激光对DNA作用机理的AFM研究

激光作用质粒DNA和小牛胸腺DNA产生损伤效应,导致DNA结构变化,利用一种改进的试样制备过程和纳米显微镜－－原子力显微镜（AFM）能够获得可重现的激光作用质粒DNA和小牛胸腺DNA的AFM图像,显示它们的特殊的表达结构,讨论了激光辐照导致DNA链断裂的作用机理。     

大肠杆菌topA-菌株中质粒pBR322的超凝集结构

超凝集DNA是质粒DNA的一种特殊拓扑结构形式,最初在大肠杆菌SD108(topA gyrB225)细胞中被发现.现在大肠杆菌DM800(topA-gyrB225)细胞中也发现了这种结构,这说明超凝集DNA的形成与细胞内旋转酶活性降低有直接关系,而与拓扑异构酶Ⅰ的存在与否无关.体外实验的结果显示,具有很强的正超螺旋松弛活性的拓扑异构酶Ⅳ可以将超凝集DNA完全松弛,这也证明质粒DNA的超凝集结构与超螺旋结构在细胞内是可以互变的.使用原子力显微镜对分离到的pBR322DNA超凝集结构进行分析,并与普通超螺旋进行比较,结果表明,超凝集DNA分子的结构发生了巨大的变化,其分子长度比正常超螺旋分子缩短了约30%,宽度和高度则增加了60%,结构更接近于A型DNA.另外,原子力显微镜研究结果表明,氯喹的嵌入并非改变了超凝集DNA的超螺旋状态,而是使其打结并最终压缩成一团.     

DNA分子结构的多态性研究——Ⅲ阳离子型表面活性剂诱导λ-DNA由线圈型向球型的转变

应用圆二色光谱 (CD)、扫描电子显微镜 (SEM )和原子力显微镜 (AFM )方法 ,研究了噬菌体λ DNA在阳离子型表面活性剂胶束介质中结构形态的变化 .结果表明 :在λ DNA溶液体系中随着阳离子型表面活性剂十六烷基三甲基溴化铵(CTAB)浓度的增加 ,其CD谱表现出λ DNA由B型DNA向DNA缩拢的构象变化过程 .SEM和AFM同时观测到λ DNA在CTAB诱导下 ,其结构形态由线圈状向小球状 (球径 =1.2 5 μm)最终至球状 (球径 =5 .4μm)的凝聚过程 .     

原子力显微镜拉伸吸附在金膜表面的短单链DNA分子

对单根DNA分子的操纵和拉伸可以直接研究DNA的弹性等力学性质. 首先通过将金沉积到云母表面制备了表面粗糙度小于0.3 nm的金膜,然后一段硫代的单链DNA (100 bases) 吸附到金膜表面. 利用原子力显微镜观察不同浓度的DNA吸附在金膜上的表面形貌. 进一步用原子力显微镜的力曲线模式拉伸DNA分子,在50％的情况下DNA可以被针尖拉伸,观察到了由于针尖和DNA分子间作用力的不同导致的多种不同力曲线.     

大肠杆菌topA－菌株中质粒pBR322的超凝集结构

超凝集DNA是质粒DNA的一种特殊拓扑结构形式，最初在大肠杆菌SD108 (topA⁺ gyrB225)细胞中被发现. 现在大肠杆菌DM800(topA^－ gyrB225)细胞中也发现了这种结构，这说明超凝集DNA的形成与细胞内旋转酶活性降低有直接关系，而与拓扑异构酶Ⅰ的存在与否无关. 体外实验的结果显示，具有很强的正超螺旋松弛活性的拓扑异构酶Ⅳ可以将超凝集DNA完全松弛，这也证明质粒DNA的超凝集结构与超螺旋结构在细胞内是可以互变的. 使用原子力显微镜对分离到的pBR322 DNA超凝集结构进行分析，并与普通超螺旋进行比较，结果表明，超凝集DNA分子的结构发生了巨大的变化，其分子长度比正常超螺旋分子缩短了约30%，宽度和高度则增加了60%，结构更接近于A型DNA. 另外，原子力显微镜研究结果表明，氯喹的嵌入并非改变了超凝集DNA的超螺旋状态，而是使其打结并最终压缩成一团.     

纳米-TiO2对变异链球菌和白色假丝酵母菌抗菌机制的研究

目的 采用原子力显微镜对应用抗菌剂纳米Ag-Ti02作用后的口腔两种常见致病菌的分子形貌进行观测,为研究其抑菌机制提供有力的直观影像科学依据和可靠、直观的实验方法.方法 选择两种菌种:白色假丝酵母菌、变形链球菌,采用液体稀释法将纳米Ag-TiO2与两种菌相互作用,分别使用光学显微镜、原子力显微镜观察两种菌的细胞微观形态变化.结果 抗菌剂与两种菌作用后,细菌形态均有不同程度的改变,甚至是死亡.结论 原子力显微镜能直观地显示白色假丝酵母菌,变形链球菌的分子结构,通过本实验在研究纳米Ag-TiO2抗菌剂对白色假丝酵母菌,变形链球菌的抑菌机理形态学改变方面做了进一步的完善.     

