应用分子梳技术对DNA与组蛋白相互作用的研究

利用分子梳技术对λ DNA和组蛋白的相互作用进行了研究. 通过这种简单有效的方法,我们将λ DNA分子拉伸到26—28 μm,相当于其原长（约162 μm）的16—17倍. 当组蛋白与DNA结合后,DNA分子发生凝聚现象,复合体的拉伸长度明显变短,其峰值分布在10—14 μm之间. DNA 组蛋白复合体的拉伸长度与组蛋白的浓度、与碱基对和荧光染料的比例有显著的关系.关键词：分子梳组蛋白DNA荧光显微     

拉直的单个DNA分子的全内反射荧光实时成像研究

全内反射荧光(TIRF)成像技术利用穿透深度仅200 nm左右的隐失波来激发诱导荧光,探测灵敏度和图像信噪比大大提高,成为单分子研究的有力工具。分子梳技术利用DNA末端与固体表面的结合力和周围流体流动产生的侧向力将DNA分子拉伸并平铺在表面上。结合这两种技术,对分子梳拉直的单个DNA分子进行了清晰的实时荧光成像,发现TIRF成像条件下DNA分子与荧光探针YOYO-1组成的复合体可自然避免发生光敏断裂现象;实时监测了单个DNA-YOYO-1复合体的光漂白过程,通过对激发光照射时间与探测器曝光时间进行同步控制,可大幅降低光漂白程度,为拉直的单个DNA分子的长时间实时观察和成像研究优化了实验条件,为实时、可视化地研究其与蛋白质相互作用的动力学过程奠定了基础。     

Poly－3BCMU薄膜光漂白的实验研究

报道了Ｐｏｌｙ－３ＢＣＭＵ薄膜光漂白过程的实验研究结果。测量了薄膜光漂白前后的可见及紫外光谱的变化，表明在光漂白过程中聚合物的长链被逐渐打断。实验表明，被漂白的聚合物薄膜可以被某些有机溶剂溶解，导致所谓的显影过程。实验研究了氧对光漂白过程的影响，并发现在Ｐｏｌｙ－３ＢＣＭＵ薄膜表面涂复光刻胶薄层可阻止氧分子向薄膜内的扩散，从而大大减缓光漂白过程。本文同时对光漂白在制备光波导元件中的应用进行了研究。     

三氯六氨络合钴导致DNA凝聚的分子梳研究

利用分子梳技术和动态光散射研究了三氯六氨络合钴和DNA分子之间的相互作用. 从荧光显微镜中可直接观察到经YOYO-1染色后?-DNA分子的平均伸直长度比未染色的DNA分子增长约30%. 这是由于荧光染料YOYO-1分子插入双链DNA的碱基对中从而使DNA分子的长度增加. 另外研究了染色后的DNA-[Co(NH₃)₆]³⁺混合物的伸直长度随三氯六氨络合钴浓度的变化关系,实验结果表明当钴离子浓度从0增加到3 μmol/L,其混合物的伸直长度由原来的20.9 μm减小到5.9 μm. 利用动态光散射技术证实了这个结果,发现在相同的实验条件下加入钴离子前后DNA-Co(NH₃)₆]³⁺混合物的流体力学半径从203.8 nm减小到39.26 nm.     

聚合物材料PMMA/DR1光漂白过程中折射率的变化

用双光束扫描法研究了在488nm的Ar^ 激光照射下,聚合物材料RMMA／DR1的光漂白过程,为精确测量光漂白模型中的参数提供了一种简单实验方法。这对于今后用光漂白的方法制备光波导器件具有重要意义。     

激光单分子探测的实验研究

单分子探测技术是一项超高灵敏度的探测技术,在生物、医学和环境等领域有着广泛的应用。在这里,本文简要介绍了基于激光诱发荧光方法的单分子探测谱仪的原理和装置,以及用ＣＣ５染料分子在该探测谱仪上进行的若干实验研究结果,包括样品流速的影响、能量特性、浓度线性、检测限等方面,并讨论了光漂白现象对实验结果的影响。     

DNA分子力学性质的测量

利用单分子实验技术测量了DNA分子的力学性质.通过生化手段将单根DNA分子连在顺磁小球与玻璃侧壁之间,利用磁铁对顺磁球施加力进而拉伸DNA.图像分析得到小球运动的轨迹以及DNA的长度,利用均分定理可以求得施加的外力.测量发现在外力作用下DNA分子的力学反应与高分子物理学的蠕虫状链模型符合良好.     

