单分子操纵与单分子生物物理

文章介绍了近年来发展起来的一些单分子操纵实验技术如光镊、磁镊、微针、斯托克斯拖曳技术，以及应用这些技术拉伸、旋转、解链DNA分子，从而研究其力学性质所取得的研究进展．各种蛋白质如T7 DNA聚合酶、拓扑异构酶，SWI／SNF染色质重建复合体、RNA聚合酶与DNA的作用在生化过程中十分重要，因此，文章也介绍了这些蛋白质与DNA在单分子的水平上相互作用所取得的研究进展．     

基于片层光照明的新型单分子横向磁镊

磁镊是一种高精度的单分子技术,它用磁场对连有生物大分子的超顺磁球产生磁力,通过追踪磁球的位置来测量生物大分子的长度信息.磁镊包括横向磁镊和纵向磁镊.纵向磁镊空间精度高,但昂贵;横向磁镊简单便宜,但由于受其成像原理的限制,一般情况下只能连接较长的DNA等生物大分子,且其空间精度较差,进而限制了其应用范围.为了解决这个问题,本文改进了横向磁镊,用片层光照明的方法使光线主要被磁球散射,从而能够直接观察到吸附在样品槽侧壁上的磁球,这使得测量短连接的底物成为可能.对于实际应用的检测,首先测试了包含270 bp发卡结构的0.5μm双链DNA,用其中发卡结构的"折叠-去折叠"跳变过程证明了改进后的横向磁镊的确可以追踪短DNA等生物大分子.然后,进一步用16μm的λ-DNA检验了实验系统.最后,将新型横向磁镊与普通横向磁镊及纵向磁镊在小力和大力条件下拉伸不同长度DNA的噪声进行了比较,发现改进后的横向磁镊在空间精度上明显优于普通横向磁镊,与纵向磁镊相比也无明显差异.以上结果证明了改进后的横向磁镊的精度优势,并扩展了横向磁镊的应用范围.     

磁镊结合DNA发夹的方法在RecA蛋白介导的同源重组机制研究中的潜在应用

同源序列识别与链交换过程是同源重组领域的重要研究方向.RecA蛋白作为重组酶家族的重要成员而一直被广泛研究.利用smFRET以及传统磁镊、光镊等技术,人们对同源重组过程的分子机制有了较深入的了解,然而,这些技术无法同时兼顾大量程与高精度的需求.本文提出一种传统磁镊结合DNA发夹结构的研究方案,并以大肠杆菌中的RecA介导的同源重组过程为例来阐述该方法的优点.使用本实验方案,我们实时观察到以下过程:1)RecA介导的链交换平均速度与已有结果一致,但并非匀速,而是以台阶式的跳变进行;2)直接观察到RecA第二结合位点与被置换链的动态相互作用过程,测量到第二结合位点与被置换链之间的结合力为3.0 pN,与光镊结合磁镊测量出的结果相符;3)能够区分链交换的方向性并观察到按照不同方向进行链交换的反应细节.本文提供了一个可以兼顾精度和测量范围的实验方法,并以RecA蛋白为例设计实验验证了其可靠性.磁镊结合DNA发夹结构的方法具备用于研究RecA或其他同源重组蛋白工作机理的潜质.因此,本文的工作有望成为单分子生物学领域研究同源重组过程的一个重要方法.     

用单分子技术研究抗癌药物顺铂对DNA结构的影响

文章作者用磁镊与原子力显微镜研究了抗癌药物顺铂对单个DNA分子结构的影响.当顺铂浓度较低时,DNA链变得柔软,驻留长度从～52 nm显著缩短到～15 nm;当顺铂浓度较高时,DNA表现出凝聚现象.基于单分子拉伸和原子力显微镜(AFM)成像两方面的实验结果,文章作者提出一个顺铂导致的DNA变软(softening)-成环(looping)-缩短(shortening)-凝聚(condensing)模型(简写为SLSC模型)来解释观察到的DNA凝聚,并认为通过远程交联使DNA形成小环结构是铂类抗癌药物作用的重要特征.     

