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Open Access Dissertation
Doctor of Philosophy (PhD)
Year Degree Awarded
Anthony D. Dinsmore
Biological and Chemical Physics
The material related with orientation of Cyanine dyes and their behavior at the ends of duplex RNA is also documented in . Cyanine dyes are widely used to study the folding and structural transformations of nucleic acids using fluorescence resonance energy transfer (FRET). The extent to which FRET can be used to extract inter- and intra-molecular distances has been the subject of considerable debate in the literature; the contribution of dye and linker dynamics to the observed FRET signal is particularly troublesome. We used molecular dynamics (MD) simulations to study the dynamics of the indocarbocyanine dyes Cy3 and Cy5 attached variously to the 3 or 5 terminal bases of a 16 base-pair RNA duplex. We then used Monte Carlo modeling of dye photophysics to predict the results of single-molecule sensitive FRET measurements of these same molecules. Our results show that the average value of FRET depends on both the terminal base and on the linker position. In particular, 3 attached dyes typically explore a wide region of configuration space and
the relative orientation factor, κ2, has a distribution that approaches that of free- rotators. This is in contrast to 5 attached dyes, which spend a significant fraction of their time in one or more configurations that are effectively stacked on the ends of the RNA duplex. The presence of distinct dye configurations for 5 attached dyes is consistent with observations made by others of multiple fluorescence lifetimes of Cy3 on nucleic acids. While FRET is frequently used as a molecular “ruler” to measure intramolecular distances, the unambiguous measurement of distances typically relies on the assumption that the rotational degrees of freedom of the dyes can be averaged out, and that the donor lifetime in the absence of the acceptor is a constant. We demonstrate that even for the relatively free 3 attached dyes, the correlation time of κ2 is still too long to justify the use of a free-rotation approximation. We further explore the consequences of multiple donor lifetimes on the predicted value of FRET.
While providing detailed information about the individual members of a molecular ensemble, FRET technique is always limited by fluorophore brightness and stability. In the case of diffusing molecules, the experiment is further limited by the number of photons that can be collected during the time it takes for a molecule to diffuse across the detection volume. To maximize the number of photons it is common to either increase the detection volume at the expense of increased background, or increase the diffusion time by adding glycerol or sucrose to increase viscosity. As an alternative to current methods, here we demonstrated that water in oil nano-emulsions in perfluorinated compounds FC40 and FC77 can be used to confine biomolecules which results a dramatic increase in signal to noise ratios. To efficiently use these attoliter volume (130 nm radius) aqueous containers, their characterization in terms of physical and chemical properties is necessary for both un- derstanding the droplet environment and making better statements for single molecule experiment results within them. Characterization in terms of size is not an easy task because droplet system is a macroemulsion, so that droplets are kinetically stable.
Therefore in addition to conventional dynamic light scattering (DLS) measurements, we proposed a new method to measure droplet sizes using Mie scattering . The method was originally used in atmospheric physics and medical imaging and in our knowledge it was the first time of its use on an emulsion system. We also measured the average pH within droplets by this new method using an absorptive dye (bromothymol blue). These measurements in combination with the emission measurements using another pH sensitive dye (fluorescein) showed that the droplet environment is acidic. Our initial attempts for changing the pH by adding a strong base to bulk aqueous samples prior to emulsification were succesful but the interpretation of result were not accurate because of working pH range of pH indicators.
Single molecule experiments within attoliter aqueous droplets provided supporting results for the low pH argument. We have seen that Cy3 only labelled duplex RNA ,in bulk experiments, was sensitive to ambient pH in the solution. This sensitivity appeared as a shift in the mean value of proximity histograms. The mean value of proximity ratio histograms at low pH (less than pH 7) was similar to the mean value of proximity ratio histograms we observed in droplet data. In addition, using photon counting histograms we identified a third brighter species in low pH solution data and in droplet data. All these materials related with single molecule works within droplets is also documented in . In our knowledge, there is no analytical function given in the literature for the autocorrelation function of fluorescence correlation spectroscopy (FCS) data within droplets. Thus, we put effort for simulating the FCS of droplets. Although, this ap- proach didn’t provide us an analytical form, it was helpful for further understanding the internal environment of droplets. As a result of simulations, we observed that dif- fusion time within droplets long (on the order of a millisecond). In addition, we were able identify the source of different correlation times observed in FCS experimental data.
MILAS, PEKER, "SINGLE MOLECULE STUDIES OF A SHORT RNA" (2014). Doctoral Dissertations. 269.