Real time observation of the photocleavage of single DNA molecules

A method for real time observation of photo-cleavage of stratched λDNA at single molecular level by a fluorescent microscope coupled with CCD is developed.DNA molecules stained with YOYO-1 are stretched by the mo-lecular combing technique and fixed on a modified slide.Then the Process of Photocleavage and relaxation of DNA under radiation of blue light is observed.We speculate that the conformation change of stretched DNA and the effect of water are likely to facilitate the effect of YOYO photocleav-age DNA molecules.The photocleavage effect of YOYO for stretched DNA may be useful to study DNA elasticity,cancer research as well as the interaction between DNA and dyes.     

DNA分子在云母表面拉直技术的研究

对DNA分子的生物、物理和化学性质的很多研究,都要求对单个DNA分子的性质进行深入探索,实现DNA分子在基底表面上的拉直是对其深入研究的物理基础。文章在APS溶液处理过的云母片表面,通过不同的方法完成了对DNA分子的拉直操作,利用原予力显微镜对拉直后的DNA分子进行了表征,获得了DNA分子拉直后的形貌,并对每种拉直方法进行了比较和讨论。     

Optical mapping of a rice BAC clone using restriction endonuclease and imaging with fluorescent microscopy at single molecule level

A method of constructing restriction map by optical mapping and single molecule fluorescent microscopy is described. DNA molecules were aligned and adsorbed on a glass coverslip surface by a modified “molecular combing” technique, and then the surface-immobilized DNAs were cleaved in situ with a restriction endonuclease. Individual DNA molecules digested by the endonuclease EcoRⅠ were observable with fluorescent microscopy. Using optical mapping, a physical map of a rice bacterial artificial chromosome clone was constructed. This method will facilitate genomic mapping and tracing the dynamic process in real time at a single molecule level with fluorescence microscopy.     

Study of the interaction of DNA and histones by spin-stretching and droplet evaporation

Molecular combing is a powerful and simple method for aligning DNA molecules onto a surface. Using this technique combined with fluorescence microscopy, DNA-histone complexes are stretched on a hydrophobic polymethyl methacrylate (PMMA) surface and observed directly. We have developed a new method to stretch single DNA-histone complexes, termed spin-stretching. The results show that the histones markedly enhance DNA binding to the PMMA surface. DNA winds around the histones and therefore decreases in length. The number of histones that bind to each DNA molecule is found to correlate with the histone concentration. The combed DNA-histone complexes are found to depend on two factors: the binding force on the surface and the centrifugal force at its local position. Na+ ions should compete with histones for binding to DNA; however, the observed competitive binding effect of Na+ ions at low concentrations was negligible.     

Direct measurement of compression spring constant of single DNA molecule with AFM

The understanding of mechanical responses of individ-ual macromolecules under a small external load (usually F<0.5 nN) is of profound importance because the induced conformational transition can result in the change of bio-logical functions[1?3]. The advent of new instrumentations, especially the development of Pico-Newton force tech-nology, has made it possible to investigate the dynamic behavior of single biomolecule and manipulate it to make direct measurements of its elastic properties[4?…     

Study the effects of metallic ions on the combination of DNA and histones with molecular combing technique

The effects of monovalent (Na^ , K^ ) and divalent (Mg^2 , Ca^2 , Mn^2 ) ions on the interaction between DNA and histone are studied using the molecular combing technique. λ-DNA molecules and DNA-histone complexes incubated with metal cations (Na^ , K^ , Mg^2 , Ca^2 , Mn^2 ) are stretched on hydrophobic surfaces, and directly observed by fluorescence microscopy. The results indicate that when these cations are added into the DNA solution, the fluorescence intensities of the stained DNA are reduced differently. The monovalent cations (Na^ , K^ ) inhibit binding of histone to DNA. The divalent cations (Mg^2 , Ca^2 , Mn^2 ) enhance significantly the binding of histone to DNA and the binding of the DNA-histone complex to the hydrophobic surface. Mn^2 also induces condensation and aggregation of the DNA- histone complex.     

