Monitoring of Basic Biochemical Processes at Single Mole

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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.