The archetypical fluorescent nucleoside analog, 2-aminopurine (2Ap) has been used in countless assays, though it suffers from very low quantum yield, especially when included in double strands, and from the fact that its residual emission frequently does not represent biologically relevant conformations. To conquer 2Ap's deficiencies, deoxythienoguanosine (dthG), was recently developed. Here, steady-state and time-resolved fluorescence spectroscopy was used to compare the ability of 2Ap and dthG, to substi-tute and provide relevant structural and dynamical infor-mation on a key G residue in the (-) DNA copy of the HIV-1 Primer Binding Site, (-)PBS, both in its stem loop confor-mation and in the corresponding (+)/(-)PBS duplex. In con-trast to 2Ap, this fluorescent nucleoside when included in (-)PBS or (-)/(+)PBS duplex fully preserves their stability and exhibits a respectable quantum yield and a simple fluores-cence decay, with marginal amounts of dark species. In further contrast to 2Ap, the fluorescently detected dthG species reflect the predominantly populated G conformers, which allows exploring their relevant dynamics. Being able to perfectly substitute G residues, dthG will transform nucle-ic acid biophysics by allowing, for the first time, to selective-ly and faithfully monitor the conformations and dynamics of a given G residue in a DNA sequence.