抗体修饰DNA测序酶的开发及其应用

【目的】开发一种新型DNA测序酶,解决一代测序技术应对复杂DNA结构模板所面临的主要挑战,如测序信号中断或测序信号快速衰减。【方法】从NCBI数据库中挖掘到了Taq DNA聚合酶和单链结合蛋白SSB基因信息,利用遗传融合、定点突变及基因设计技术获得了一种新型的测序酶Sso-Sequenase。利用亲和层析和离子交换层析,获得了纯化的测序酶。利用抗体修饰技术改良了Sso-Sequenase的性能,并且利用STR技术对其热启动性能进行了表征。选取多组不同类型的复杂模板,采用一代技术对比分析了Sso-Sequenase测序酶试剂盒和传统BigDye测序试剂盒的测序表现。【结果】Sso-Sequenase在大肠杆菌中稳定表达,纯度达到95%以上,产量高达10.5 mg/L。当温度低于35℃时,Sso-Sequenase表现出热启动活性。在复杂模板的一代测序反应中,如重复序列、高GC和发夹结构等样本,Sso-Sequenase测序酶成功完成了所有样本的测序,序列的平均碱基质量QV大于20。相比而言,BigDye测序试剂盒在处理这些复杂样本时,多数样本测序信号出现了显著衰减或中断。【结论】开发了一种纯度好、产量高,兼具热启动活性的DNA测序酶Sso-Sequenase及测序试剂盒,显著提高了复杂DNA结构模板(重复序列、高GC和发夹结构)测序成功率。

吲哚羧酸类Dy(Ⅲ)配合物与DNA作用机制研究

以7-氮杂吲哚-3-羧酸(7AI_(3)CAH_(2))为配体构筑了一例新型稀土Dy(Ⅲ)配合物[Dy(7AI_(3)CAH)_(2)(NO_(3))(H_(2)O)_(2)],利用元素分析、红外光谱、热重分析等对其进行结构表征。黏度法和荧光光谱表明该配合物与小牛胸腺DNA(CT-DNA)间结合模式为嵌插结合,配合物与CT-DNA表现出良好结合作用。

Mechanism of cleaving DNA through hydrolysis of a novel complex of Mg containing dien ligand

A series of metal complexes were designed and synthesized and a novel binuclear magnesium complex has been selected, namely [Mg-2(dien)Cl(OH2)(2)]Cl-2 . H2O (dien=diethylene-triamine), which can cleave the plasmid pBR322 DNA effectively in close to physiological condition without adding any external materials. Through biological and chemical methods, especially the comparative experiments, we find the interaction between the complex and DNA belongs to hydrolytic mechanism.

Rapid, Catalytic Hydrolysis of DNA by Copper(Ⅱ) Complexes

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Synthesis, DNA-binding, and Photocleavage of 2,3-Di-2-pyridylquinoxaline-silver(Ⅰ) Complex

A new dinuclear complex, [Ag（L）（CH3CN）]2（ClO4）2·2H2O（L=2,3-di-2-pyridylquinoxaline）, was prepared and characterized by elemental analyses, IR spectroscopy, and single-crystal X-ray diffraction analyses. The interaction of the complex with calf thymus DNA（CT-DNA） was investigated by absorption, fluorescence spectroscopies, and viscosity measurement. The results suggested that the complex was bound to DNA via an intercalative mode. The intrinsic binding constant value Kb was found to be approximately 1.48×10^3 L·mol^-1. Moreover, the Ag（I） complex could cleave the plasmid pUC19 DNA from the supercoiled Form Ⅰto the nicked FormⅡ under irradiation at 365 nm.

Microwave Synthesis, Characterization and DNA-binding Properties of a New Cobalt(Ⅱ) Complex with 2,6-Bis(benzimidazol-2-yl)pyridine

A new cobalt(Ⅱ) complex with tridentate ligand 2, 6-bis (benzimidazol- 2-yl)pyridine has been synthesized by microwave irradiation method and characterized by elemental analysis, electrochemical and spectral methods. The binding of the complex with calf thymus DNA has also been investigated by absorption and fluorescence spectra.

