Monitoring viral DNA release with capillary electrophoresis

Viral DNA injection into host cells is one of the primary mechanisms of viral propagation. Drug development that targets viral propagation requires fast and sensitive methods for monitoring the release of viral DNA in vitro. Here we demonstrate the use of capillary electrophoresis (CE) for monitoring DNA release from virus particles. As a model for this study, we used T5 bacteriophages that infect the bacterium Escherichia coli K-12 by binding to the outer membrane FhuA receptor and then injecting DNA. DNA release from the T5 phages in vitro was induced by either elevated temperature or by interaction with the purified FhuA receptor. After DNA release, the viral samples were stained with the high affinity fluorescent dye YOYO-1, injected into the capillary and subjected to electrophoresis. YOYO-1-stained DNA generated a well-defined peak, allowing reliable detection of viral DNA from as few as 10(5) viral particles. The staining to track T5 phage DNA release exemplifies the great versatility that CE offers in studying viral systems. This CE-based method can be used to study molecular mechanisms of viral infections and to evaluate anti-viral drug candidates.     

毛细管电泳结合电喷雾质谱分析人类癌基因c-myb四链体核酸与活性天然产物的相互作用

药物与靶点间的作用关系直接影响到药理和药效。药物-靶点结合能力、结合计量关系等信息是药物研发过程中必需的表征数据。人类癌基因c-myb在结直肠癌等多种癌症组织中存在过度表达,目前已成为结直肠癌、白血病等癌症疾病潜在的治疗靶点。位于癌基因c-myb启动子区的一段富含鸟嘌呤（G）的DNA序列,通过阳离子的诱导可自发折叠形成分子内G-四链体,而小分子的特异性识别可以稳定该G-四链体,进而调节基因的转录和表达过程。该文采用压力辅助毛细管电泳前沿分析（PACE-FA）结合电喷雾质谱（ESI-MS）研究人类癌基因c-myb启动子G-四链体（G4）与天然产物分子间的相互作用。PACE-FA法在毛细管电泳前沿分析（CE-FA）过程中施加一个与分析物迁移同向的压力,在保证结果准确度的前提下,能够大大加快分析速度。同时结合ESI-MS,可快速解析结合分子与靶点的亲合力和化学计量关系。首先,利用ESI-MS快速筛选出3种有亲合力的天然产物,亲合力大小依次为：土荆皮乙酸>丁溴东莨菪碱>荷叶碱。考虑到溶液相中存在特异性与非特异性结合,接着用PACE-FA法准确分析溶液相中结合的特异性和结合常数。结果发现：丁溴东莨菪碱能够特异性结合靶点G4 DNA,结合比为1：1,结合常数为1.18×10⁵ L/mol;荷叶碱属于非特异性结合,而土荆皮乙酸并未与靶点G4 DNA形成复合物。该组合方法不仅分析速度快,而且能够提高亲和分析的准确度和特异性,有望应用于靶向药物先导结构的发现和作用机制评价。     

Study on the interaction of 5-pyridine-10,15,20-tris-(p-chlorophenyl)porphyrin with cyclodextrins and DNA by spectroscopy

The interaction of 5-pyridine-10,15,20-tris-(p-chlorophenyl)porphyrin (PyTPP) with beta-CD and TM-beta-CD were examined by UV-vis absorption, fluorescence and (1)H NMR spectroscopy. PyTPP prefers to form the 1:1 inclusion complex with TM-beta-CD but hardly form inclusion complex with beta-CD. An inclusion constant (K) for the formation of PyTPP-TM-beta-CD inclusion complex has been evaluated to be 4.4x10(3)L/mol from the absorbance changes. This K value is nearly the same as that 4.5x10(3)L/mol obtained from the fluorescence intensity changes. Compared to beta-CD, the inclusion ability of TM-beta-CD with PyTPP is stronger. It indicates that the hydrophobic effect plays an important role in the inclusion procedure. The mechanism of inclusion interaction was carried out by 1H NMR technique. Furthermore, the interaction of PyTPP with DNA is shown here. It can bind DNA by out-side stacking along the DNA helix but not by intercalation because of the high electron density in the porphyrin core. The binding constant and binding number of PyTPP to DNA are 4.3x10(3) and 1.3, respectively. The interaction of PyTPP with DNA was further carried out in the presence of TM-beta-CD. The significant decrease of the binding constant and binding number were observed and the interaction of porphyrin-bound DNA has been inhibited, which was due to the fact that PyTPP inter into the cavity of TM-beta-CD and influence binding affinity of PyTPP to DNA.     

