荧光光谱技术在生物大分子相互作用以及解旋酶酶促反应研究中的应用
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摘要
本研究选取当前生物学领域中广泛采用的荧光光潜检测技术(如:荧光交联相关光谱技术,FCCS:荧光偏振检测技术,Florescence anisotropy以及荧光强度衰退检测技术,Photoluminescence decay)并深入开展其在生物大分子相互作用机制中的应用研究,研究结果与结论如下:
     一.荧光交联相关光谱技术及其在RecQ解旋酶活性作用研究中的应用
     实验选用双荧光标记互补链小片断寡聚核苷酸链作为研究底物,利用荧光交联相关光谱(Fluorescence Cross-Correlation Spectroscope,FCCS)荧光检测技术实时监控大肠杆菌RecQ以及人类RecQ5β解旋酶蛋白的酶促反应活性。结果表明荧光交联相关光谱技术FCCS适用于实时监测RecQ解旋酶蛋白的酶促反应活性。且该检测体系具有较高的灵敏性与稳定性。
     1)应用FCCS技术监测E.Coli RecQ解旋酶蛋白的双链DNA解链活性
     本实验选取高度保守的大肠杆菌E.Coli RecQ解旋酶(Xu et al.,2003)作为研究对象,并采用FCCS技术作为检测手段,监测E.Coli RecQ蛋白的酶促反应活性。研究结果表明:在一级反应条件下,E.Coli RecQ蛋白对底物的作用方式具有温度依赖性;此外,DNA底物核苷酸链的长度也会影响蛋白对底物的协同作用程度,进而引发酶促反应活性的显著差异。在多极反应条件下,DNA底物链长度不会影响酶蛋白对底物协同作用效应,主要由于反应体系中底物浓度远远大于酶蛋白浓度,该结果再次印证了一级反应条件下蛋白酶促活性的差异是由于酶蛋白对底物协同作用效应差异而引起的。
     2)应用FCCS技术监测RecQ5β解旋酶蛋白的互补单链DNA链“退火”活性
     本研究采用FCCS技术作为检测手段,监测人类RecQ5β解旋酶蛋白的酶促反应活性。研究结果表明RecQ5β酶蛋白能够引发互补单链DNA链“退火”反应,且该反应活性与DNA底物链长度无关。通过研究RecQ5β酶蛋白在不同孵育温度下对DNA的底物亲和力,结果表明RecQ5β酶蛋白对底物DNA结合能力不受孵育温度的影响,进而印证了较高温度下较强的互补单链DNA链“退火”程度归因为RecQ5β酶蛋白自身具有的酶促反应活性。该研究还检测了反应体系中镁离子浓度对互补单链DNA链“退火”程度的影响,结果表明人类RecQ5β解旋酶蛋白的互补单链DNA链“退火”活性不具有镁离子依赖性。
     二.稳态荧光偏振检测技术在HIV病毒整合酶蛋白酶促反应活性研究中的应用
     本研究采用稳态荧光偏振检测仪Beacon 2000(PanVera,Madison,WI)来实时监控HIV病毒整合酶蛋白与底物DNA结合过程中稳态偏振荧光数值的变化。本试验研究了两类主要的针对HIV病毒复制以及重组HIV整合酶蛋白催化特性的遗传抑制通路(N155H以及G140S/Q148H)。该研究采用稳态荧光偏振检测技术,成功实时监测了野生型以及突变型HIV整合酶蛋白的DNA结合活性以厦3'端处理活性。结果表明N155H,G140S,Q148H以及G14QS/Q148H每种整合酶遗传突变体都会对整合酶蛋白活性产生抑制作用,然而抑制程度依据突变类型的不同存在不同程度的差异。其中Q148H突变在单独存在的情况下对抗病毒药物RAL具有最强的抗药性;G140S则表现出较弱的抗药性;然而G140S/Q148H双重突变体却表现出极强的抗药性,且此时病毒复制水平类似于野生型HIV病毒。但是研究表明Q148H突变不仅抑制了重组病毒整合酶的催化活性,同时该突变体也严重抑制了病毒的复制能力。尽管G140S未表现出强烈的抗药性,但该突变可以帮助Q148H突变体恢复病毒复制能力。这与体外试验研究结果一致;即G140S/Q148H双重突变体能够恢复至野生型病毒整合酶的活性,但Q148H单突变体不具备该能力。该结果很好的诠释了为何艾滋病患者在服用RAL抗病毒药物后体内会很快形成G140S/Q148H双重突变整合酶。本研究还表明较之N155H突变体,Q148H突变体具有更强的抗RAL抗药性,进而解释了为何艾滋病患者在服用RAL抗病毒药物后体内的N155代谢通路会很快转变为Q148H代谢通路。
     三.荧光强度衰减检测技术在纳米量子点荧光特性研究中的应用
     本试验采用荧光衰减检测技术以及常用的光谱技术来研究实验室合成的具有MPA处壳CdTe纳米量子点荧光特性。分析了量子点体积依赖性和温度依赖性相关的时间相关荧光衰减特征曲线,结果表明:最小体积纳米量子点(直径约为2纳米)的激发光谱在较长的波长处具有一个小峰值。CdTe量子点的荧光寿命峰值分布在0.1ns,1-3ns,10-20ns以及40-50 ns。此外,研究结果表明量子点体积越大,其荧光寿命衰减越慢。且荧光寿命的数值在单光子激发与双光子激发情况下无显著差异。温度梯度研究结果表明,在较高温度下,纳米量子点的荧光衰减寿命越慢。上述研究结果表明纳米量子点的荧光光谱特性相当复杂,且与量子点的体积大小密切相关。因而量子点在荧光共振能量转移研究技术中的应用需要多阶分析函数模型的辅助。纳米量子点表面电荷对其荧光衰减特性影响的研究仍有待发展。
