Microheterogeneity of Telomeric DNA Guanine Residues: pH Dependent Spectroscopic Studies of Fluorescently Labeled Model Trinucleotides

Abstract

Guanine-rich telomeric DNA (TTAGGG)4 (hT4) folds through Hoogsteen base pairing to form G-quadruplex structures, which inhibit the activity of telomerase, a key enzyme in tumorigenesis. Using a fluorescent guanine analog, 6-methylisoxanthopterin (6MI), we previously observed varying fluorescence intensity and emission wavelength shifts when incorporated into selective quadruplex sequence guanine positions, either in the folded or unfolded conformations. Our data suggest that quadruplexed guanine residues may play differing roles in maintaining global conformational stability through varying base-base, hydrogen bonding and/or solvent interactions. Previously long-wavelength emission spectral shifts of 6MI (Hawkins et al., Analytical Biochemistry (1997) 244, 86) were proposed to arise from possible deprotonation of 6MI at position N3, a key residue involved in Hoogsteen base-pairing. Here the effect of pH on the fluorescence properties of 6-MI (F) has been studied, when incorporated into model trinucleotides of hT4 with varying flanking bases: -AFG-; -GFG-; -GFT-; and -GGF-. All 6MI trinucleotides showed intensity quenching over 6MI, with a greater effect for purine neighbors (GFG>GGF>GFT>AFG) as noted previously (Poulin et al., Biochemistry (2009) 48, 8861). Increasing pH resulted in fluorescence intensity quenching for the parent 6-MI and a red wavelength fluorescence shift (excited-state pKa ∼8.3). Interestingly fluorescence lifetime values appeared invariant over the corresponding pH range (τav ∼ 6.2ns at pH 5 and ∼6.8ns at pH 11). Absorption spectral pH titrations revealed a ground-state pKa for 6MI of ∼9.8. Model 6MI labeled trinucleotides also revealed long-wavelength emission shifts, but associated with a dequenching of fluorescence signals. Increased excited-state pKa values corresponded closer to ground state values for the fluorescent trinucleotides. Effects of microheterogeneity on possible deactivating pathways for 6MI labeled trinucleotides, including possible electron transfer, deprotonation, and quenching mechanisms will be discussed. (1Supported by NIH 5SC3 GM095437).