Studies on Interaction Between Gallocyanine and DNA

IntroductionConsiderable attention has been focused onnew DNA- binding and DNA- modifying agents fromnatural ones to wholly synthetic designs due totheir usage as probes of deciphering the structureand the function of nucleic acids and as potentialchemotherapeutic agents[1— 4] . The application ofthose molecules must be based on the preciseunderstanding of the structural details about thebinding of the agents with the target molecule,double- helical DNA. The interaction of smallmolecules …     

Electrochemical and ultraviolet-visible spectroscopic studies on the interaction of deoxyribonucleic acid with vitamin B6

Studies on the interaction of DNA with vitamin B(6) were carried out with a DNA-modified electrode by electrochemistry and Ultraviolet-visible spectroscopy. The results showed that there exists the supra-molecule interaction between base groups on DNA and vitamin B(6) by forming hydrogen binding, the binding equilibrium constant of the interaction is equal to 115.3 M(-1), the binding ratio of nucleotide to vitamin B(6) is 5:1. Based on the electrochemical and Ultraviolet-visible spectrum studies the interaction mode of DNA with vitamin B(6) was explored.     

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.     

Capillary electrophoresis of double-stranded DNA in an untreated capillary.

Using the zwitterionic buffer N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) in the presence of a high-molecular-mass hydroxypropylmethylcellulose (HPMC) as a sieving polymer and ethidium bromide double-stranded DNA (dsDNA) was separated in an untreated capillary. The HEPES buffer shielded the DNA against the capillary wall interaction and decreased the electroosmotic flow enabling a good separation of the DNA similar to that obtained in a commercially coated capillary. In addition to the low cost of the untreated capillary it can be washed after each run. Furthermore, stacking with hydrodynamic injection filling about half of the capillary volume is demonstrated.     

Electrochemical and spectroscopic studies of the interaction of proflavine with DNA.

The interaction of proflavine with herring sperm DNA has been investigated by cyclic voltammetry and UV-Vis spectroscopy as well as viscosity measurements. Shifts in the peak potentials in cyclic voltammetry, spectral changes in UV absorption titration, an increase in viscosity of DNA and the results of the effect of ionic strength on the binding constant strongly support the intercalation of proflavine into the DNA double helix. The binding constant for the interaction between proflavine and DNA was K = 2.32 (+/- 0.41) x 10(4) M(-1) and the binding site size was 2.07 (+/- 0.1) base pairs, estimated in voltammetric measurements. The value of the binding site size was determined to be closer to that expected for a planar intercalating agent. The standard Gibbs free-energy change is ca. -24.90 kJ/mol at 25 degrees C, indicating the spontaneity of the binding interaction. The binding constant determined by UV absorption measurements was K = 2.20 (+/- 0.48) x 10(4) M(-1), which is very close to the value determined by cyclic voltammetry assuming that the binding equilibrium is static.     

Extracting stoichiometry, thermodynamics, and kinetics for the interaction of DNA with cadmium ion by capillary electrophoresis on-line coupled with electrothermal atomic absorption spectrometry

Understanding the binding of cadmium with DNA is of great importance for elucidating the mechanism of cadmium genotoxicity and carcinogenicity. In the present work, CE on-line coupled with electrothermal atomic absorption spectrometry was employed to study the binding electrophoretic behaviors, stoichiometry, thermodynamics, and kinetics for the interaction of cadmium cation (Cd(II)) with DNA. The stoichiometry (Cd(II) to DNA (as the concentration of base pairs)) for the interaction was determined to be 1:5. Two types of binding sites on DNA were observed with the binding constants of 10(6) and 10(5) L/mol, respectively, showing strong affinity of Cd(II) to DNA. The interaction of Cd(II) with both types of binding sites on DNA were driven by negative enthalpy change with a large positive entropy change. The binding of Cd(II) to DNA followed a first-order kinetics for Cd(II) with the apparent activation energy of 45.7 +/- 1.9 kJ/mol. The results obtained in present investigation would be helpful to understanding the genotoxicity and carcinogenicity of cadmium.     

