Protonation of deoxycytidine residues in dC 4 tetraloops: UV spectrophotometric study of dC 10 and d(A 14C 4T 14)

Abstract

It is shown that component analysis could be applied to study the UV difference spectra of cytidine oligomers and hairpin oligonucleotides with cytidines in the loop region in order to account for the melting and titration results in terms of cytidine stacking and protonation. Upon acid titration, the dC 10 oligomer undergoes cooperative conformational transition at pH 6.3 accompanied by protonation and formation of the i-structure with half of the residues protonated. The stability of the hemiprotonated structure increases with decreasing pH, the i-structure persisting still in the region of pH<p K of cytidine. An UV difference spectrum that reflects the stacking/unstacking of hemiprotonated cytidine residues was acquired from the melting and titration experiments of the dC 10 oligomer and used to describe the behavior of the dC 4 loop of the hairpin oligonucleotide d(A 14C 4T 14). It is shown that upon titration, the 50% level of protonation of the deoxycytidine tetraloop is attained at pH 5.0. Simultaneously, the stacking interactions of cytidine residues reach the maximum at this pH with two residues stacked, and thereafter decline again. Only marginal stabilization of the oligomer hairpin (Δ T m=1.5 °C) is found to accompany the formation of this single hemiprotonated dC·dC + base pair. We propose that at pH 5 the cytidines of the dC 4 loop form a hemiprotonated dC·dC + pair stacked with the last dA·dT base pair of the hairpin stem.