Investigation of the interactions between fluorescent base analogues and the natural DNA bases
Paterson, Kyle Andrew
DNA and RNA are integral to all life on Earth, and yet their physical properties and behaviour in their native environment are still only imperfectly understood. Using fluorescent analogues of natural DNA bases (FBAs) as a probe of local inter-base interactions is a widely employed solution-phase technique to obtain information about DNA conformation and its response to enzyme activity. Work presented in this thesis aims to show that free FBAs in solution with the natural DNA bases is a useful model of the inter-base interactions of FBAs in oligonucleotides, and that the effect of substituting DNA bases with fluorescent analogues on DNA conformation can be predicted computationally. Some results from fluorescence spectroscopy to gain further insights into the effect of conformation on electronic energy transfer will also be discussed. 2-aminopurine (2AP) is a responsive fluorescent base analogue that is widely used as a probe of the local molecular environment in DNA. However, the mechanism of this inter-base quenching remains imperfectly understood. Two previous studies of collisional quenching of 2AP by the natural nucleotides presented conflicting results. A comprehensive investigation of inter-base quenching of 2AP by the natural bases in solution is presented here, reproducing the buffer conditions used in the previous studies. Time-resolved fluorescence measurements are used to provide insight into both dynamic and static quenching, showing consistent trends across both buffer systems, and the results support a charge transfer mechanism. Time-resolved fluorescence data also provide evidence for formation of 2AP-nucleotide ground-state complexes in solution, the fluorescence lifetimes of which are comparable to that seen in 2AP-containing oligonucleotides. Collisional quenching studies were extended to a recently reported FBA, pentacyclic adenine (pA), which has red-shifted emission relative to 2AP, as well as increased brightness. However, rapid photobleaching of pA makes it difficult to use steady-state fluorescence measurements to calculate quenching efficiencies; in consequence time-resolved fluorescence data was obtained to quantify the effect of the natural monophosphate nucleotides on the fluorescence of pA. It was found that collisional interaction of pA with the purine bases increased its fluorescence lifetime (the inverse of a quenching effect), while interaction with the pyrimidine bases shortened the lifetime. These observations were consistent with previous studies of the effect of the base sequence surrounding pA in oligonucleotides. The results of these collisional quenching experiments for 2AP and pA show that measuring the fluorescence of free FBAs in solution in the presence of the natural bases is a valid technique for predicting the behaviour of FBAs in oligonucleotide strands. In order to complement the spectroscopic studies, computational techniques were employed to examine the structural impact of substituting a natural base with a base analogue in oligonucleotide sequences. Geometry optimisations of dinucleotides containing pA were carried out, using the DFT functional M06-2X, which accounts for dispersion, to model the effect of this novel FBA on inter-base stacking in DNA. DNA base-step and backbone structural parameters were extracted from the optimised structures and used to show that the substituted dinucleotides adopt conformations similar to that associated with B-form DNA. Previous studies have shown that, in 2AP-containing dinucleotides, electronic energy transfer occurs from the natural base to 2AP, on excitation of the natural base at 260 nm. It was found that there was a substantial increase in energy transfer efficiency in frozen solution at 77 K compared to room temperature. In the present study, the energy transfer process was investigated as a function of temperature over the range 5-25 °C, to examine the effect of reducing temperature while maintaining fluid conditions. A trend of decreasing quenching efficiency with increasing temperature was found, which is consistent with the previous findings. The results of this work also show that energy transfer is conformationally selective over this temperature range, as can be inferred from decay parameters obtained using time-resolved fluorescence measurements. In summary, this thesis yields deeper understanding of the effect of interactions with natural DNA bases on the photophysics of two FBAs, pA and 2AP, and presents a method for predicting the behaviour of novel FBAs without a priori preparing substituted dinucleotides.