The steady-state and time-resolved fluorescence of 2-aminopurine (2AP) have been used to monitor base dynamics and base stacking interactions in DNA single strands and dinucleotides, and to investigate the interactions between DNA and a polymerase, Pfu-Pol. A new class of base analogues has also been investigated using a combination of experiment and quantum chemical computation. In recent years, 2AP has been widely used as a fluorescent probe to study conformational changes and inter-bases interactions in duplex DNA, but the conformational behaviour of DNA in single strands has been far less investigated. In the present work, six 2AP–labelled single strands have been studied by steady-state and time-resolved fluorescence measurements. Single strands were found to show similar conformational heterogeneity (manifested by 4-exponential fluorescence decays) to duplex DNA, but highly stacked conformations, in which 2AP is rapidly quenched by inter-base charge transfer, are less populated in single strands, whereas imperfectly stacked (weakly quenched) conformations are more highly populated. The effect of base pairing in constraining base mobility is evident. To further investigate the influence of base stacking interaction on DNA conformation and the mechanism of inter-base quenching of 2AP, the time-resolved fluorescence of 2AP-containing dinucleotides was measured. The fluorescence decay of 2AP-containing dinucleotides in PBS buffer at room temperature is also multiexponential and the shortest lifetime varies with the identity of the natural base partner, in a manner consistent with quenching by inter-base electron transfer. When the dinucleotides are frozen to 77 K, the quenching of 2AP is almost eliminated, demonstrating the importance of thermal fluctuations of the bases in facilitating inter-base quenching at room temperature. In the frozen dinucleotides, an additional decay component with a lifetime significantly longer than unquenched 2AP is also observed, suggesting the formation of a new, delocalised, inter-base excited-state. Archaeal family-B DNA polymerases bind tightly to uracil and stall replication when they encounter this base in template strands, four bases ahead of the primer-template junction. If the polymerase progresses further towards the uracil, the 3′-5′ proof reading exonuclease becomes stimulated, trimming the primer and re-setting uracil to the +4 position. Uracil sensing prevents copying of the deaminated base and the introduction of mutations into the genome. Time-resolved fluorescence of 2AP has been used to investigate the role played by unwinding of primer-templates in this mechanism. 2AP-labelled primer-templates (2AP positioned next to the terminal 3′ base of the primer strand), with a misincorpated uracil at the +2 position (U+2) or +4 position (U+4) from the replication fork in the complementary template strand, were investigated in complex with the polymerase Pfu-Pol. For the U+2 primer-template, the fluorescence decay parameters show clear evidence for a decrease in the amount of double-stranded DNA on polymerase binding, manifested by marked weakening of inter-base stacking and a large transfer of population from highly stacked to poorly stacked conformations. In contrast, for the U+4 primer-template only a small perturbation to inter-base stacking is seen, together with the persistence of a high population of strongly stacked states. A new class of base analogues with selenium replacing oxygen at the 4 position of thymine and the 6 position of guanine has been investigated experimentally and computationally. These base analogues are interesting because they show a large shift (>80 nm) in their absorption spectrum compared with the natural bases, taking their absorption into the visible region, with minimal change in molecular structure. The potential of two examples of these analogues, 4-selenium thymine-3’-phosphate and 6-selenium-2’-deoxyguanosine-3-phosphate as luminescent probes has been investigated. However, they prove to have very low emission quantum yields for both fluorescence and phosphorescence. The effect of selenium-substitution on the structural and photophysical properties of the bases has been studied by various ab initio computational methods. It has been found that replacement of oxygen by selenium does not affect the ground state structure but changes the structure of the first excited-state from buckled to nearly planar. The shift in the absorption spectrum on introduction of selenium is successfully predicted by the calculations; the red-shifted absorption band of selenium-substituted thymine is due to a new electronic transition that is not present in the natural base, whereas that of selenium-substituted guanine is from red-shifting of a guanine-like transition.