采用电活性标记物N-(2-乙基-二茂铁)马来酰亚胺检测与表面固定的 一致性双链DNA特异性作用的p53蛋白质

建立了电化学检测表面固定捕获的野生型p53蛋白质的方法. 首先在金电极表面形成巯基化的单链DNA探针/己硫醇(HT)混合自组装膜, 随后巯基化的单链DNA探针与溶液中序列匹配的靶点DNA杂交, 所形成的一致性双链DNA捕获溶液中的野生型p53蛋白质. p53分子表面的半胱氨酸残基采用巯基特异性试剂N-(2-乙基-二茂铁)马来酰亚胺(Fc-Mi)进行衍生. 通过检测二茂铁的电化学信号来指示p53与一致性双链DNA之间的特异性相互作用. p53蛋白质与双链DNA的键合程度取决于双链DNA的序列. 该方法可检测的p53最低浓度为1.33 nmol•L－1.     

DNA发夹结构自组装信号放大策略应用的研究进展

DNA发夹结构自组装因具有无酶参与、等温以及识别序列能力强等优点,在生物分子和金属离子检测方面展现了良好的发展前景。该文梳理了DNA发夹结构自组装信号放大策略的类型,综述了近年来该策略在致病菌、核酸肿瘤标记物、蛋白质、无机金属离子,以及生物小分子检测中应用的研究进展,并对其未来发展趋势进行了展望,旨在为基于DNA发夹结构自组装检测生物分子提供一定的参考。     

微流路中利用DNA选择性固定蛋白质

报道了一种可控的通过DNA复合物在微流路中杂交固定蛋白质的方法. 微流路系统中的玻璃基底上固定寡聚核苷酸, 其中的层流提供了不同的DNA-蛋白质复合物. DNA的特异性识别可以将蛋白通过表面寻址固定在基底上. 并且在体系中引入了全内反射荧光技术来追踪整个过程. 此方法的特异性和灵敏度均较高, 且蛋白质的固定和去除可重复. 实验结果显示, 同时检测特异性和非特异性的识别, 可以有效提高生物检测的准确性. 这项技术可以提高具有微流路结构的生物传感器装置的检测质量.     

手性钌(Ⅱ)配合物对DNA的立体选择性断裂

八面体钌(Ⅱ)多吡啶配合物与双螺旋DNA插入结合后具有较强的结合能力,并且含有一个具有氧化一还原活性的中心金属离子．它们对氧化剂相对比较稳定,但对光比较敏感,因此可利用光辐射使之产生单线态氧或羟基自由基等而使DNA裂解．此外,这些配合物具有左手∧-和右手△-两种构型,与同样具有手性的DNA作用时,存在着立体选择性结合．并且在对DNA的断裂反应中也存在一定的立体选择性,可作为不同构型DNA的结构探针．     

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.     

“发夹”结构DNA用于生物分析传感器研究进展

由于链内碱基互补配对作用形成的"发夹"结构DNA分子被广泛用于生物分子传感分析。双链或多链"发夹"结构DNA分子参与的杂交链式反应信号记录方式多样,主要有荧光法、比色法、电化学方法等。基于杂交链式反应的检测方法具有快速、方便、成本低、准确度高、灵敏度高、特异性强的优点,在分析传感研究中的应用尤其受到人们的关注,近些年发展迅速。本文综述了"发夹"结构DNA与杂交链式反应应用于生物传感分析的原理、信号记录方式及其在蛋白质、重金属离子、小分子、疾病标志物、DNA等检测中的研究进展。     

无标记增强型检测Hg2+的荧光DNA传感器

基于Hg2+与T–T错配碱基对能特异性结合形成T-Hg2+-T结构以及结晶紫(CV)与单、双链DNA作用后荧光信号的差异,构建了一种荧光增强型检测Hg2+的DNA生物传感器。实验考察了不同序列DNA及溶液的pH、DNA与CV浓度比、稳定时间等因素对灵敏度的影响。在优化的条件下体系荧光效率和Hg2+浓度在2～40 nmol/L范围内呈良好的线性关系,检出限为0.7 nmol/L。并且高浓度的Ca2+,Mg2+等常见金属离子对Hg2+的检测没有干扰。方法为重金属Hg2+的检测提供了一个较好的荧光分析方法。     