DNA-binding Studies of Two Silver(I)-sulfonate Complexes Constructed by In Situ Reaction

Two silver（I）-sulfonate complexes,[Ag（NO3）（4,4＇-bipy）][Ag（HL）（4,4＇-bipy）]·2H2O（1） and[Ag（H2O）（4,4＇-bipy）][AgL（4,4＇-bipy）]·2H2O（2）,have been synthesized by in situ reaction（H2L = 2-formylbenzenesulfonic acid,4,4-bipy = 4,4-bipyridine）.1 displays two parallel 1D chains expending to a 3D supramolecule by n—n interactions and O-H…O hydrogen bonds,in which the[Ag（NO3）]or[Ag（HL）]units are joined by bridging 4,4＇-bipy molecules,respectively.The interactions between the complexes and DNA were studied by means of fluorescence spectra and surface-enhanced Raman scattering（SERS） spectra.The special configuration and intercalation effects between 1 and DNA are stronger than that between 2 and DNA.

Synthesis, Structure, and DNA Binding of a Platinum(Ⅱ) Complex Based on the 3,4,5-Trimethoxy-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline

A new platinum（Ⅱ） complex of [Pt（chda）（3,4,5-triopip）]Cl·2H2O（1） based on 3,4,5-trimethoxy-phenyl-1 H-imidazo[4,5-f][1,10]phenanthroline（3,4,5-triopip） has been synthesized by hydrothermal methods and its consequences are characterized by elemental analysis, IR, single-crystal X-ray diffraction, UV-Vis absorption and fluorescent spectrum. The UV-Vis absorption studies reveal that the molecule undergoes considerable interaction with the nucleic acid. In this mononuclear structure, the platinum adopts a four-coordinated square planar geometry, which may favor the intercalation between the neighboring bases of the G-quadruplex（G4） DNA.

Synthesis, structure, DNA binding and cleavage activity of a new copper(II) complex of bispyridylpyrrolide

A copper-bispyridylpyrrolide complex [Cu(PDPH)Cl](PDPH = 2,5-bis(2′-pyridyl)pyrrole) was synthesized and characterized. The complex crystallizes in the orthorhombic system with space group Pccn, a = 0.9016(3) nm, b = 1.0931(4) nm, c =2.5319(8) nm, and V = 2.4951(15) nm3. The copper center is situated in a square planar geometry. The interaction of the copper(II)complex with calf thymus DNA(CT-DNA) was investigated by electronic absorption, circular dichroism(CD) and fluorescence spectra. It is proposed that the complex binds to CT-DNA through groove binding mode. Nuclease activity of the complex was also studied by gel electrophoresis method. The complex can efficiently cleave supercoiled p BR322 DNA in the presence of ascorbate(H2A) via oxidative pathway. The preliminary mechanism of DNA cleavage by the complex with different inhibiting reagents indicates that the hydroxyl radicals were involved as the active species in the DNA cleavage process.

Interaction between Eu(bpy)_3^(3+) Complex and DNA by Fluorophotometry

The interaction of complex Eu(bpy)_3 (3+) with DNA was studied by fluorophotometry. The binding constant ( K ) and size ( n ) of the binding site were calculated according to the changes of the fluorescent intensity at the adding process of DNA. The model for the complex Eu(bpy)_3 (3+) interacted with DNA solution is found to be electrostatic action. The system Eu(bpy)_3 (3+)-DNA was studied by the voltammetry. The interaction of the complex Eu(bpy)_3 (3+) with the calf thymus DNA was investigated by fluorophotometry,then K and n were obtained.

Catalytic hydrolysis of phosphate diester (BNPP) and plasmid DNA by mononuclear macrocyclic polyamine metal complexes

The activities of the catalytic hydrolysis of phosphate diester （BNPP） [bis（p-nitrophenyl）phosphate diester] and plasmid DNA （pUC 18） by mononuclear macrocyclic polyamine metal complexes have been investigated in this paper. The results showed that the highest activity in hydrolysis of BNPP was obtained with le--Zn（II） complex （composed of lipophilic group） as catalyst. The hydrolysis rate enhancement is up to 3.64 × 10^4 fold. These metal complexes could effectively promote the cleavage of plasmid DNA （pUC18） at physiological conditions.

