铜配合物促进的DNA氧化断裂

本文首先对铜配合物催化DNA氧化断裂的机制及相关因素包括DNA结合能力与方式、活性氧物种形成和活性氧物种对底物的损伤展开了讨论;其次结合我们课题组的工作对配合物的结构对其DNA切割性能的影响分别进行了总结,分别探讨了核数、核的种类、位阻、电荷、结合方式以及氧化还原电位等因素的影响;同时还对光激发铜配合物断裂DNA的机制和性能进行分析;最后对特异性含铜DNA断裂试剂的不同设计策略及其相关切割行为进行的系统地总结. 这些总结不仅有利于对铜配合物促进DNA 断裂行为的系统理解,而且对于进一步设计高效含铜人工核酸酶实现DNA的特异性断裂具有良好的指导意义.     

两亲性大环多胺La(III)配合物的合成、表征及催化质粒DNA水解的研究

近年来,人工核酸切割试剂的研究一直是化学生物学、生物化学和分子生物学中最为活跃的前沿领域之一。最近的研究结果表明大环多胺金属配合物在磷酸二酯水解方面表现出独特的催化性能,能作为化学核酸酶有效的催化DNA和RNA的磷酸二酯键的水解[1-2]。尤其是电荷较高的金属阳离子形     

具有DNA切割功能的新型多聚酰胺/丝组缀合物

为得到具有核酸切割功能的人工核酸酶, 设计合成了一种新型多聚酰胺/丝组缀合物, 并研究了其DNA切割活性. 合成的目标化合物在pH=6.0的BR缓冲溶液中对pBR322 DNA切割活性的初步实验结果表明, 于37 ℃保温6 h后, pBR322 DNA基本上被完全从Form Ⅰ切割为Form Ⅱ, 保温36 h后, pBR322 DNA几乎被切割完全.     

金属有机配合物对DNA的断链作用

过渡金属配合物催化DNA、RNA的断链反应研究是近年来最为活跃的前沿研究领域之一,因某些金属配合物具有核酸酶特异性催化DNA、RNA断链的功能,因而该研究对新型抗肿瘤、抗艾滋病化学药物的定向设计及其基因治疗和分子生物学研究中DNA、RNA的高度专一性定点断裂、染色体图谱分析及DNA定位诱变、基因工程中足迹技术(footprinting)以及DNA构象识别等方面均具有重要意义和应用前景,文献已报道了一些具有断裂DNA、RNA功能的金属配合物,但其中很少有含金属-碳σ键的金属有机化合物,本文首次报道了二茂铁鎓离子三氯乙酸盐和二氯二茂钛对DNA的断链作用。     

大环配合物在核酸切割中的应用

核酸切割试剂的研究是化学和分子生物学中最为活跃的前沿领域之一.研究核酸切割试剂,不仅是因为对核酸酶催化机理进行深入了解之需,同时还因为人工核酸酶在基因治疗中有着诱人的前景.大环配合物的合成、结构及其对核酸的切割作用已有广泛的文献报道.本文综述了作为重要的核酸切割试剂的大环多胺配合物和氮杂冠醚配合物对核酸的切割作用及其规律性,并展望了今后的发展与应用方向.     

N,N'-双(2-氨基乙基)-2,6-吡啶二甲酰胺铜配合物催化DNA裂解

本文通过凝胶电泳分析研究了N,N'-双(2-氨基乙基)-2,6-吡啶二甲酰胺铜配合物(BAP)和DNA的相互作用。结果表明BAP-Cu^2＋配合物能够有效催化裂解超螺旋质粒DNA, 它是DNA自然裂解的3.2×10⁶倍,成功地用简单化合物模拟了核酸酶水解DNA的功能。同時本文也初步提出铜离子和氨基协同催化水解DNA磷酸二酯键的裂解机理。     

