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 (dh-G) was recently,developed. Here, steady-state and time-resolved fluorescence spectroscopy was used to compare the ability of 2Ap and dthG, to substitute and provide relevant structural and dynamical information on a key G residue in the () DNA copy of the HIV-1 primer binding site, (-)PBS, both in its stem loop conformation and in the corresponding (-)/(+)PBS duplex. In contrast 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 fluorescence decay, with marginal amounts of dark species. In further contrast to 2Ap, the fluorescently detected dthG species reflect the pre-dominantly populated G conformers, which allows exploring their relevant dynamics. Being able to perfectly substitute G residues, dthG will transform nucleic acid biophysics by allowing, for the first time, to selectively and faithfully monitor the conformations and dynamics of a given G residue in a DNA sequence.