A Stably Protonated Adenine Nucleotide with a Highly Shifted p<i>K</i>_a Value Stabilizes the Tertiary Structure of a GTP‐Binding RNA Aptamer

Abstract

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.