厦门湾有色溶解有机物光漂白的三维荧光光谱研究

利用荧光激发-发射矩阵光谱(excitation-emission matrix spectroscopy,EEMs)研究了厦门湾九龙江河口中、低盐度区表层水样中有色溶解有机物在秋季天然太阳辐照下的光化学漂白。结果表明,光漂白未引起水样中类腐殖质(C, A, M)和类蛋白质(T,B)荧光峰位置的移动。5个峰的荧光强度均随辐照时间的增加而减少,其中低盐度水样的降低幅度更大,并以指示陆源河流输入的特征荧光峰C的光漂白程度最大。根据漂白速率可将各荧光团区分出易漂白和难漂白两类组分。光漂白导致T、C峰及A、C峰之间的荧光强度比值增加,说明光漂白可引起海水中溶解有机物性质的明显改变,并且也是近海类蛋白质荧光相对类腐殖质荧光占优势的一个重要控制因素。研究结果对于探究陆源有机物在近海的转化及去除过程以及海洋光学有一定的参考价值。     

DNA分子链的电子局域性质及电导的研究

将一维随机二元固体模型应用于DNA分子链,利用传输矩阵方法来研究系统电子态的局域性质并进而讨论系统的导电性质.对一个链长为50000个碱基对的DNA序列,数值分析了局域长度和电导随碱基对的摩尔百分数、本征能量和无序度的变化关系.结果表明,系统的局域长度和电导强烈地依赖于能量,在能带中心部分局域长度大于边沿部分.无序度也在一定程度上影响着局域长度,双方成反向变化的关系.对有限长度的DNA分子链,局域长度体现出明显的对碱基对摩尔百分数的依赖关系,对正常成分比例的随机DNA序列,在所有能量范围内系统的态都是局域的,系统的电导很小,系统呈现绝缘体行为.仅当一种碱基对在序列中所占比例很小时,系统中可以发现与特定分立能量值相对应的“扩展态”存在,处在这些态下的系统有较大的电导,但这些扩展态是不稳定的,在热力学极限之下会消失.关键词：DNA分子链电子局域局域长度电导     

Fluorescence Recovery Under Decaying Photobleaching Irradiation: Concept and Experiment

A novel modification of photobleaching method for measurement of lateral diffusion is developed. In this approach fluorescence recovery kinetics is measured under decaying photobleaching irradiation, termed as fluorescence recovery under decaying photobleaching (FRDP). The time evolution of fluorescence intensity normalized to input irradiation starts from the photobleaching kinetics and transforms into the kinetics of fluorescence recovery at a later stage resulting in appearance of minimum. The analytical solution for the kinetics of fluorescence for Gaussian lineshape of laser beam and hyperbolic decay of irradiation in the first order approximation on bleaching rate was obtained. The accuracy of the analytical function was evaluated with exact numerical solution computed with finite differentiates method. The FRDP method was successfully applied to fluorescein solution in the glycerol/water mixture (80%) under various experimental settings using home-made experimental set-up. The FRDP approach demonstrated 25–30 fold enhancement in signal intensity over classical fluorescence recovery after photobleaching (FRAP) method at 3–5 fold increase in total irradiation. Among other advantages of the FRDP is the opportunity to perform measurements on varying time scales under constant size of the bleaching spot, including “safe” long time measurements. The potential extra advantage of FRDP method for analysis of complex diffusion in the biological system is discussed.     