基于SPHD及FRET技术的蛋白质传感器

蛋白质传感器作为一种分析蛋白质与其他分子之间作用力的有效手段已经越来越显示出其优点, 但是蛋白质在基底表面的变性问题对于研究者来说仍然是一个挑战. 作者介绍了一种具有高灵敏度的蛋白质传感器, 即利用DNA引导固定(DDI)方法, 将DNA- 蛋白质复合物通过DNA双链特异性杂交固定在硅基底表面, 从而有效保持了蛋白质的活性, 并且引入荧光共振能量传递(FRET)技术以及电势扫描发卡去杂交(SPHD)技术降低非特异性吸附带来的荧光信号. 这种蛋白质传感器在蛋白质识别以及DNA杂交方面具有显著区分不同体系荧光信号的能力.     

DNA原位自组装用于细胞荧光成像分析研究

DNA自组装由于其低毒性、高生物相容性和内置特性,在生物传感、药物递送和临床治疗中发挥着越来越重要的作用.与大多数天然聚合物或合成纤维相比,这些DNA分子相对坚固,可通过序列变异进行修饰.作为侧链连接到聚合物上形成的二级结构赋予DNA结构可设计的响应特性,如对金属离子、蛋白质、pH、DNA、RNA和其他一些小信号分子(...     

基于G-四链体-血红素和链式放大反应的基因检测传感器的构建

该文以特殊设计的DNA序列为捕获探针,以G-四链体-血红素复合物作为信号分子,利用链式反应实现目标DNA的灵敏检测。在目标DNA存在时,捕获探针与目标DNA相互识别,同时目标DNA能与辅助探针发生连续的链式反应,从而在电极表面引入大量G-四链体结构。血红素存在下,G-四链体可与血红素结合形成具有很强电化学信号的G-四链体-血红素复合物。用差分脉冲伏安法(DPV)扫描得到的电化学信号与体系中的目标DNA浓度存在对应关系,从而实现对目标DNA的检测。在各组分浓度最适的情况下,电流响应值与目标DNA浓度在0.01～10 pmol/L内具有良好的线性关系,检出限可达8 fmol/L。该传感器灵敏度高、特异性好,具有良好的应用前景。     

Biosynthesis of a site-specific DNA cleaving protein

An E. coli catabolite activator protein (CAP) has been converted into a sequence-specific DNA cleaving protein by genetically introducing (2,2'-bipyridin-5-yl)alanine (Bpy-Ala) into the protein. The mutant CAP (CAP-K26Bpy-Ala) showed comparable binding affinity to CAP-WT for the consensus operator sequence. In the presence of Cu(II) and 3-mercaptopropionic acid, CAP-K26Bpy-Ala cleaves double-stranded DNA with high sequence specificity. This method should provide a useful tool for mapping the molecular details of protein-nucleic acid interactions.     

Tuning the Reactivity of a Substrate for SNAP-Tag Expands Its Application for Recognition-Driven DNA-Protein Conjugation

Recognition-driven modification has been emerging as a novel approach to modifying biomolecular targets of interest site-specifically and efficiently. To this end, protein modular adaptors (MAs) are the ideal reaction model for recognition-driven modification of DNA as they consist of both a sequence-specific DNA-binding domain (DBD) and a self-ligating protein-tag. Coupling DNA recognition by DBD and the chemoselective reaction of the protein tag could provide a highly efficient sequence-specific reaction. However, combining an MA consisting of a reactive protein-tag and its substrate, for example, SNAP-tag and benzyl guanine (BG), revealed rather nonselective reaction with DNA. Therefore new substrates of SNAP-tag have been designed to realize sequence-selective rapid crosslinking reactions of MAs with SNAP-tag. The reactions of substrates with SNAP-tag were verified by kinetic analyses to enable the sequence-selective crosslinking reaction of MA. The new substrate enables the distinctive orthogonality of SNAP-tag against CLIP-tag to achieve orthogonal DNA-protein crosslinking by six unique MAs.     