环三聚磷腈多齿配体的合成及DNA的切割活性

为了得到具有核酸切割功能的人工核酸酶, 设计合成了5种环三聚磷腈多齿配体, 并初步检测了其对DNA的切割活性. 目标化合物的结构由IR, 1H NMR, 31P NMR, 13C NMR和ESI-MS确认. 在生理条件下对pUC19 DNA切割活性的初步实验结果表明, 在化合物5a～5e的Cu(Ⅱ)配合物存在下, 保温24 h后, pUC19 DNA由Form Ⅰ断裂为Form Ⅱ, 即合成目标化合物有明显的DNA切割活性. 同时, 考察了配合物5b+Cu在不同时间下对DNA的切割活性的影响.     

丝组二肽对DNA的切割作用的研究

近20年来,人工核酸切割试剂的研究一直是生物化学中最为活跃的前沿领域之一,研究人工核酸切割试剂的主要目的是合成定点切割试剂,后者是一种重要的分子生物学工具,在疾病的基团治疗、反义PCR技术等领域中有着重要的应用价值。此外,人工核酸切割试剂还可以在足迹技术和核酸高级结构的研究中用作高分辨率的化学探针。     

利用人工模拟酶环糊精催化羧酸酯水解反应的研究进展

环糊精因其结构和性质的特殊性在人工模拟酶方面发挥着重要的作用．概述了 利用简单的环糊精、带有催化基团的环糊精和二聚体环糊精催化羧酸酯水解反应的 研究进展．     

负载型廉价金属催化剂在低温催化氧化甲醛中的应用

甲醛是室内常见的挥发性有机污染物之一,长期接触会严重危害人体健康。负载型廉价金属催化剂在甲醛去除和实际应用方面表现出优异性能,引起研究人员的广泛关注。本文阐述了低温条件下负载型廉价金属催化剂在甲醛热催化氧化、光催化氧化和等离子协同催化氧化方面的研究进展,介绍了甲醛低温催化的影响因素,并讨论了反应机理。反应条件、载体类型和制备方式是影响甲醛低温催化活性的重要因素。虽然负载型廉价金属催化剂在甲醛光催化氧化和热催化氧化方面均表现出良好性能,但仍须进一步探究提升其在可见光和室温下的催化活性。对于甲醛等离子协同催化氧化,降低反应过程所产生的副产物和能耗仍是研究重点。此外,本文还对负载型廉价金属催化剂在甲醛催化应用中的发展方向进行了展望。     

Design of artificial nucleases and studies of their interaction with DNA

The design of artificial nucleases and nuclease mimics has attracted extensive attention and made great progress due to their significant scientific meanings and potential application in the field of gene medicine and molecular biology. This paper reviews recent progress in the investigation of artificial nuclease, including “bifunctional cooperative catalysis”, “dinuclear synergistic catalysis”, “metal-free catalysis”, and especially, the studies of aza-crown ethers as artificial nucleases and their interaction with DNA. Supported by the National Natural Science Foundation of China (Grant Nos. 20872061 & 20372032) and the National Basic Research of China (Grant No. 2007CB925103)     

A simple, high sensitivity mutation screening using Ampligase mediated T7 endonuclease I and Surveyor nuclease with microfluidic capillary electrophoresis

Mutation and polymorphism detection is of increasing importance for a variety of medical applications, including identification of cancer biomarkers and genotyping for inherited genetic disorders. Among various mutation-screening technologies, enzyme mismatch cleavage (EMC) represents a great potential as an ideal scanning method for its simplicity and high efficiency, where the heteroduplex DNAs are recognized and cleaved into DNA fragments by mismatch-recognizing nucleases. Thereby, the enzymatic cleavage activities of the resolving nucleases play a critical role for the EMC sensitivity. In this study, we utilized the unique features of microfluidic capillary electrophoresis and de novo gene synthesis to explore the enzymatic properties of T7 endonuclease I and Surveyor nuclease for EMC. Homoduplex and HE DNAs with specific mismatches at desired positions were synthesized using PCR (polymerase chain reaction) gene synthesis. The effects of nonspecific cleavage, preference of mismatches, exonuclease activity, incubation time, and DNA loading capability were systematically examined. In addition, the utilization of a thermostable DNA ligase for real-time ligase mediation was investigated. Analysis of the experimental results has led to new insights into the enzymatic cleavage activities of T7 endonuclease I and Surveyor nuclease, and aided in optimizing EMC conditions, which enhance the sensitivity and efficiency in screening of unknown DNA variations.     