Single-molecule fluorescence imaging of membrane-bound proteins for studies of cell signal transduction

Fluorescence imaging of single molecules is becoming a powerful tool to examine biological processes at the molecular level.Using total internal reflection fluorescence microscopy (TIRFM),it has been possible to study the dynamic behavior of single molecules on living cell membranes.Herein,we briefly review the application of TIRFM-based single-molecule imaging in studies of membrane receptors involved in signal transduction.Furthermore,we discuss several examples of our own research on growth factor receptors,including TGF-β receptors,HER2,and EGFR,and speculate possible applications of this technique to investigate other cellular events occurring on or near the plasma-membrane.     

Video imaging of walking myosin V by high-speed atomic force microscopy

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative 'visual evidence' for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action.     

Detecting CD20-Rituximab interaction forces using AFM single-molecule force spectroscopy

The invention of atomic force microscopy(AFM) has provided new technology for measuring specific molecular interaction forces.Using AFM single-molecule force spectroscopy(SMFS) techniques,CD20-Rimximab rupture forces were measured on purified CD20 proteins,Raji cells,and lymphoma patient B cells.Rimximab molecules were linked onto AFM tips using AFM probe functionalization technology,and purified CD20 proteins were attached to mica using substrate functionalization technology.Raji cells(a lymphoma cell line) or lymphoma patient cells were immobilized on a glass substrate via electrostatic adsorption and chemical fixation.The topography of the purified CD20 proteins,Raji cells,and patient lymphoma cells was visualized using AFM imaging and the differences in the rupture forces were analyzed and measured.The results showed that the rupture forces between the CD20 proteins on Raji cells and Rituximab were markedly smaller than those for purified CD20 proteins and CD20 proteins on lymphoma patient B cells.These findings provide an effective experimental method for investigating the mechanisms underlying the variable efficacy of Rituximab.     

黄原胶分子形貌的原子力显微镜研究

通过溶胶凝胶的方法制备了黄原胶水溶液,并采用轻敲模式原子力显微镜(AFM)对不同浓度的黄原胶分子溶液的表面微观结构形态进行了观察.实验结果表明,轻敲模式下的AFM能够观测到黄原胶分子的微观结构,浓度为0.1g/L时分子类似于球状体的颗粒,以多聚体形式存在.浓度达到0.01g/L时能够形成稳定的网状结构,当浓度达到0.001g/L的情况下形成单个纤维分子.利用AFM截面软件对网状结构和单个纤维进行分析,发现了云母表面出现的黄原胶分子分别为双链和单链.本文中提出了黄原胶分子从网状结构到单个纤维形成过程的假设模型,很好地解释了实验观察结果,对以后的实验研究有很大意义.     

原子力显微镜针尖与样品间的材料转移

研究氮化硅针尖在十八烷基三甲氧基硅烷 (OTE) /云母表面的修饰过程。使用原子力 /摩擦力显微镜 ,以云母作为参考样品 ,研究了针尖在样品表面的修饰效应和修饰后针尖的清洁过程 ,并考察了湿度和载荷对针尖修饰效应的影响。修饰过程不是一个渐进的过程 ,在最初几次摩擦扫描中修饰较快 ,然后在 10～ 2 0次扫描后达到平衡态。在 OTE/云母表面修饰后的针尖在云母表面的摩擦力信号比修饰前针尖在云母表面的摩擦力信号小 ,并且大部分吸附在针尖表面的 OTE分子在云母表面的前 10次扫描中就被磨掉。相对湿度对针尖的修饰效应影响不大。在研究不同样品的摩擦性能时 ,尽量使用清洁针尖 ,并使用摩擦性能稳定的参考样品 (如云母 )来检测针尖的表面状态     

Mechanism and development of dip-pen nanolithography （DPN）

Dip-pen nanolithography is a new scanning probe lithography （SPL） technique based on atomic force microscopy （AFM） , and now has made a great progress. The process of dip-pen lithography involves the adsorption of ink molecules on AFM tip, the formation of water meniscus, the transport of ink molecules, and diffusion of ink molecules on the substrate. More factors such as temperature, humidity, tip, scanning speed and so on will influence the process of dip-pen lithography. The paper analyzes in detail the mechanism of this technique, introduces synthetically the latest development, including electrochemical DPN, more-mode DPN, multiple DPN, multi-probe array DPN and so on. Finally, the paper describes the characteristics and the application of DPN.     