Synthesis and DNA cleavage activity of diiron(Ⅲ) complex bearing pyrene group

As DNA cleavage agent, a new diiron（Ⅲ） complex （Fe2Lb）, in which a DNA-intercalator （pyrene） is covalently linked to a diferric moiety, has been synthesized and characterized. The DNA-binding property and DNA cleavage activity of the complex have also been investigated.

Synthesis of a New Cobalt(Ⅱ)Complex and its Interaction with DNA

A new complex of bis(N-benzyl benzotriazole-N3) dichloro Co(II) (Co(II)L2Cl2) was synthesized and its crystal was characterized. The interaction of this new complex with fs-DNA was studied by electrochemical and UV spectrophotometric method. In 0.01 mol/L pH 4.2 Tris-HCl buffer solution Co(II)L2Cl2 has two reductive peaks with peak potentials at 0.38 V and 0.27 V, respectively, and an oxidative peak at 1.05 V. After the addition of DNA, the peak currents decrease and the peak potential at 0.27 V shifts negatively. The UV spectrophotometric experiment indicates that the maximum absorbance of Co(II)L2Cl2 at 204 nm decreases and exhibits bathochromic shift after the addition of DNA. All the results reveal that Co(II)L2Cl2 binds with DNA by intercalation as well as electrostatic interaction.

Synthesis,Characterization,and DNA Binding Properties of Dinuclear Copper(Ⅱ) Complex [Cu_2(TATP)_2(L-Leu)_2(ClO_4)_2]_2·2H_2O

A dinuclear copper（Ⅱ） complex[Cu2（TATP）2（L-Leu）2（CIO4）2]2·2H2Owas synthesized and characterized, where, TATP=1,4,8,9-tetraazatriphenylene, and L-Leu=L-leucinate. The complex was crystallized in the triclinic space group P1, with two independent molecules in a unit cell. Two Cu（Ⅱ） ions in each complex [Cu2（TATP）2（L-Leu）2（CIO4）2] molecule were found to be in different coordination geometries, i.e., Cu2 or Cu4 of a distorted square-pyramidal geometry coordinated with two nitrogens of TATP, the amino nitrogen and one carboxylate oxygen of L-Leu and one oxygen of perchlorate, and Cul or Cu3 with an octahedral geometry coordinated with the above stated similar coordinated atoms, and another carboxylate oxygen of L-Leu coordinating to Cu2 or Cu4. The complex can interact with CT-DNA by an intercalative mode and cleave pBR322 DNA in the presence of ascorbate.

Synthesis,Crystal Structure and DNA Interaction of a Chiral Platinum(Ⅱ)Complex with 2-(2,3-Dimethoxyphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline

A new platinum（Ⅱ） complex of [Pt（chda）（2,3-dmopip）]Cl·H2O（1, chda = cyclohexan-ediamine, 2,3-dmopipH = 2-（2,3-dimethoxyphenyl）-1H-imidazo[4,5-f][1,10]phenanthroline） has been synthesized by hydrothermal methods and characterized by elemental analysis, IR, single-crystal X-ray diffraction, UV-vis and fluorescent spectrum. In complex 1, the platinum adopts a four-coordinated square planar geometry. The DNA-binding interaction has been investigated. The complex can bind to DNA by an intercalative mode, presenting high DNA-binding affinity.

Synthesis and Crystal Structure of a Dinuclear Cu(Ⅱ) Complex Based on a Carboxyl-substituted 1H-1,2,3-Triazole and Its DNA Cleavage Activity

