We present theoretical characterization of fluorescent non-natural nucleobases, ^tzA, ^tzG, ^tzC, and ^tzU, derived from the isothiazolo[4,3-d]pyrimidine heterocycle. Consistent with the experimental evidence, our calculations show that the non-natural bases have minimal impact on the geometry and stability of the classical Watson–Crick base pairs, allowing them to accurately mimic natural bases in a RNA duplex, in terms of H-bonding. In contrast, our calculations indicate that H-bonded base pairs involving the Hoogsteen edge are destabilized relative to their natural counterparts. Analysis of the photophysical properties of the non-natural bases allowed us to correlate their absorption/emission peaks to the strong impact of the modification on the energy of the lowest unoccupied molecular orbital, LUMO, which is stabilized by roughly 1.0–1.2 eV relative to the natural analogues, while the highest occupied molecular orbital, HOMO, is not substantially affected. As a result, the HOMO–LUMO gap is reduced from 5.3–5.5 eV in the natural bases to 4.0–4.4 eV in the modified ones, with a consequent bathochromic shift in the absorption and emission spectra.