Monitoring of Basic Biochemical Processes at Single Mole

大分子生物化学和分子生物学的知识主要基于体外技术，如超离心、凝胶电泳、足迹法等，所有这些都基于大分子集合的行为。在大多数情况下，所获得的信息在很大程度上是通过分析许多分子的平均响应来推断的。例如，凝胶阻滞测定能够从凝胶中条带强度的定量确定平衡结合常数。然而，不能直接观察到解离或缔合事件。由于这种技术不能使人们在真实的时间内和以高的时间分辨率跟踪分子过程，因此失去了诸如凝胶笼化的效果以及最重要的关于动力学的信息。迄今为止，强纳米尺寸的荧光团，称为量子点（Qdots），仅用于一次一个地监测单个种类的复合物，或可视化活细胞中的过程。最近发展的低噪声探测器的高量子效率，如雪崩光电二极管，增强相机和计算机编程的显微镜提供了潜在的可视化和计算分析的分子过程在单分子和“小群体的分子”的水平。迄今为止，这些进展的应用几乎没有得到开发。我们选择了通过将Qdot附着到DNA、RNA和蛋白质上而不干扰其生物活性来监测单个大分子事件的先驱。该方法提供了体内转录复合物中复杂相互作用的分析（ChIP分析）与通过传统批量生物化学获得的结果之间的缺失环节。该系统也可以适用于蛋白质组学和药物发现。

Knowledge in biochemistry and molecular biology of macromolecules is based mostly on in vitro techniques such as ultracentrifugation, gel electrophoresis, footprinting, and others, all based on the behavior of large ensembles of molecules. In most of these cases, the information obtained is inferred to a large extent by analyzing the average response from many molecules. For example, gel retardation assay enables the determination of equilibrium binding constants from quantification of the intensity of bands in a gel. Dissociation or association events are, however, not observed directly. Effects such as gel caging and, most importantly, information about the kinetics is lost since such techniques do not enable one to follow molecular processes in real time and with high temporal resolution. To date, strong nano-size fluorophores, known as Quantum Dots (Qdots), have been only used either for monitoring individual species of complexes one at a time, or visualizing processes in live cells. Recent developments of low noise detectors of high quantum efficiency such as avalanche photodiodes, intensified cameras and computer programmed microscopes offer the potential of visualization and computational analyses of molecular processes at single molecule and "small population of molecules" levels. To date, the application of these advances has barely been tapped. We have chosen to pioneer the monitoring of single macromolecular events by the attachment of Qdots to DNA, RNA and proteins without interfering with their biological activity. This method provides a missing link between analysis of complex interactions in transcription complexes in vivo (ChIP analysis) and results obtained by traditional bulk biochemistry. This system can also be adapted for proteomics and drug discovery.