确计算分子结构和能量的计算方法,它通过原子核的位置来计算体系能量。最初的分子力场都是针对某一特定体系的,它们的许多参数要由观测数据拟合得到。当时要建立新的分子力场是十分困难的,因为实验归属振动谱带需要花费大量的时间。所以此后大多数工作者都致力于发展涵盖尽可能多体系的“求全”型分子力场,这种趋势一直延续至今。但是随着各个学科研究的不断深入,所需要研究的体系越来越复杂,要求的精度也越来越高。在保证相当精度的条件下,“求全”型的分子力场要想涵盖所有需要研究的体系常常是十分困难的事情。2003年问世的Direct Force Field软件包能够便捷的建立针对某一特定分子体系,并且有相当精度的分子力场。它的出现为分子力场从“求全”转为向“求精”发展提供了可能。     

Molecular modeling study of an abasic DNA undecamer duplex: d(GCGTGOGTGCG) · d(CGCACTCACGC)

Molecular mechanics calculations were performed with the JUMNA program on d(GCGTGOGTGCG) · d(CGCACTCACGC) where “O” is a modified abasic site: 3-hydroxy-2-(hydroxymethyl)tetrahydrofuran. From energy minimizations, for intrahelical or extrahelical positions of the unpaired thymine, various structures with different curvatures were obtained. Dynamical properties of this abasic sequence were also investigated through the controlled studies of DNA bending. Poisson-Boltzmann calculations were used to mimic the electrostatic effect of solvent on this sequence. The lowest energy structures show an acceptable agreement with experimental data. Received: 1 June 1998 / Accepted: 17 September 1998 / Published online: 10 December 1998     

分子动力学研究亚铁血红素激活蛋白转录激活机理

以3种亚铁血红素激活蛋白(Heme activator protein, HAP)-DNA 复合物(野生型HAP1-wt, Ser63/Arg63突变HAP1-18 和 Ser63/Gly63突变HAP1-PC7)为对象对亚铁血红素激活蛋白的转录激活机理进行了分子动力学研究. 对3个复合物分子动力学轨迹的比较性分析显示, 涉及到上游活化序列(Upstream activation sequences, UAS)识别的蛋白质-DNA 相互作用分布与实验观测到的3种蛋白转录活性一致. 进一步对3个复合物进行柔性分析显示, 3个DNA分子具有相似的柔性, 而又有所不同, 特别在涉及UAS识别的N-端和Zn2Cys6结构域前部有明显的柔性差异. 蛋白质柔性的差别导致不同的蛋白质-DNA相互作用. 因此亚铁血红素激活蛋白的N-端和Zn2Cys6结构域前部的柔性大小能够调节亚铁血红素激活蛋白转录激活功能.     

Molecular modeling studies on the urease active site and the enzyme-catalyzed urea hydrolysis

Summary These studies are an attempt to gain better insight into the pharmacophore requirements of urease. On the basis of published information on this enzyme (EXAFS, amino acid sequence, essential groups at the active site) a hypothetical nickel-tripeptide complex, as preliminary substitute for the urease active site was modeled using computer-aided molecular modeling techniques. The results suggest two alternative docking modes of urea and reaction intermediates, corresponding to two different reaction mechanisms. Both binding modes are compatible with the docking of known potent inhibitors such as selected hydroxamic acids and phosphorodiamides. The results can be used to help in the design of new potential inhibitors of urease.