Reversibly switchable DNA nanocompartment on surfaces: experiments, applications, and theory

This paper summarizes our studies of DNA nano-compartement in recent years. Biological macromolecules have been used to fabricate many nanostructures, bio-devices, and biomimetics because of their physical and chemical properties. But dynamic nanostructure and bio-machinery that depend on collective behavior of biomolecules have not been demonstrated. Here, we report the design of DNA nanocompartment on surfaces that exhibit reversible changes in molecular mechanical properties. Such molecular nanocompartment is served to encage molecules, switched by the collective effect of Watson-Crick base-pairing interactions. This effect is used to investigate the dynamic process of nanocompartment switching and molecular thermosensing, as well as perform molecular recognition. Further, we found that ‘fuel’ strands with single-base variation cannot afford an efficient closing of nanocompartment, which allows highly sensitive label-free DNA array detection. Theoretical analysis and computer simulations confirm our experimental observations, which are discussed in this review paper. Our results suggest that DNA nanocompartment can be used as building blocks for complex biomaterials, because its core functions are independent of substrates and mediators.      

Poly-3BCMU 薄膜光漂白过程的理论模型

本文报道了使用两种不同漂白光源时,Poly-3BCMU薄膜的光漂白过程,并建立了描述漂白过程的理论模型.最后讨论了采用光漂白方法制备导波微光学元件的可能性.     

Heterogeneous photobleaching in confocal microscopy

Photobleaching was studied during recording of confocal scanning laser microscopy. Studies on fluorescent gels of FITC-labeled dextran were used to evaluate differential bleaching along thez-axis. Differential bleaching along the z-axis was observed and it was seen that this was related to the numerical aperture of the objective in use. This points to the conclusion that photon energy flux density is an important parameter in photobleaching. To check if photon energy flux density heterogeneity is affected by local variation in the refractive index of the sample, photobleaching rates were calculated for different fluorescent objects (sections of seeds, animal cells stained with nuclear stains, immunocytochemistry preparations) and a pronounced similarity was found between photobleaching rates and DIC images.     

沢吨染料的光化漂白动力学及全息特性研究

研究了以三乙醇胺作为引发剂,五种沢吨染料的光化漂白过程.实验结果表明五种染料的光化漂白速率大小顺序是藻红B(ErB)＞曙红Y(EY)＞孟加拉玫瑰红B(RsB)＞罗丹明B(RoB)＞荧光素(F).并研究了曝光强度及染料浓度对漂白过程的影响,以及这五种染料的全息记录性能.     

Photoexpansion and photobleaching effects in oxysulfide thin films of the GeS2+Ga2O3 system

Oxysulfide systems undergo structural transformations upon illumination with laser light of near bandgap energy, as well as chalcogenide materials (glasses and films). In this paper, photoinduced effects such as photoexpansion and photobleaching were observed in GeS₂+Ga₂O₃ (GGSO) films synthesized by electron beam evaporation. A surface expansion of the thin films and a shift to shorter wavelengths of the optical absorption edge were observed as a result of UV laser irradiation (wavelength of 351 nm) and they are dependent on laser power density, exposure time and film composition. These parameters were varied to evaluate and enhance the observed effects. In addition, the irradiated GGSO samples exhibited a decrease in refractive index, measured with a prism-coupling technique, which makes these films suitable candidates for applications as gratings and waveguides in integrated optics.     

肿瘤细胞中表达的GFP蛋白的荧光漂白特性的研究

GFP作为生物源性荧光探针具有其他荧光标记物所无法比拟的优势，目前已广泛应用于生物学研究的各个领域。利用常规转染方法将带有EGFP基因的质粒载体导入人肺腺癌肿瘤细胞(ASTC-a-1)，并得到GFP稳定表达的细胞株。研究中发现，肿瘤细胞中表达的GFP长时间暴露于强激发光中会发生非常强列的荧光漂白作用，并且这种漂白作用是不可恢复的。对不同强度的激发光(饱和光源、阻断片ND4／ND8／ND16)对GFP的漂白作用进行了研究，并对冷冻保存样品的光漂白作用进行了初步的探讨。