打开DNA双链的物理新方法——激光微流法

文章报道了一种打开DNA双链的物理方法——激光微流法.DNA双链分子被固定在石英片的表面,在一定强度的激光衰逝波和微流振荡共同作用下,DNA双链间的氢键被迅速打开.利用全内反射单分子荧光共振能量转移技术,可以观察到激光微流法打开单个DNA的动力学过程.通过对激光进行控制,可以准确地打开连接在石英表面特定位置的DNA双链.DNA分子打开的比例与激光功率的关系表明微流产生的机械能与激光光能在DNA双链打开过程中缺一不可.     

全内反射瞬逝场照明高精度磁镊及其在DNA解旋酶研究中的应用

传统磁镊的测量精度受限于磁球的布朗涨落, 当磁力小于约10 pN时, 磁球的布朗涨落明显增大, 对应磁镊的空间分辨率显著下降. 为了提高传统磁镊在小力条件下的测量精度, 本文将全内反射荧光技术引入到磁镊技术中, 并建立相适应的“磁球-手柄-荧光微球-待测生物分子”单分子连接系统, 在小力条件下(小于10 pN)获得纳米量级的测量精度. 应用改进的磁镊对DNA发卡的折叠-去折叠态的转变过程进行了研究, 依据DNA发卡的折叠-去折叠态转变的性质对全内反射场的穿透深度进行了校正, 并结合实验结果对改进后的磁镊的测量精度进行分析. 观察了Bloom解旋酶的解旋动力学过程, 获得初步实验结果, 证实了改进的磁镊在单分子研究中的实用性. 关键词： 磁镊 全内反射荧光 DNA发卡 解旋酶     

一种改进型单分子操纵装置及其应用

改进了单分子横向磁镊装置,可以直接用于观察单个DNA分子的伸长和扭转并随时更换样品池内的溶液,还可以同时操纵多个DNA分子,大大提高实验效率.此方法可以提供01到40pN范围的拉力,足以满足大部分单分子DNA实验的要求.用该装置实时观测到了DNA分子在拉力作用下的伸长以及在旋转磁场作用下的扭转,并成功地观察到了分子马达拉动DNA的动力学过程,从而验证了该装置的实用性. 关键词： 横向磁镊 单分子操纵 DNA拉伸 DNA扭转     

抗生物素蛋白与DNA相互作用的单分子研究

抗生物素蛋白(avidin)在生物单分子实验中被广泛用于DNA与修饰表面的连接,同时avidin也可作为一种DNA载体用于基因治疗中.本文利用原子力显微镜(AFM)、动态光散射(DLS)、单分子磁镊(MT)技术系统地研究了avidin与DNA之间的相互作用,以及avidin引起DNA凝聚的机理.首先通过AFM对avidin-DNA复合体形貌进行观察,发现不但有avidin导致DNA凝聚的环状形貌,同时也存在avidin自身聚集引起的DNA凝聚现象,通过定量分析,发现其凝聚尺寸越来越小,而当avidin浓度大于2 ng·μL~(-1)时,其凝聚尺寸又突然变大.DLS实验结果也显示了同样的规律,伴随着avidin浓度的升高,DNA的粒径大小从大约170 nm减小到125 nm左右,其电泳迁移率由-2.76(10~(-4)cm~2·V~(-1)·s~(-1))变化到-0.1(10~(-4)cm~2·V~(-1)·~(-1)).此外,通过MT技术的力谱曲线变化,发现avidin导致的DNA凝聚与其他多价离子相比,长度的变化曲线几乎呈线性变化,偶尔存在少而小的阶跃,这种变化趋势与组蛋白的变化曲线更相似.因此可以判断,avidin导致DNA凝聚是由avidin与DNA的静电吸引和avidin自身聚集两种相互作用引起的.     

谐振势阱中的布朗运动——磁镊实验与模拟

从磁镊实验和模拟角度研究了处于谐振势阱中的布朗运动. 利用实验和模拟的结果验证了理论.然后通过理论与实验的对照, 对磁镊实验中DNA分子的持久长度大小对小球位移分布的影响, 以及磁镊实验中的测力误差作了相关分析.分析指出:持久长度的变化对沿 DNA链方向上的布朗运动影响更大;小的外力作用下力的测量会出现较大误差.     

