Modelling fluorescence quenching in systems with restricted diffusion: applications to oligonucleotides and polypeptides

The objectives of the present thesis are 1) to systematically develop a series of models and theoretical expressions for the diffusion-controlled reaction, which can be used to analyze the time-resolved fluorescence data in systems where the diffusion is restricted, and 2) to investigate the structural and dynamic properties of oligonucleotides and polypeptides by applying fluorescence-based methods and theoretical models. Firstly, the diffusion-controlled reaction in one- to three-dimensional systems was analyzed with the Smoluchowski approach. The analytical expressions containing diffusion coefficient in different systems were summarized for time-resolved fluorescence data fitting. Some of these expressions were drawn from literature sources and presented here in a unified form and new expressions have also been derived to fill some gaps found in the literature. This work is very useful not only for my own project but also for the global research framework in our group. ("Biomolecular and Supramolecular Kinetics in the Submicrosecond Time Range: The Fluorazophore Approach", W. M. Nau and X. Wang, ChemPhysChem, 2002, 3, 393-398 (Appendix II)). The diffusion-controlled intrachain fluorescence quenching was also analyzed. With the help of Prof. E. N. Bodunov, equilibrium conformational distributions of short polymer chains were simulated with Monte Carlo techniques. The kinetics of intramolecular end-toend collisions of short biopolymer chains that are labelled with a probe and a quencher at opposite ends was numerically simulated and the survival probability of the excited endattached probe, which reacts with the quencher at the other end upon contact, has been calculated. The results were compared with the experimental work on polypeptides carried out by other group members, suggesting that the reduced mobility of the ends of shorter chains was attributed to an increased steric hindrance, which results in an “internal friction” during intrachain motion. ("Fluorescence Quenching Kinetics in Short Polymer Chains: Dependence on Chain Length". X. Wang, E. N. Bodunov, and W. M. Nau Opt. Spectrosc. 2003, 95, 560-570 (Appendix IV)). Furthermore, two novel FRET energy donor/acceptor pairs with small critical radius, Trp/DBO and Nal/DBO, were employed to experimentally recover the end-to-end distance distribution and the intramolecular diffusion coefficient in Gly-Ser peptides. This work
offered an independent approach to verify the theoretical analysis and compare it with the
previous study based on collision-induced quenching systems. ("Application of FRET
donor/acceptor pairs with small critical radius to recover the structural and dynamic
properties in short flexible peptides", F. Huang, X. Wang, E. Haas, and W. M. Nau, 2004, in
preparation (Appendix VII)).
At the same time, some experimental projects were also carried out to investigate the
structural and dynamic properties in DNA, RNA and 2′-O-methyl RNA single-stranded
oligonucleotides. A phosphoramidite DBO derivative was synthesized, which can be directly
applied in the automated solid-phase synthesis to obtain 5'-DBO-labeled oligonucleotides.
The desirable properties of DBO, such as long lifetime, good solubility in water as well as
efficient quenching by guanine upon direct contact, make it possible to extract the kinetics of
molecular fluctuations from the intrachain fluorescence quenching. The end-to-end collision
rates in short single-stranded oligodeoxyribonucleotides were successfully measured for the
first time, which provided a strong support for the configurational diffusion model of hairpin
formation. ("Kinetics of End-to-End Collision in Short Single-Stranded Nucleic Acids". X.
Wang and W. M. Nau, J. Am. Chem. Soc. 2004, 126, 808-813 (Appendix VI)). The
investigation on RNA and 2′-O-methyl RNA oligomers showed that the 2′ substitutions could
result in different sugar puckering and fluctuational freedom in these analogues.
Consequently, the conformational and dynamic properties of different oligonucleotides can be
predicted, which will be useful for the research efforts in the area of antisense agents.
Additionally, a kinetic hopping model for one- and two-directional charge migration in a
one-dimensional system was developed and applied to the analysis of charge transfer
processes in DNA strands. ("Kinetics of one- and two-directional charge hopping in onedimensional
system: application to DNA". X. Wang and W. M. Nau, ChemPhysChem 2001,
2, 761-766 (Appendix I))
The mathematic methods obtained from these projects were also applied in the data
analysis of cyclodextrin host-guest complexation and diffusion-controlled fluorescence
quenching in biopolymer chains. ("A Joint Structural, Kinetic, and Thermodynamic
Investigation of Substituent Effects on Host-Guest Complexation of Bicyclic Azoalkanes by β-Cyclodextrin", X. Zhang, G. Gramlich, X. Wang, and W. M. Nau, J. Am. Chem. Soc. 2002,
124, 254-263 (Appendix III) and "Exploiting Long-Lived Molecular Fluorescence" W. M.
Nau, F. Huang, X. Wang, H. Bakirci, G. Gramlich, and C. Marquez, Chimia 2003, 57, 161-
167 (Appendix V)).