Probing structure and dynamics of DNA with 2-aminopurine: Effects of localenvironment on fluorescence

2-Aminopurine (2AP) is an analogue of adenine that has been utilized widely as a fluorescence probe of protein-induced local conformational changes in DNA. Within a DNA strand, this fluorophore demonstrates characteristic dec reases in quantum yield and emission decay lifetime that vary sensitively w ith base sequence, temperature, and helix conformation but that are accompa nied by only small changes in emission wavelength. However, the molecular i nteractions that give rise to these spectroscopic changes have not been est ablished. To develop a molecular model for interpreting the fluorescence me asurements, we have investigated the effects of environmental polarity, hyd rogen bonding, and the purine and pyrimidine bases of DNA on the emission e nergy, quantum yield, and intensity decay kinetics of 2AP in simple model s ystems. The effects of environmental polarity were examined in a series of solvents of varying dielectric constant, and hydrogen bonding was investiga ted in binary mixtures of water with 1,4-dioxane or N,N-dimethylformamide ( DMF). The effects of the purine and pyrimidine bases were studied by titrat ing 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine (rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosph ates ATP and GTP in neutral aqueous solution. The nucleosides and NTPs each quench the fluorescence of d2AP by a combination of static (affecting only the quantum yield) and dynamic (affecting both the quantum yield and the l ifetime, proportionately) mechanisms. The peak wavelength and shape of the emission spectrum are not altered by either of these effects. The static qu enching is saturable and has half-maximal effect at approximately 20 mM nuc leoside or NTP, consistent with an aromatic stacking interaction. The rate constant for dynamic quenching is near the diffusion limit for collisional interaction (k(q) approximate to 2 x 10(9) M-1 s(-1)). Neither of these eff ects varies significantly between the various nucleosides and NTPs studied. In contrast, hydrogen bonding with water was observed to have a negligible effect on the emission wavelength, fluorescence quantum yield, or lifetime of 2AP in either dioxane or DMF. In nonpolar solvents, the fluorescence li fetime and quantum yield decrease dramatically, accompanied by significant shifts in the emission spectrum to shorter wavelengths. However, these effe cts of polarity do not coincide with the observed emission wavelength-indep endent quenching of 2AP fluorescence in DNA. Therefore, we conclude that th e fluorescence quenching of 2AP in DNA arises from base stacking and collis ions with neighboring bases only but is insensitive to base-pairing or othe r hydrogen bonding interactions. These results implicate both structural an d dynamic properties of DNA in quenching of 2AP and constitute a simple mod el within which the fluorescence changes induced by protein-DNA binding or other perturbations may be interpreted.