Fluorescence properties of nucleobase analogues in DNA under one- and two-photon excitation
Creators: name: Bailie, Alexandra Elizabeth
Publisher: name: The University of Edinburgh
Year: 2023
Subjects: fluorescence
DNA

description: Since the mapping of the euchromatic human genome in 2003, in many ways, the mysteries of DNA and its cellular processes only got deeper. The complexities of epigenetic modifications which are passed to offspring and dictate fundamentals of an embryo’s development and even viability cannot be fully expressed by the genetic code alone. As DNA interacts with enzymes to be read, switched on and off, modified, or copied, the way this interaction occurs and how it can slip up is of great interest to the scientific community. The investigation of these DNA and enzymatic processes can be carried out using fluorophores inside the DNA duplex, such as 2-aminopurine (2AP), where changes in DNA conformation, such as unwinding or base-flipping, may be observed for ensembles of molecules using time-resolved fluorescence measurements. This approach has been used in this thesis to explore the interaction of two enzymes from the ModA family with 2AP-labelled duplexes. This work epitomises the conformational complexity of DNA and DNA-enzyme complexes which limits the interpretation of fluorescence decay parameters obtained in ensemble measurements and prevents a full picture of the interactions taking place from being uncovered. To fully explain the mechanism of epigenetic modifications, a single-molecule approach is needed, and with this, a new generation of fluorescent base analogues (FBAs) is required. Isomorphic FBAs (those with structures close to those of the natural bases) such as 2AP, have excitation wavelengths in the ultraviolet and are not suitable for single-molecule spectroscopy. There is a need for new FBAs with longer excitation wavelengths, but this requires extended structures that do not resemble those of the natural bases. An alternative approach, which is explored in this thesis, is the use of two-photon excitation, at wavelengths around 800 nm, to enable single-molecule detection of isomorphic FBAs. This demands the development of FBAs with high two-photon cross sections combined with high fluorescence quantum yields when introduced into oligonucleotides. In pursuit of this, two newly developed FBAs have been investigated, a 6-aza-uridine derivative (MeOthaU) and a tri-cyclic thymidine analogue (ABN). The photophysical properties of these FBAs have been determined as free nucleosides, and in a range of oligonucleotides where the influence of surrounding bases can be seen. Fluorescence spectra, quantum yields and decay parameters have been determined (under one-photon excitation) and two-photon cross-sections have been measured. MeOthaU is a responsive fluorophore, with its quantum yield depending strongly on the polarity of the environment. It has been found that the photophysical behaviour of the nucleoside in polar and non-polar solvents may be used as a predictor of its behaviour when incorporated in DNA. Under two-photon excitation, the fluorescence brightness of MeOthaU in duplex oligonucleotides is exceptionally high compared with established, responsive FBAs. ABN shows a high fluorescence quantum yield as the free nucleoside, and retains this when incorporated in oligonucleotides. Under two-photon excitation, its fluorescence brightness in duplexes exceeds that of MeOthaU and is the highest yet observed for a FBA in DNA. Time-resolved fluorescence measurements have revealed the existence of ground-state tautomers and the occurrence of excited-state tautomerisation, in both the free ABN nucleoside and within duplexes. The existence of ABN as thymine-like and cytosine-like tautomers enables it to form base pairs with both adenine and guanine. In summary, this thesis explores in detail the photophysical behaviour of new FBAs under one- and two-photon excitation, as nucleosides and in oligonucleotides, with the aim of achieving single-molecule detection. Time-resolved fluorescence of 2AP, the archetypal, isomorphic FBA, has been used to explore whether the mechanism of ModA enzymes involves base flipping.