     综上所述,本论文成功将多种现代荧光相关光谱检测技术用于酶动力学参数监测以及化学合成纳米量子点荧光特性的研究当中,并阐明了该检测手法较之传统微量元素检测技术的优越性以及该检测手法的生物适应性。这不仅在理论上完善了荧光光谱检测技术在生命科学领域中应用的可行性,同时该研究为酶动力学参数计算,核酸分析,化学扩散常数计算,活细胞分析,肿瘤早期诊断以及高通量药物筛选等研究提供了数据支持。
This thesis presents the applications of fluorescence detection approaches in understanding the fundamental principles of the light activation of biomolecules, bioassemblies, and their catalytic mechanisms. In this context, three frequently used fluorescent methods have been discussed.
     The first technique, the fluorescence cross-correlation spectroscopy, based on measurements in micro-volumes with weak molecular concentration, has been essentially applied to monitor the cross-correlation of the fluorescence fluctuations of the two complementary DNA strands. In particular, the helicase activity of E.Coli RecQ enzyme and the strand annealing activity of human RecQ5βhelicase have been monitored. Results proved that the FCCS approach is particularly well-suited for monitoring the RecQ helicase enzymatic activity.
     The second technique, the fluorescence steady-state anisotropy measurements, has been adopted to analyse impact of the two main Raltegravir resistance pathways (N155H and G140S/Q148H) on HIV viral replication and the catalytic properties of recombinant integrase (INs). Results demonstrated the Q148H mutation is responsible for predominant resistance to Raltegravir whereas the G140S mutation increases viral fitness in the context of double mutant G140S/Q148H.
     The third technique, the time-resolved photoluminescence decay measurement, has been conducted to characterise the fluorescent properties of MPA capped CdTe quantum dots (QDs). Results confirmed the advantages of QDs and their promising applications in fluorescent labelling.
     In conclusion, this thesis encompasses the fundamentals and various applications involving the integration of light, photonics and biology into biophotonics.
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