Combined-dynamic mode “dip-pen” nanolithography and physically nanopatterning along single DNA molecules

Atomic force micriscope (AFM)-based dip-pen nanolithography (DPN) is an emerging approach for constructing nanostructures on material surfaces such as gold, silicon and silicon oxide. Although DPN is a powerful technique, it has not shown its ability of direct-writing and patterning of nanostructures on surfaces of soft materials, for example biomacromolecules. Direct depositing on soft surfaces becomes possible with the introduction of a combined-dynamic mode DPN rather than mostly used contact mode DPN or tapping mode DPN. In this report, the combined dynamic mode DPN is used for direct depositing protein ink on DNA molecules at the nanometer scale.     

DNA与CdS-COOH-EcoRI复合物相互作用的研究

采用荧光光谱、原子力显微镜、琼脂糖凝胶电泳等技术探究CdS-COOH-EcoRI复合物与DNA的相互作用.研究发现,小粒径的CdS-COOH-EcoRI复合物与DNA除了有特异性结合,还发生非特异性结合.DNA相对浓度比较大以及恒温孵育时间较短时,以特异性结合为主;而随着DNA相对浓度的减小以及恒温孵育时间的延长,会伴有非特异性结合.Ca2+存在时,可以通过延长孵育时间“激活”酶切反应,使得DNA被剪切.因此可以通过改变DNA相对浓度和孵育时间来调控EcoRI对DNA的剪切.     

Micro-friction of diazoresin/polyacrylic acid self-assembly films in water with atomic force microscopy

Ultrathin films composed of diazoresin（DR）and polyacrylic acid（PAA）were fabricated.The surface morphology of the films in water was measured using an atomic force microscopy（AFM）.The self-assembly technique makes the surface rather flat and uniform.The friction force and its dependence on the velocity differ from the surface charge of the thin films.The friction force of repulsive DR/PAA film increases linearly with velocity and has lower values than that of attractive DR film over the full range of velocity.As the velocity increases,the attractive friction of DR film first decreases to a minimum at a velocity of 2 line/s and then increases all the way.When the surface is repulsive to the friction substrate,the friction of thin films that is determined by hydrated lubrication of polymer chains that is ultralubricated;when it is adhesive to the friction substrate,the friction is mainly contributed from the elastic deformation of adsorbed polymer chains in the low velocity region and from viscous sliding in the presence of hydrated-layer lubrication of the polymer chains in the higher velocity region.     

原子力显微镜与磷脂双层膜作用实验结果理论分析

 利用原子力显微镜研究生物膜力学性质,已经成为研究生物膜稳定性的重要技术。通常,原子力显微镜探针和磷脂双层膜相互作用力谱曲线包括非线性部分和线性部分,其中线性部分的物理机制仍不完全清楚。将磷脂双层膜看作弹性半空间,基于相互作用原理,系统地研究了原子力显微镜探针与磷脂双层膜相互作用过程,理论结果包含了能够表征磷脂双层膜稳定性的重要参数：杨氏模量和泊松系数,以及原子力显微镜探针尖端半径。不仅合理地揭示了H. J. Bütt的实验结果曲线中线性部分的物理机制,还指出,通过增加离子浓度,磷脂双层膜的杨氏模量增大和泊松系数减小,从而导致原子力显微镜探针与磷脂双层膜之间的相互作用力随着离子浓度增加而增加。     

不同体积分数二甲基亚砜对DNA变性的影响

利用原子力显微镜和动态光散射研究不同体积分数二甲基亚砜(DMSO)对质粒及线性DNA变性的影响。结果表明,体积分数1%的DMSO也能导致DNA发生局部变性,这个结果可以用原子力显微镜直接观测。同时,由于质粒DNA连接数的守恒以及DNA单链的产生,通过原子力显微镜和动态光散射发现,DNA随DMSO体积分数的增加逐渐形成超螺旋结构,DNA聚拢程度增加;DMSO体积分数从0增加到10%时,pBR322 DNA、5 000 bp DNA及λ-DNA的平均粒径分别从(474±10) nm、(554±11) nm和(871±17) nm降低到(257±8) nm、(282±18) nm和(449±21) nm。