用全内反射瞬逝场照明磁镊研究Bloom解旋G-四联体

G-四联体(G-quadruplex,G4)是广泛存在于细胞基因组中的一种DNA结构,在DNA的代谢如复制、转录、同源重组等过程中起重要作用.G4解旋酶近年来受到广泛研究,其中Bloom(BLM)解旋酶的研究已经相当丰富,但仍有一些基本问题不清楚.我们应用全内反射瞬逝场照明磁镊对BLM解旋G4的动力学过程进行了深入研究,观察到了BLM解旋G4的分步过程.相对于单分子荧光共振能量转移技术而言,借助磁镊的长时间观测性能,我们在近饱和三磷酸腺苷(ATP)浓度的实验体系中观察到BLM长时间反复解开G4或者长时间维持G4于打开状态的两种作用方式.最后,使用相同的实验条件做了单分子荧光共振能量转移实验,确定了加载2-3 pN的外力对BLM解旋G4没有显著影响.     

Insights into the Discrepancy between Single Molecule Experiments

Sharp bending as one of the mechanical properties of double-stranded DNA(dsDNA) on the nanoscale is essential for biological functions and processes. Force sensors with optical readout have been designed to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA(ssDNA). Recent FRET singlemolecule experiments were carried out based on the same force sensor design, but provided totally contrary results. In the current work, Monte Carlo simulations were performed under three conditions to clarify the discrepancy between the two experiments. The criterion that the work done by the force exerted on dsDNA by ssDNA should be larger than the nearest-neighbor(NN) stacking interaction energy is used to identify the generation of the fork at the junction of dsDNA and ssDNA. When the contour length of dsDNA in the sensor is larger than its critical length, the fork begins to generate at the junction of dsDNA and ssDNA, even with a kink in dsDNA. The forces inferred from simulations under three conditions are consistent with the ones inferred from experiments, including extra large force and can be grouped into two different states, namely, fork states and kink states. The phase diagrams constructed in the phase space of the NN stacking interaction energy and excited energy indicate that the transition between the fork state and kink state is difficult to identify in the phase space with an ultra small or large number of forks, but it can be detected in the phase space with a medium number of forks and kinks.     

Equilibrium folding and unfolding dynamics to reveal detailed free energy landscape of src SH3 protein by magnetic tweezers

Src SH3 protein domain is a typical two-state protein which has been confirmed by research of denaturant-induced unfolding dynamics. Force spectroscopy experiments by optical tweezers and atomic force microscopy have measured the force-dependent unfolding rates with different kinds of pulling geometry. However, the equilibrium folding and unfolding dynamics at constant forces has not been reported. Here, using stable magnetic tweezers, we performed equilibrium folding and unfolding dynamic measurement and force-jump measurement of src SH3 domain with tethering points at its N-and C-termini. From the obtained force-dependent transition rates, a detailed two-state free energy landscape of src SH3 protein is constructed with quantitative information of folding free energy, transition state barrier height and position,which exemplifies the capability of magnetic tweezers to study protein folding and unfolding dynamics.     

Biophysical characterization of DNA binding from single molecule force measurements

Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.     

Ashkin-Teller Formalism for Elastic Response of DNA Molecule to External Force and Torque

We propose an Ashkin-Teller-like model for elastic response of DNA molecule to external force and torque. The base-stacking interaction is described in a simple and uniform way. We obtain the phase diagram of dsDNA, and in particular, the transition from 13 form to the S state induced by stretching and twisting. The elastic response of the ssDNA is presented also in a unified formalism. The close relation of dsDNA molecule structure with elastic response is shown clearly. The calculated folding angle of the dsDNA molecule is 59.2°.     

Forces of interaction between DNA-grafted colloids: an optical tweezer measurement

Optical tweezers are employed to measure the forces of interaction between single DNA-grafted colloids. Parameters to be varied are the length of the DNA, the grafting density, and the ion concentration of the surrounding medium. From the measured force-separation dependence an interaction length at a given force is deduced. It shows in the mushroom regime a scaling with the grafting density which levels off for brushes. For the latter the transition from an osmotic to a salted brush can be traced in detail by varying the ion concentration in accordance with mean field theories.