The Essential Adenosine Stacking in a Two-Base-Pair Minimal Kissing Complex

Abstract

A stable RNA helix requires at least three base pairs. Surprisingly, a tertiary kissing complex formed between two GACG hairpin loops contains only two GC pairs. In the NMR structure of this complex, the two flanking adenosines stack on the kissing GC pair. This observation raised a possibility that the 5'-dangling adenines contribute to the formation and stability of the kissing interaction. To test this hypothesis, we took a two-pronged approach to examine the effects of various mutational and chemical modifications of the flanking adenosines on the folding of the kissing complex. using mass spectrometry, we studied dimerization of various kissing hairpins. using optical tweezers, we monitored mechanical unfolding of intramolecular kissing complexes at single-molecule level. In both experiments, replacing either adenine with a uridine abolished the kissing interaction, suggesting that a minimal kissing complex must contain two GC pairs flanked by inter-strand stacking adenines. The stabilizing effect by the adenines can be explained by the fact that the stacking purine nucleobases shield the hydrogen bonds of the adjacent GC pairs, preventing them from fraying. Unlike in the context of secondary structure, the 5'-unpaired adenines in the tertiary structure are structurally constrained in a way that allows for effective stacking onto the adjacent base pairs.