The novel dinuclear copper complex [Cu2（H20）2（DMF）2（L）2] （1, H2L = 5-phenyl- 2H-1,2,3-triazole-4-carboxylic acid, DMF = N,N-dimethyl-formamide） has been synthesized and characterized by X-ray single-crystal diffraction. The compound crystallizes in triclinic system, space group P1 with a = 9.591, b = 10.508, c = 15.515A,β = 75.11°, V= 1446.2 A3, Z= 2, Mr = 683.62, Dc = 1.570 g/cm3, μ = 1.531 mm^-1, F（000） = 700, the final R = 0.0404 and wR = 0.1130 for 5327 observed reflections with I 〉 2σ（I）. In each unit of the complex, two Cu2＋ ions coordinated with two triazole ligands to form a dimeric [5,6,5] tricyclic structure. The central Cu atom is five-coordinated, and each copper atom shows a square pyramidal geometry. The crystal structure is stabilized by the inversion-related O-H…O hydrogen bond and C-H…O hydrogen bonding interactions to form a layer structure. Fluorescent spectra show an obvious quenching of fluorescence compared with free 1,2,3-triazole ligand. The results of agarose gel electrophoresis indicate that this complex can cleave the plasmid supercoiled DNA within shorter time in the 50-folds excess of ascorbate under physiological conditions, providing a new example in the research for artificial metal nucleic acid enzyme.

Synthesis, Crystal Structure and DNA Binding Studies of a Nickel(II) Complex with 2-Aminomethylbenzimidazole and Demethylcantharate

A new mixed-ligand nickel（Ⅱ） complex, [Ni（L）（DCA）（H2O）]·2H2O （L = C8H9N3, 2-aminomethyl-benzimidazole, DCA2- = 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, demethylcantharate, C8H8O5）, has been synthesized and characterized. The structure of the complex was determined by single-crystal X-ray diffraction. It is of monoclinic system, space group P21/c with a = 7.7211（7）, b = 12.0799（12）, c = 19.7867（19）, β = 100.390（6）°, V = 1815.2（3） nm3, Dc = 1.625 g·cm-3, Ζ = 4, F（000） = 928, R = 0.0314 and wR = 0.0822. In addition, the interaction between the complex and DNA was studied by means of fluorescence spectra and viscosity. The results indicate that the complex binds to DNA by the mode of partial intercalation with the Stern-Volmer constants Ksv of 3.81 × 104 mol·L-1.

Efficient Cleavage of DNA by the Novel Copper (II) Complex Derived From 2,2'-Pyridil Ligand

Copper (II) complex derived from 2, 2'-pyridil ligand has been, demonstrated to cleave efficiently DNA at micromolar concentrations at room temperature and pH 7.25.

DNA Cleavage by Copper (Ⅱ) Complexes Without added Coreactants

The cleavage activity of complexes between copper (Ⅱ) and four different amino acidor amino acid methyl ester on DNA was tested at physioloical pH and temperature., It was found that Cu (Ⅱ)-L-His and Cu (Ⅱ)-L-His methyl ester complexes could cleane DNA. The extent ofDNA cleavage is largely dependent on the metal ion-to-ligand ratio. The cleavage of doublestranded DNA mediated by Cu (Ⅱ)-L-His complexes occurs via a hydrolytie mechanism.

A Novel Mononuclear Copper(Ⅱ) Complex:Crystal Structure and DNA Cleavage Ability

The reaction of N-tosylatirdine with 1,2-diaminopropane in dry benzene solution yields an intermediate H2L, N,N,N＇,N＇-tetrakis（2-（p-tolylsulfonyl）aminoethyl） propane-1,2-diamine. The mononuclear copper（Ⅱ） complex, [CuLH2O]·H2O, was synthesized by the reaction between the intermediate and copper（Ⅱ） in absolute methanol. The complex has been characterized by IR, UV-vis and X-ray diffraction technology, and its crystal crystallizes in the orthorhombic system, space group Pbca with a = 15.589（1）, b = 21.897（2）, c = 27.645（2） A, V = 9436.4（1）A^3, Dc = 1.352 g/cm^3, Z = 8, Mr = 960.68, F（000） = 4040, μ（MoKa） = 0.698 mm-1, S = 0.98, R = 0.0537 and wR = 0.1180 for 5804 observed reflections （I 〉 2σ（I））. In the crystal structure, a one-dimensional chain is formed by abundant hydrogen bond interactions. The interaction of the complex with DNA was monitored using agarose gel electrophoresis. The result shows that the complex can transform the supercoiled to nicked and liner forms, and has a concentration-dependent cleavage activity.