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 decreases in quantum yield and emission
decay lifetime that vary sensitively with base sequence, temperature, and helix conformation
but that are accompanied by only small changes in emission wavelength. However, the
molecular interactions that give rise to these spectroscopic changes have not been
established. To develop a molecular model for interpreting the fluorescence measurements,
we have investigated the effects of environmental polarity, hydrogen bonding, and
the purine and pyrimidine bases of DNA on the emission energy, quantum yield, and
intensity decay kinetics of 2AP in simple model systems. The effects of environmental
polarity were examined in a series of solvents of varying dielectric constant, and
hydrogen bonding was investigated 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 titrating 2AP deoxyriboside (d2AP) with the nucleosides adenosine (rA), cytidine
(rC), guanosine (rG), and deoxythymidine (dT), and the nucleoside triphosphates 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 lifetime, proportionately) mechanisms. The
peak wavelength and shape of the emission spectrum are not altered by either of these
effects. The static quenching is saturable and has half-maximal effect at approximately
20 mM nucleoside or NTP, consistent with an aromatic stacking interaction. The rate
constant for dynamic quenching is near the diffusion limit for collisional interaction
(k(q) approximately 2 x 10(9) M(-1) s(-1)). Neither of these effects 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 lifetime and quantum yield decrease dramatically, accompanied by
significant shifts in the emission spectrum to shorter wavelengths. However, these
effects of polarity do not coincide with the observed emission wavelength-independent
quenching of 2AP fluorescence in DNA. Therefore, we conclude that the fluorescence
quenching of 2AP in DNA arises from base stacking and collisions with neighboring
bases only but is insensitive to base-pairing or other hydrogen bonding interactions.
These results implicate both structural and dynamic properties of DNA in quenching
of 2AP and constitute a simple model within which the fluorescence changes induced
by protein-DNA binding or other perturbations may be interpreted.