miR-93通过靶定PTEN基因增加肝癌细胞对奥沙利铂的耐药性

微小RNA（miRNA）参与了肿瘤的耐药过程.本研究通过建立对奥沙利铂 (oxaliplatin,Oxa)耐药的肝癌细胞系BEL-7402/Oxa和Hep-3B/Oxa,利用miRNA芯片结合实时荧光定量PCR的方法,筛选得到数个参与肝癌细胞对奥沙利铂耐药的miRNA 分子,其中miR-93表达增加最为明显.MTT实验发现,增加肝癌细胞株中miR-93的表达可以增强其对奥沙利铂的耐药性.进一步结合生物信息学、荧光报告载体及免疫印迹实验,证实miR-93通过靶定抑癌基因PTEN增加肝癌细胞对奥沙利铂的耐药性.总之,肝癌耐药细胞系的建立及其miRNA差异表达谱的分析,以及miRNA分子对肝癌细胞发生奥沙利铂耐药的具体作用及其分子机制的研究,不仅有助于理解肝癌细胞发生耐药的分子机制,而且为探索克服肝癌对奥沙利铂耐药性的有效途径提供可靠依据.     

用原子力显微技术研究受体-配体间的相互作用

原子力显微镜(AFM)不仅能对纳米生物结构进行实时动态的形态和结构观察,而且还能以10^-12N(pN)的精度对溶液中生物分子表面的相互作用力进行直接测量,逐渐成为一种研究受体-配体间相互作用的良好工具。本简要综述用AFM研究受体-配体间作用力、受体-配体间相互作用的影响因素及对这些因素的处理方法。     

原子力显微镜及其在肿瘤研究中的应用

原子力显微术是一种利用原子、分子间的相互作用力来观察物体表面超微结构的新型实验技术.介绍了原子力显微镜作为一种显微探测和操纵工具的主要特点及其在肿瘤研究中的优势,评述了国内外有关原子力显微镜在肿瘤的诊断、治疗、抗肿瘤药物开发等研究中的应用情况,展望了原子力显微镜应用于肿瘤单细胞研究的前景.     

HeLa细胞内DNA的抗酶(Dnase Ⅰ)组分和AFM直接观察

从HeLa细胞提取的DNA经DNaseⅠ酶解后,发现有两种抗酶组分(B和C).凝胶电泳结果显示组分B的分子大小要超过20000bp,而组分C的分子大小相当于40～50bp.这两种组分在原子力显微镜下的形态都明显不同于标准的双链DNA,其中组分B与以前观察到的λ-DNA变异结构较为相似,组分C则未见报道,根据其分子的表观宽度和高度推测可能为四链结构.此外,荧光实验还表明这两种组分可与EB相互作用,使其荧光增强.     

Insight into the cooperative DNA binding of the O6-alkylguanine DNA alkyltransferase

The O⁶-alkylguanine DNA alkyltransferase (AGT) is a highly conserved protein responsible for direct repair of alkylated guanine and to a lesser degree thymine bases. While specific DNA lesion-bound complexes in crystal structures consist of monomeric AGT, several solution studies have suggested that cooperative DNA binding plays a role in the physiological activities of AGT. Cooperative AGT–DNA complexes have been described by theoretical models, which can be tested by atomic force microscopy (AFM). Direct access to structural features of AGT–DNA complexes at the single molecule level by AFM imaging revealed non-specifically bound, cooperative complexes with limited cluster length. Implications of cooperative binding in AGT–DNA interactions are discussed.     

The structural peculiarities of condensed DNA micro- and nanoparticles formed in PCR

Studies of DNA condensation have opened new perspectives in biotechnology and medicine. DNA condensation induced by polyamines or trivalent metal ions in vitro at room temperature has been investigated in detail. Our recent studies have demonstrated Mg²⁺-mediated formation of DNA condensates during the PCR. In this study, we report the unique morphology and fine structure of PCR-generated condensed DNA particles using electron and atomic force microscopy. The principal morphologies of studied DNA condensates are 3D particles of micrometer dimensions, oval microdisks of nanometer thickness, filaments, and compact nano-sized particles. SEM examinations have revealed a new structural type of spherical and elliptical 3D microparticles formed by numerous definitely oriented microdisks and their segments. AFM revealed a granular structure of the microdisk surface and the smallest nano-sized disks and thinnest nanofibrils – that appear to be the primary products of DNA condensation during the PCR. We suggest that the formation of DNA nanofibrils and nanodisks in PCR occurs due to Mg²⁺ – mediated intermolecular (lateral) and intramolecular condensation of ssDNA. Aggregation of elementary nanodisks in the course of thermal PCR cycles, occurring both by magnesium cations and via complementary interactions, give a rise to large nano-sized aggregates and more complex microparticles.     