Bromoacetophenone-based photonucleases: photoinduced cleavage of DNA by 4'-bromoacetophenone-pyrrolecarboxamide conjugates

[formula: see text] 4'-Bromoacetophenone derivatives which upon excitation can generate monophenyl radicals capable of hydrogen atom abstraction were investigated as photoinducible DNA cleaving agents. Pyrrolecarboxamide-conjugated 4'-bromoacetophenones were synthesized, and their DNA cleaving activities and sequence selectivities were determined.     

Experimental and DFT studies on DNA binding and photocleavage of two cationic porphyrins. Effects of the introduction of a carboxyphenyl into pyridinium porphyrin

The DNA-binding affinities and DNA photocleavage abilities of cationic porphyrin, 5-(4-carboxyphenyl)-10,15,20-tris(4-methylpyridiniumyl)porphyrin (CTMPyP), and its reference compound meso-tetrakis(N-methyl-4-pyridiniumyl)porphyrin (H2TMPyP) have been investigated. The DNA-binding behaviors of the two compounds in NaH2PO4 buffer were compared systematically by using absorption, fluorescence and circular dichroism (CD) spectra, thermal denaturation as well as viscosity measurements. The experimental results show that CTMPyP binds to DNA in an outside binding mode, while H2TMPyP in an intercalative mode. Photocleavage experiments reveal that both two compounds employ 1O2-mediated mechanism in cleaving DNA and H2TMPyP can cleave DNA more efficiently than CTMPyP. Theoretical calculations were carried out with the density functional theory (DFT), and the calculated results indicate that the character and energies of some frontier orbitals of CTMPyP are quite different from those of H2TMPyP. These theoretical results can be used to explain their different DNA-binding modes and affinities to a certain extent.     

Structure–affinity relationships for the binding of actinomycin D to DNA

Molecular models of the complexes between actinomycin D and 14 different DNA hexamers were built based on the X-ray crystal structure of the actinomycin–d(GAAGCTTC)2 complex. The DNA sequences included the canonical GpC binding step flanked by different base pairs, nonclassical binding sites such as GpG and GpT, and sites containing 2,6-diamino- purine. A good correlation was found between the intermolecular interaction energies calculated for the refined complexes and the relative preferences of actinomycin binding to standard and modified DNA. A detailed energy decomposition into van der Waals and electrostatic components for the interactions between the DNA base pairs and either the chromophore or the peptidic part of the antibiotic was performed for each complex. The resulting energy matrix was then subjected to principal component analysis, which showed that actinomycin D discriminates among different DNA sequences by an interplay of hydrogen bonding and stacking interactions. The structure–affinity relationships for this important antitumor drug are thus rationalized and may be used to advantage in the design of novel sequence-specific DNA-binding agents.     

Double strand DNA cleavage with a binuclear iron complex

Covalently linking two single strand DNA cleaving agents resulted in a new biomimetic binuclear iron complex capable of effecting oxidative double strand DNA cleavage.     

新型有机小分子DNA切割试剂的合成

根据活性基团的协同催化原理,设计合成了有机小分子核酸切割剂1-(N-胍乙基)-4-(N-羟乙基)哌嗪盐酸盐(4),并通过核磁共振和液相色谱-质谱联用技术对其结构进行了表征.利用琼糖凝胶电泳研究了pH值对其切割pUC 19 DNA效率的影响,通过自由基猝灭实验研究其切割DNA的反应类型.运用密度泛函理论,利用Gaussian软件进行了理论计算,研究其裂解DNA的反应方式.研究结果表明,在pH=7.2时化合物4的裂解效率最高,且能通过非氧化还原反应以磷酯转移的方式裂解DNA的磷酸二酯键.