Design of a colicin E7 based chimeric zinc-finger nuclease

Colicin E7 is a natural bacterial toxin. Its nuclease domain (NColE7) enters the target cell and kills it by digesting the nucleic acids. The HNH-motif as the catalytic centre of NColE7 at the C-terminus requires the positively charged N-terminal loop for the nuclease activity—offering opportunities for allosteric control in a NColE7-based artificial nuclease. Accordingly, four novel zinc finger nucleases were designed by computational methods exploiting the special structural features of NColE7. The constructed models were subjected to MD simulations. The comparison of structural stability and functional aspects showed that these models may function as safely controlled artificial nucleases. This study was complemented by random mutagenesis experiments identifying potentially important residues for NColE7 function outside the catalytic region.     

化学核酸酶及其作用机理

化学核酸酶是一类人工设计、合成的DNA 或RNA 定位断裂工具, 由核酸识别结合系统和化学断裂系统组成。它们能够在任何位点断裂单链、双链DNA 或RNA , 不受限制性内切酶的天然专一性限制。本文除介绍了一些新的化学核酸酶体系外, 着重对它们的作用方式及作用机理进行了讨论。     

Synthesis,crystallographic and spectroscopic characterization and magnetic properties of dimer and monomer ternary copper(II) complexes with sulfonamide derivatives and 1,10-phenanthroline. Nuclease activity by the oxidative mechanism

Three dinuclear and one mononuclear copper(II)-1,10-phenanthroline ternary complexes, [Cu(L1)(phen)(OH)]₂ (1), [Cu(L2)(phen)(OH)]₂·3H₂O (2), [Cu(L3)(phen)(OH)]₂ (3) and [Cu(L4)₂(phen)(H₂O)] (4), with thiadiazole sulfonamide derivative ligands: HL1 (N-(5-ethyl-1,3,4-thiadiazol-2-yl)naphthalene-1-sulfonamide), HL2 (N-(5-ethylthio)-1,3,4-thiadiazol-2-yl)-4-methylbenzenesulfonamide), HL3 (N-(5-ethyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide) and HL4 (N-(5-ethyl-1,3,4-thiadiazol-2-yl)-4-methylbenzenesulfonamide) have been synthesized and characterized. In the four complexes each copper atom is five-coordinated. The structure of complexes 1, 2 and 3 consists of a dimeric unit with a C2 symmetry axis, where both coppers are bridged by two hydroxo anions. Magnetic measurements show that the dimer complexes are ferromagnetic according to the Cu–O–Cu angles. Cleavage experiments using pUC18 plasmid DNA in the presence of H₂O₂/ascorbic acid as an activating agent show that the title complexes are potent artificial chemical nucleases, the order of efficiency being 3 > 2 ∼ 1 > 4. Control cleavage experiments indicated that the dimer complexes are stronger artificial nucleases than the [Cu(phen)₂]²⁺ complex under the same experimental conditions, while the monomer 4 has a lower nuclease activity than the [Cu(phen)₂]²⁺ complex. The inhibition of the cleavage process in the presence of reactive oxygen intermediate scavengers suggests that the hydroxyl radical and the superoxide anion are reactive species for the breakage of the DNA strands.     

