Differential fluorescence quenching of fluorescent nucleic acid base analogues by native nucleic acid monophosphates

Fluorescent nucleic acid base analogues (FBAs) are used widely as probes of DNA and RNA structure and dynamics. Of increasing utility are the pteridone adenosine analogues (6MAP, DMAP) and pteridine guanosine analogues (3MI, 6MI). These FBAs (collectively referred to as PTERs) are useful, in part, because their fluorescence quantum yields, Phi(f), are modulated by base stacking with native bases (NBs), making them sensitive reporters of DNA structure. The quenching mechanism has been hypothesized to be photoinduced electron transfer following selective excitation of the FBA, but hard evidence for this has been lacking. The degree of quenching shows some dependence on the neighboring bases, but there has been no real determination as to whether FBA*:NB complexes satisfy the basic thermodynamic requirement for spontaneous PET: a negative free energy for the electron transfer reaction. Indeed, quenching may result from entirely different mechanisms. To address these questions, Stern-Volmer (S-V) experiments were performed using the native-base monophosphate nucleotides (NMPs) GMP, AMP, CMP, and dTMP in aqueous solutions as quenchers to obtain quenching rate constants, k(q). Cyclic voltammetry (CV) and optical absorption and emission data of the PTERS were obtained in aprotic organic solvents. These data were used to obtain excited-state redox potentials from which electron transfer free energies were derived using the Rehm-Weller equation. The reorganization energies for PET were obtained using the Scandola-Balzani equation, taking into account the free energy contribution due to water. 6MAP*, DMAP*, and 3MI* gave negative free energies between -0.1 and -0.2 eV and reorganization energies of about 0.13 eV. They all displayed ET activation energies below the accessible thermal energy (0.038 eV = 3/2k(B)T, where k(B) is Boltzmann's constant) for all NMPs with the exception of CMP, whose activation barrier was only about 35% higher (approximately 0.05 eV). Thus, we conclude that these PTERs act as electron acceptors and promote NMP oxidation. However, 6MI* had positive ET free energies for all NMPs with the exception of GMP (and then only for nucleobase oxidation). The magnitudes of these free energies (> or = 0.45 eV for AMP, CMP, and dTMP) suggest that 6MI* may not quenched by PET.