Direct Monitoring of Cytosine Protonation in an Intramolecular DNA Triple Helix

Potential applications in molecular biology and medicine have sparked renewed interest in DNA triple helices recently. Binding of a single stranded DNA or RNA to the major groove of a double helical target results in the formation of specific Hoogsteen hydrogen bonds with the Watson-Crick purine bases. Thus, in triple helices of the pyrimidine motif a third pyrimidine strand interacts with the double helix forming C super(+)GC and TAT base triplets. However, the requirement for protonation of the cytosine bases in the third strand results in decreased stabilities of the triplex with increasing pH. Studies on the pH dependence of triple helix formation with a third pyrimidine strand by ethidium bromide fluorescence, by UV absorption spectroscopy, and by CD spectroscopy yielded semiprotonation points for oligonucleotide triplexes. However, the determination of such apparent pK sub(a) values by the above methods is based on a two-state model with the cytosine protonation being the only pH-dependent process for the duplex-triplex transition. Moreover, analysis is restricted to the influence of pH on a global equilibrium process. Protonation of individual bases within a sequence is not resolved. In a recent NMR study a uniformly super(13)C- and super(15)N-labeled RNA third strand was used for the unambiguous structural characterization of the triplex formed with a DNA hairpin. To assess the effect of cytosine protonation on triple helix formation at single sites we decided to perform heteronuclear NMR experiments on a specifically super(15)N-labeled oligonucleotide. The oligonucleotide was designed to form an intramolecular triple helix with two (T) sub(4) loops and seven base triplets by folding back on itself under appropriate conditions. Except for the 3'-terminal cytosine all cytosine bases within the Hoogsteen bound "third strand" were specifically super(15)N labeled at the 4-amino group (Figure 1). Although this group is not directly protonated, studies on monomers indicate a downfield shift of the super(15)N amino resonance by more than 10 ppm upon cytidine protonation. The specifically isotope-labeled cytidine was synthesized according to published procedures, protected, and subsequently used as its phosphoramidite in the automated DNA synthesis.