RecA蛋白介导同源重组的步进式链交换

同源重组过程由重组酶介导,对维持细胞的遗传稳定性有极大的作用.链交换是同源重组的关键过程,研究链交换发生的基本步长对理解整个反应机制有着重要的作用. Rec A作为原核生物重组酶的重要成员,近年来持续受到广泛关注,但Rec A介导的同源重组链交换的步长目前还有争议.现在主流的观点认为链交换步长为3 bp,而我们的前期工作测得步长的最可几值为9 bp.为了进一步验证我们的结论,进而为更深层次的机理研究提供基础,本文采用酶切保护实验和单分子磁镊,配合使用不同的错配碱基序列从侧面验证了链交换步长不为3 bp,而更倾向于9 bp,并分析了一个步长内的错配碱基数目和分布对链交换进程的影响.该结论为进一步探索重组酶工作的分子机理提供了基础和新的思路.     

用荧光显微示踪方法研究RecA在DNA同源识别过程中的工作机理

RecA是原核生物体内参与DNA同源识别过程的一种关键蛋白,长期以来一直是同源重组相关课题的重要研宄对象.通过荧光显微示踪方法,发现在同源识别过程中RecA与单链DNA形成的核蛋白丝与模板DNA的结合是短时(τ=0.2 s)和短程(l=1.05μm)的,结合后搜寻模板DNA上的同源位点的过程可分为布朗运动和定向运动两种模式.结合时核蛋白丝并不是缠绕在模板DNA上,而是以一种更弱的方式结合在模板DNA外侧进行位点搜寻.如果在该过程中没有找到同源位点,核蛋白丝就会脱离模板DNA,并寻找下一次与模板DNA结合的机会,重复以上过程.     

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     

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.     

阿霉素与DNA作用的共振光散射研究及在DNA分析中的应用

研究了抗癌药物阿霉素与DNA相互作用的吸收光谱、荧光光谱和共振光散射光谱,发现阿霉素与DNA相互作用产生强烈增强的共振光散射信号,共振光散射技术在研究DNA与阿霉素的相互作用时,其灵敏度远远高于吸收光谱和荧光光谱。DNA与阿霉素作用在322与564 nm处产生两个共振散射峰,在弱酸性条件下(pH 5.72),DNA的浓度在0～8.0 μg·mL-1范围内与散射强度呈良好的线性关系,对小牛胸腺DNA和鱼精子DNA的检出限分别为36.8和40.1 ng·mL-1。由此建立了一种选择性好,灵敏度高的DNA共振光散射分析方法。     

一种互补型自适应滤波最小均方法在SQUID测量的心磁信号中的应用

提出了一种互补型自适应滤波最小均方算法在SQUID测量的心磁信号中的应用，快的收敛速度和很好的跟踪能力通过根据最小均方原则在两个滤波器之间来回转换加权参量来实现     

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.     

An atomic force microscopy study of DNA hairpin probes monolabelled with gold nanoparticle: Grafting and hybridization on oxide thin films

First and original results are reported regarding the surface evolution of two kinds of oxide film after covalent grafting and hybridization of hairpin oligonucleotide probes. These hairpin probes were monolabelled with a 1.4 nm gold nanoparticle. One kind of oxide film was rough Sb doped SnO₂ oxide film and the other kind was smooth SiO₂ film. Same process of covalent grafting, involving a silanization step, was performed on both oxide surfaces. Atomic force microscopy (AFM) was used to study the evolution of each oxide surface after different steps of the process: functionalization, probe grafting and hybridization. In the case of rough SnO₂ films, a slight decrease of the roughness was observed after each step whereas in the case of smooth SiO₂ films, a maximum of roughness was obtained after probe grafting. Step height measurements of grafted probes could be performed on SiO₂ leading to an apparent thickness of around 3.7 ± 1.0 nm. After hybridization, on the granular surface of SnO₂, by coupling AFM with SEM FEG analyses, dispersed and well-resolved groups of gold nanoparticles linked to DNA duplexes could be observed. Their density varied from 6.6 ± 0.3 × 10¹⁰ to 2.3 ± 0.3 × 10¹¹ dots cm⁻². On the contrary, on smooth SiO₂ surface, the DNA duplexes behave like a dense carpet of globular structures with a density of 2.9 ± 0.5 × 10¹¹ globular structures cm⁻².     

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.     

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.     

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.     

Single Molecule Fluorescence Imaging and Its Application to the Study of DNA Condensation

Single molecule fluorescence imaging incorporated with optical tweezers and a laminar flow cell has been used to monitor the kinetic process of DNA condensation induced by spermidine. It was found that at least two steps were involved in the condensation process of the hydrodynamically-stretched linear DNA; a lag period followed by a rapid collapse of DNA. The lag time increased with the flow speed and the collapse time remained short within the range of the flow speed studied. The effect of salt concentration on the condensation process was examined, and the results suggest that the longer lag time observed in the higher salt buffer probably results from the displacement of bound cations and rearrangement of spermidine on the DNA. The flow-speed dependence of the lag time suggests that a nucleation event at the free end of the DNA, i.e. formation of a loop, may play a vital role in the kinetic process of condensation.     

线电流与无限长磁介质圆柱系统的磁矢势的计算及讨论

用镜象电流法和分离变量法计算了线电流与无限长磁介质圆柱系统的磁矢势,指出线电流与无限大磁介质分界平面、线电流与超导体圆柱、均匀外磁场中有磁介质圆柱系统时的磁矢势都可以由线电流与磁介质圆柱系统的磁矢势的极限情形给出.     

Methylene blue as a DNA probe for a comparative study of Cd, Pb and Cr ions binding to calf thymus DNA

Methylene blue (MB) was developed as a sensitive DNA probe for a comparative study of Cd²⁺, Pb²⁺ and Cr³⁺ ions binding with calf thymus DNA (ctDNA). The fluorescence intensity of the MB-ctDNA system increased dramatically when heavy metal ions (Cd²⁺, Pb²⁺ and Cr³⁺ ions) were added, which indicated that some of the bound MB molecules were released from the ctDNA base pairs. To compare the binding affinity of these three different heavy metal ions with ctDNA, the relationships between the fluorescence intensity of the MB-ctDNA-M (Metal ions) system and the concentration ratio of [M]/[DNA(p)] were investigated. The results showed that the order of the binding affinity of heavy metal ions with ctDNA had the following sequence: Cr³⁺> Cd²⁺>Pb²⁺. This order was further proved by the effects of heavy metal ions on the number of MB bound to ctDNA, the measurements of binding constants of these heavy metal ions to ctDNA, and the effects of heavy metal ions on the absorption of the MB-ctDNA system. In addition, the interaction mechanisms of Cd²⁺, Pb²⁺ and Cr³⁺ ions with ctDNA were also discussed in detail. These results indicated that their interaction mechanisms are related to the concentration ratios of heavy metal ions to DNA.