Investigating the structure and dynamics of DNA with fluorescence and computational techniques
Smith, Darren Andrew
Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.