Nonequilibrium capillary electrophoresis of equilibrium mixtures--a single experiment reveals equilibrium and kinetic parameters of protein-DNA interactions

We introduce a novel electrophoretic method, nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), and demonstrate its use for studying protein-DNA interactions. The equilibrium mixture of protein and DNA contains three components: free protein, free DNA, and the protein-DNA complex. A short plug of such a mixture is injected into the capillary, and the three components are separated under nonequilibrium conditions. The resulting electropherograms are composed of characteristic peaks and exponential curves. An easy nonnumerical analysis of a single electropherogram reveals two parameters: the equilibrium binding constant and the monomolecular rate constant of complex decay. The bimolecular rate constant of complex formation can then be calculated as the product of the two experimentally determined constants. NECEEM was applied to study the interaction between single-stranded DNA binding protein and a fluorescently labeled 15-mer oligonucleotide. It allowed us to measure for the first time the rate constant of complex decay for this important protein-DNA pair, k-1 = 0.03 s-1. The value of the equilibrium binding constant, Kb = 3.6 x 10-6 M-1, was in good agreement with those measured by other methods. As low as 10-18 mol of the protein was sufficient for the measurements. Thus, the new method is simple, informative, and highly sensitive. Moreover, it can be equally applied to other noncovalent protein-ligand complexes. These features of NECEEM make this method an indispensable tool in studies of macromolecular interactions. They also emphasize the potential role of NECEEM in the development of extremely sensitive protein assays using nucleotide aptamers.     

Evaluation of the DNA Alkylation Properties of a Chlorambucil-Conjugated Cyclic Pyrrole-Imidazole Polyamide

Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP-Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP-Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP-Chb conjugates 1 and 2 . Subsequent HPLC analysis revealed that the alkylation site of conjugate 3 , which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP-Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2 . These results suggest that cPIP-Chbs could be novel DNA alkylating anticancer drugs.     

一种新型发光探针与DNA相互作用的研究

本文以稳态荧光光谱、紫外-可见吸收光谱、荧光偏振、热变性、阴离子猝灭等手段,研究了一种具有强电荷转移能力的化合物2,7-二[(N-乙基咔唑-3-基)丙烯-1-酮基]芴与DNA的相互作用。研究结果表明,2,7-二[(N-乙基咔唑-3-基)丙烯-1-酮基)芴与DNA的作用方式是混合模式,以嵌插作用为主,同时存在沟槽相互作用,其咔唑基团可能插入到DNA的碱基对之间,结合常数K为8 123.48mol/L,结合位点n为0.71。该发光探针灵敏度高,结合稳定。     

一种苊并杂环有机小分子嵌入DNA的几何学模式研究

利用圆二色谱(CD)、紫外-可见吸收光谱以及荧光光谱等方法对苊并杂环化合物8-氧-8H-苊并[1,2-b]吡咯-9-腈(A1) 与小牛胸腺DNA(CT DNA) 的相互作用进行了研究. 结果表明, 随着n(A1)/n(CT DNA)的变化存在两种不同的几何学结合构型. 当n(A1)/n(CT DNA)值低于0.20时, A1分子与DNA的结合方式是不均一的, 化合物分子以多种角度嵌入到DNA碱基对之间. 表现为A1-DNA复合物的诱导圆二色光谱图上较小的正峰和紫外吸收光谱图缺省等吸收点. DNA的特征圆二色谱图表明, 在n(A1)/n(CT DNA)≤0.20范围内, CT DNA的构象从标准的B型转化为A-like型; 当n(A1)/n(CT DNA)>0.20时, 诱导圆二色光谱由正峰转变为强度大、波形复杂的负峰, 表明A1分子开始堆积到DNA螺旋的表面, 同时DNA的二级结构发生了进一步变化.     

