Incorporation of fluorescent nucleoside analogues into duplex DNA usually leads to a reduction in quantum yield, which significantly limits their potential use and application. We have identified two pentamer DNA sequences containing 6-methylisoxanthopterin (6-MI) (ATFAA and AAFTA, where F is 6-MI) that exhibit significant enhancement of fluorescence upon formation of duplex DNA with quantum yields close to that of monomeric 6-MI. The enhanced fluorescence dramatically increases the utility and sensitivity of the probe and is used to study protein-DNA interactions of nanomolar specificity in this work. The increased sensitivity of 6-MI allows anisotropy binding measurements to be performed at DNA concentrations of 1 nM and fluorescence intensity measurements at 50 pM DNA. The ATFAA sequence was incorporated into DNA constructs to measure the binding affinity of four different protein-DNA interactions that exhibit sequence-specific and non-sequence-specific recognition. In all cases, the K(d) values obtained were consistent with previously reported values measured by other methods. Time-resolved and steady-state fluorescence measurements demonstrate that 6-MI fluorescence is very sensitive to local distortion and reports on different degrees of protein-induced perturbations with single-base resolution, where the largest changes occur at the site of protein binding.