Intrinsically fluorescent nucleoside analogues with minimally perturbing architecture offer a non-disruptive method for optically studying nucleic acid structure, dynamics, and metabolism. These nucleoside analogues have inherently fluorescent nucleobases, thus replacing the natural canonical bases of DNA, but retain Watson-Crick base-pairing. Minimally perturbing structures are those that induce few or mild adverse deviations from native, unmodified biomolecular conformations. Using fluorescent nucleobases eliminates fluorophore conjugation chemistry often employed in conventional labeling strategies and their minimally perturbing structure better preserves the natural biomolecular topography and function of DNA. Since the discovery of 2-aminopurine 51 years ago, a multitude of nucleoside analogue scaffolds have been synthesized, including the tricyclic cytidine (tC) structure that the Purse Lab uses as a platform to develop novel derivatives with varied photophysical properties. Applying these novel derivatives towards biological applications requires characterization of their fluorescent, biophysical properties in DNA helices, and their compatibility with polymerase enzymes for metabolic labeling. This dissertation describes the biophysical characterization of a fluorescence turn-on probe in DNA-RNA hybrids, the polymerization kinetics exhibited by viral reverse transcriptase when incorporating the fluorescent nucleotides into nascent DNA strands, and preliminary live cell studies with fluorescent nucleotides. To better understand environmental factors modulating the turn-on response in the derivative 8-diethylamino tC, spectroscopic experiments with the probe in DNA-RNA hybrids were performed. Shielding from bulk water attenuated excited-state proton transfer and was identified to be a major factor increasing the brightness by up to 37-fold compared to the nucleoside in water. Michaelis-Menten kinetics for viral reverse transcriptase inserting the nucleotide analogues during DNA polymerization show efficiencies from 0.09 – 5 times that of natural dCTP across from G, with continued strand elongation. In a simplified reverse transcription cycle model, HIV-1 RT effectively recognized the tC analogues as the incoming nucleotide and as the templating base during complementary DNA synthesis. Live cell work with bacterial and human cells has suggested that tC nucleotides are retained in cells following delivery and that cytotoxicity is not a major concern. The findings of these studies suggest these tC-derivatives are appropriate biophysical tools for applications including sequence detection, DNA/RNA structural changes, and metabolic labeling.