Visualizing Invisibles with Single-molecule Techniques: from Protein Folding to Clinical Applications

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.Ph