A three-point method for evaluations of AMBER force field parameters: an application to copper-based artificial nucleases

We present the theoretical evaluation of new AMBER force field parameters for 12 copper-based nucleases with bis(2-pyridylmethyl) amine, 2,2′-dipyridylamine, imidazole, N,N-bis(2-benzimidazolylmethyl) amine and their derivative ligands based on first-principles electronic structure calculations at the B3LYP level of theory. A three-point approach was developed to accurately and efficiently evaluate the force field parameters for the copper-based nucleases with the ligands. The protocol of RESP atomic charges has been used to calculate the atomic charge distributions of the studied copper-based nucleases. The evaluated force field parameters and RESP atomic charges have been successfully applied in the testing molecular mechanics calculations and molecular dynamics simulations on the nucleases and the nuclease–DNA complexes, respectively. It has been demonstrated that the developed force field parameters and atomic charges can consistently reproduce molecular geometries and conformations in the available X-ray crystal structures and can reasonably predict the interaction properties of the nucleases with DNA. The developed force field parameters in this work provide an extension of the AMBER force field for its application to computational modeling and simulations of the copper-based artificial nucleases associated with DNA.     

Synthesis,structure, DNA binding studies and nuclease activities of two luminescent neodymium complexes

Two neodymium(III) complexes, [Nd(Phen)(NO₃)₃(DMF)₂] (1) and [Nd(Phen)₂(NO₃)₃] (2) (phen = 1,10-phenanthroline; DMF = dimethylformamide), have been synthesized with a view to design artificial luminescent nucleases and nuclease mimics. The complexes were characterized by spectroscopic, powder, and single crystal XRD studies. The complexes, as expected, have luminescent properties. The DNA binding studies of both complexes have been carried out by spectroscopic studies e.g. electronic absorption (UV–Vis), fluorescence emission as well as viscosity measurements. The nuclease activity of the complexes has been established by gel electrophoresis using pUC19 circular plasmid DNA. The results of DNA binding as well as DNA cleavage activity and the model studies of interaction with pNPP indicate that both neodymium complexes demonstrate nuclease activity through phosphoester bond cleavage.     

席夫碱大环铜配合物的化学核酸酶活性研究

对3种结构相近的席夫碱四氮大环草酰胺铜配合物（CuL^1～3）的化学核酸酶活性进行比较研究。结果表明：这类配合物的化学核酸酶活性与中心金属离子的类型、配体的结构、溶液的pH值、离子强度及配合物的浓度等都有关系。3种配合物表现出来的化学核酸酶活性顺序为CuL³＞CuL²＞CuL¹。CuL³的DNA切割反应表现为典型的假一级连续反应。在80 μmol·L^-1 CuL³和2 mmol·L^-1 H₂O₂的存在下,就超螺旋DNA向切口开环型DNA进而向线型DNA的转化而言,反应速率常数分别为0.044 0±0.001 5 min^-1(k₁)和0.003 52±0.000 18 min^-1(k₂)。     

Preorganized bis-zinc phosphodiester cleavage catalysts possessing natural ligands: a lesson pertinent to bimetallic artificial enzymes

Two preorganized bis-zinc receptors (2 and 3) were synthesized wherein the metals were ligated with ligands present in natural phosphodiesterases: imidazoles and carboxylates. The intrametallic distance is near 4.5 A, that found in natural nucleases and other successful artificial nucleases. With only two imidazoles (2), the zinc binding affinities were not high enough to achieve cooperativity. Yet, with a third ligand, a carboxylate (3), cooperativity was found in the cleavage of HPNPP. The preorganization of 3 was achieved using a "steric gearing" strategy. The enhancement was 80-fold for cooperation between the two metals relative to a mono-metallic analogue (5). However, there was no observable enhancement in the hydrolysis of RNA using 3 relative to 5. Therefore, we conclude that placing two zinc atoms that are ligated with natural ligands at the appropriate distance for catalysis is not sufficient to enhance the cleavage of RNA, but is successful for activated RNA substrate mimics.