Sweeping capillary electrophoresis: a non-stopped-flow method for measuring bimolecular rate constant of complex formation between protein and DNA

We introduce sweeping capillary electrophoresis (SweepCE), a non-stopped-flow method for directly measuring the bimolecular rate constant of complex formation, and demonstrate its use for studying protein-DNA interaction. The capillary is prefilled with a solution of DNA, and electrophoresis is then carried out from a solution of the protein in a continuous mode. Because the electrophoretic mobility of the protein is greater than that of DNA, the protein continuously mixes with DNA and forms the protein-DNA complex. The complex migrates with a velocity higher than that of DNA and causes sweeping of DNA, which gave the name to the method. The bimolecular rate constant, kon, of complex formation can be determined from the time profile of DNA concentration using a simple mathematical model of the sweeping process. In this proof-of-principle work, we used SweepCE to directly measure kon = (3.4 +/- 0.6) x 106 M-1 s-1 for the interaction between single-stranded DNA-binding protein and a 15-mer DNA oligonucleotide. Along with nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM), SweepCE establishes a universal and comprehensive platform for studying kinetic and equilibrium parameters of complex formation between biopolymers.     

Affinity capillary electrophoresis and isothermal titration calorimetry for the determination of fatty acid binding with beta-cyclodextrin

Affinity capillary electrophoresis (ACE) and isothermal titration calorimetry (ITC) were used to investigate the binding interaction between several fatty acids (FAs) and beta-cyclodextrin (beta-CD). Within each method, steps taken to obtain accurate binding constants are discussed. The stoichiometry of interaction was revealed to be 1:1 regardless of FA chain length. The binding constants obtained using ACE were: octanoate, 6.4x10(2); 2-octenoate, 4.7x10(2); decanoate, 3.7x10(3); 9-decenoate, 1.8x10(3) and dodecanote, 1.4x10(4). The binding constants obtained from ITC were of the same order of magnitude, but were consistently greater than those from ACE. Thermodynamic data obtained using ITC are used to explain the observed trends in binding strength.     

亲和毛细管电泳法研究锌离子与人血清白蛋白的结合反应机制

建立了研究金属离子与人血清白蛋白(Human serum albumin,HSA)相互作用的亲和毛细管电泳(Affinity capillary electrophoresis,ACE)方法。生理条件下,构建配体(Zn2+)-受体(HSA)相互作用模型,以N,N-二甲基甲酰胺(N,N-Dimethylformamide,DMF)为内标物,基于Scatchard方程,依据有效淌度的变化,通过非线性模拟方程计算Zn2+-HSA结合反应的表观结合常数KB,定量表征了Zn2+-HSA相互作用的强度,并解析电泳谱图获得了Zn2+-HSA结合反应为一快平衡体系的结论。结果表明,建立的ACE方法简捷、有效,Zn2+-HSA相互作用的强度与Zn2+浓度之间存在明显的量效关系。     

Evaluation of interactions between RAW264.7 macrophages and small molecules by capillary electrophoresis

In this study, the affinity interactions between RAW 264.7 macrophages and three small molecules including naringin, oleuropein and paeoniflorin were evaluated by affinity capillary electrophoresis (ACE), partial filling affinity capillary electrophoresis (PFACE) and frontal analysis capillary electrophoresis (FACE), respectively. The result indicated that ACE (varying concentrations of cell suspension were filled in the capillary as receptor) may not be suitable for the evaluation of interactions between cell and small molecules due to the high viscosity of cell suspension; PFACE can qualitatively evaluate the interaction, but the difference in viscosity between RAW264.7 suspension and buffer effects on the liner relationship between filling length and injection time, which makes the calculation of binding constant difficult. Furthermore, based on the PFACE results, naringin showed stronger interaction with macrophages than the other two molecules; taking advantage of the aggregation phenomenon of cell induced by electric field, FACE was successfully used to determine the stoichiometry (n = 5×10⁹) and binding constant (K_b = 1×10⁴ L/mol) of the interaction between RAW264.7 and naringin.     

Interaction of an antidepressant buzepide methiodide with DNA immobilized on the glassy carbon electrode

In the present study, a DNA-biosensor was prepared using immobilization technique to investigate the interaction between an antidepressant, buzepide methiodide (BZP) and calf thymus DNA. BZP showed a quasireversible peak in Britton-Robinson (BR) buffer of pH 5 at bare glassy carbon electrode (GCE). At DNA modified GCE, the peak potential of BZP was observed to be shifted towards positive potential revealing intercalative mode of binding. The binding constant and stoichiometry between DNA and BZP are calculated to be 1.908×10(5)M(-1) and 0.982, respectively. The spectroscopic techniques viz., spectrofluorescence and UV-vis absorption have also been employed to understand the interaction between BZP and DNA. The results serve as a reference for the interaction of BZP with DNA base pairs in the natural environment of living cells.