We propose to develop a novel general methodology for studying intra-molecular conformational changes occurring in biological macromolecules in a single cell. Our novel approach is based on the combination of Fluorescence Correlation Spectroscopy (FCS) with Fluorescence Resonance Energy Transfer (FRET), and will allow the study of a small number of molecules at the time in individual living cells. We will use a simple DNA hairpin as a """"""""proof of principle"""""""" system to validate our novel methodology. The completion of this research will open up new avenues of research for the investigation of other dynamic biochemical processes in the cell. Although current technologies allow the observation of a single fluorescent molecule at the time, the extremely low signals obtained in such measurements limit their application to the study of slow process in solution. Fluorescence Correlation Spectroscopy (FCS), has recently emerged as an alternative to study the dynamics of processes in a wide range of timescales (?s-s). In this technique, which has been successfully applied in solution, the fluctuations in fluorescence intensity are analyzed statistically to obtain dynamic information of the system. However, the applicability of FCS to reactions occurring in biological samples is limited by the fact that the fluctuations caused by the reaction one wishes to study are coupled to the fluctuations caused by diffusion of the macromolecule in and out of the confocal volume. Thus, the current methodologies require that the diffusion properties are evaluated in an independent experiment using a reference sample. To overcome this issue, we propose a new methodology that involves the simultaneous measurement and analysis of the fluctuations in intensity of a donor- acceptor FRET pair. We performed a preliminary theoretical study that shows that the auto (donor-donor and acceptor-acceptor) and cross-correlation (donor-acceptor) functions can be analyzed in a way that uncouples diffusion from kinetics. In this way, the dynamics of biochemical reactions will be able to be studied in environments where diffusion is difficult or impossible to characterize. We will study the opening-closing kinetic rates of a DNA hairpin using our novel FCS-FRET method. We propose to start by studying this system in solution to validate the method, and then optimize the methodology in more complex environments such as vesicles and cells. The ability to study a single cell is critical in order to understand processes like aging, and diseases like cancer. These processes are originated in a single cell due to often unknown environmental and chemical stresses. Thus, in order to understand how a given type of stress triggers the formation of an anomalous cell, it is necessary to follow the fate of the progeny of individual cells as a function of time. Furthermore, the understanding of conformational dynamics in living cells is critical to understand all its biological functions. For instance, gene accessibility to the cell's protein machinery depends on the conformational dynamics of DNA-protein interactions in chromatin. Understanding chromatin dynamics is a key step in the process of elucidating the mechanisms of development, differentiation and cancer. ? ? ?
| Levitus, Marcia (2010) Relaxation Kinetics by Fluorescence Correlation Spectroscopy: Determination of Kinetic Parameters in the Presence of Fluorescent Impurities. J Phys Chem Lett 1:1346-1350 |
| Gurunathan, Kaushik; Levitus, Marcia (2010) FRET fluctuation spectroscopy of diffusing biopolymers: contributions of conformational dynamics and translational diffusion. J Phys Chem B 114:980-6 |
