A Stably Protonated Adenine Nucleotide with a Highly Shifted pKa Value Stabilizes the Tertiary Structure of a GTP-Binding RNA Aptamer

RNA tertiary structure motifs are stabilized by a wide variety of hydrogen-bonding interactions. Protonated A and C nucleotides are normally not considered to be suitable building blocks for such motifs since their pK(a) values are far from physiological pH. Here, we report the NMR solution structure of an in vitro selected GTP-binding RNA aptamer bound to GTP with an intricate tertiary structure. It contains a novel kind of base quartet stabilized by a protonated A residue. Owing to its unique structural environment in the base quartet, the pK(a) value for the protonation of this A residue in the complex is shifted by more than 5 pH units compared to the pK(a) for A nucleotides in single-stranded RNA. This is the largest pK(a) shift for an A residue in structured nucleic acids reported so far, and similar in size to the largest pK(a) shifts observed for amino acid side chains in proteins. Both RNA pre-folding and ligand binding contribute to the pK(a) shift.