Effect of Supporting Substrates on the Structure of DNA and DNA–Trivaline Complexes Studied by Atomic Force Microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA–TV complexes onto this surface. On mica, both purified DNA and DNA–TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA–TV displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

The Interaction of DNA with Bacteriophage φ29 Connector: A Study by AFM and TEM

The connector of bacteriophage φ29 is involved in DNA packaging during viral morphogenesis and we have studied itsin vitrobinding to DNA using either linear or circular DNA. The protein–DNA complexes have been analyzed by transmission electron microscopy (TEM) and by atomic force microscopy (AFM) of samples directly deposited on mica. TEM showed the presence of a specific binding due to the interaction of the protein with the free ends of the DNA. The study of these samples by AFM showed two major types of morphologies: The interaction of the connector with circular DNA revealed that the strands of DNA that enter and exit the protein complex form an angle with a mean value of 132°. Nevertheless, when the connector was incubated with linear DNA (and later circularized), there was an additional bend angle of about 168°. Further morphological analysis of the latter samples by AFM revealed a structure of the protein–DNA complex consistent with the DNA traversing the connector, probably through the inner channel. On the other hand, images from the samples obtained by incubation of the connector with circular DNA were consistent with an interaction of the DNA with the outer side of the connector.     

Effect of supporting substrates on the structure of DNA and DNA-trivaline complexes studied by atomic force microscopy

Linear DNA, circular DNA, and circular DNA complexes with trivaline (TV), a synthetic oligopeptide, were imaged by atomic force microscopy (AFM) using mica as a conventional supporting substrate and modified highly ordered pyrolytic graphite (HOPG) as an alternative substrate. A method of modifying the HOPG surface was developed that enabled the adsorption of DNA and DNA-TV complexes onto this surface. On mica, both purified DNA and DNA-TV complexes were shown to undergo significant structural distortions: DNA molecules decrease in height and DNA-TP displays substantial changes in the shape of its circular compact structures. Use of the HOPG support helps preserve the structural integrity of the complexes and increase the measured height of DNA molecules up to 2 nm. AFM with the HOPG support was shown to efficiently reveal the particular points of the complexes where, according to known models of their organization, a great number of bent DNA fibers meet. These results provide additional information on DNA organization in its complexes with TV and are also of methodological interest, since the use of the modified HOPG may widen the possibilities of AFM in studying DNA and its complexes with various ligands.     

The structure of intramolecular triplex DNA: atomic force microscopy study

We applied atomic force microscopy (AFM) for direct imaging of intramolecular triplexes (H-DNA) formed by mirror-repeated purine-pyrimidine repeats and stabilized by negative DNA supercoiling. H-DNA appears in atomic force microscopy images as a clear protrusion with a different thickness than DNA duplex. Consistent with the existing models, H-DNA formation results in a kink in the double helix path. The kink forms an acute angle so that the flanking DNA regions are brought in close proximity. The mobility of flanking DNA arms is limited compared with that for cruciforms and three-way junctions. Structural properties of H-DNA may be important for promoter-enhancer interactions and other DNA transactions.     

Atomic force microscope imaging of chromatin assembled in <Emphasis Type="Italic">Xenopus laevis</Emphasis> egg extract

Gaps persist in our understanding of chromatin lower- and higher-order structures. Xenopus egg extracts provide a way to study essential chromatin components which are difficult to manipulate in living cells, but nanoscale imaging of chromatin assembled in extracts poses a challenge. We describe a method for preparing chromatin assembled in extracts for atomic force microscopy (AFM) utilizing restriction enzyme digestion followed by transferring to a mica surface. Using this method, we find that buffer dilution of the chromatin assembly extract or incubation of chromatin in solutions of low ionic strength results in loosely compacted chromatin fibers that are prone to unraveling into naked DNA. We also describe a method for direct AFM imaging of chromatin which does not utilize restriction enzymes and reveals higher-order fibers of varying widths. Due to the capability of controlling chromatin assembly conditions, we believe these methods have broad potential for studying physiologically relevant chromatin structures.     

Toward correlating structure and mechanics of platelets

ABSTRACT

The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryo-electron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining high-resolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.     

Analysis of DNA and Zinc finger interactions using mechanical force spectroscopy

The unbinding force of Zif268-DNA complex has been studied by atomic force microscopy (AFM). DNA and Zif268 were covalently immobilized on the surfaces of an AFM tip and glass substrate, respectively. Confocal microscopy was used to confirm the successful immobilization of DNA. Because of the complexity of the protein-DNA interaction, parallel experiments were designed to discriminate specific interactions. For such experiments, a typical unbinding force of a single Zif268-DNA complex (approx 550 pN at 40 nN/s force loading rate) was evaluated.     

DNA binding and aggregation by carbon nanoparticles

Significant environmental and health risks due to the increasing applications of engineered nanoparticles in medical and industrial activities have been concerned by many communities. The interactions between nanomaterials and genomes have been poorly studied so far. This study examined interactions of DNA with carbon nanoparticles (CNP) using atomic force microscopy (AFM). We experimentally assessed how CNP affect DNA molecule and bacterial growth of Escherichia coli. We found that CNP were bound to the DNA molecules during the DNA replication in vivo. The results revealed that the interaction of DNA with CNP resulted in DNA molecule binding and aggregation both in vivo and in vitro in a dose-dependent manner, and consequently inhabiting the E. coli growth. While this was a preliminary study, our results showed that this nanoparticle may have a significant impact on genomic activities.