Monitoring of Basic Biochemical Processes at Single Mole

生物化学和大分子分子生物学的知识主要是基于体外技术，如超离心、凝胶电泳、足迹等，所有这些都是基于大分子集合的行为。在大多数情况下，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，凝胶阻滞测定可以通过定量测定凝胶中的条带强度来确定平衡结合常数。然而，分离或联想事件不能直接观察到。由于这些技术不能实时、高时间分辨率地跟踪分子过程，因此诸如凝胶笼化等效应以及最重要的动力学信息会丢失。迄今为止，强纳米尺寸的荧光团，被称为量子点（Qdots），只用于监测单个物种的复合体，或者在活细胞中可视化过程。最近发展的高量子效率的低噪声探测器，如雪崩光电二极管、强化照相机和计算机编程显微镜，提供了在单分子和“小分子群体”水平上对分子过程进行可视化和计算分析的潜力。迄今为止，这些进步的应用几乎没有被挖掘出来。我们选择在不干扰其生物活性的情况下，通过将Qdots附着在DNA、RNA和蛋白质上，率先监测单个大分子事件。该方法提供了体内转录复合物复杂相互作用分析（ChIP分析）与传统批量生物化学结果之间缺失的联系。该系统也可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.