The Transition from a Neutral-pH Double Helix to a Low-pH Triple Helix Induces a Conformational Switch in the CCCG Tetraloop Closing a Watson-Crick Stem

The CCCG-loop in a DNA fragment, which is capable of forming an intramolecular triple helix as well as a hairpin structure, was investigated by NMR and molecular modeling studies. The structure of this loop is found as a type II conformation, one of the three commonly observed folding patterns of tetraloops, irrespective of the geometry of the underlying helix. In each situation, the loop exhibits a base-pair between the first cytosine and the guanine residue of the loop. The geometry of this base-pair, however, depends upon the circumstances. At neutral pH, in the hairpin form of the molecule, a Watson-Crick C·G base-pair is formed, whereas at low pH, when the strand exists as an intramolecular triple helix, a Hoogsteen C +-G base-pair is present. We used molecular modeling to lay the foundations for understanding the observed conformational switch. A lower amount of strain, related to the short C1′ – C1′ of the base-pair, and protonation effects of the structure comprising the Hoogsteen base-pair turn out to outweigh the effects of a more stable base-pair, improved stacking and more favorable interactions in the minor groove of the structure comprising the Watson-Crick C·G base-pair. The models also provide an explanation for the general preference of loops meeting the consensus sequence -d(CYNG)- to fold into a type II conformation, i.e. with the base of second loop residue turned into the minor groove.