Fast Catalytic Activity of an in Vitro Evolved Hammerhead Ribozyme Is Mediated through Novel Tertiary Interactions

Directed evolution studies have resulted in the isolation of highly active catalytic RNAs. To better understand the sequence requirements of self-cleaving RNAs, we have explored an atypical hammerhead ribozyme, obtained through in vitro evolution, that exhibits high catalytic activity despite possessing an unusually small stem-loop II structure. This ribozyme, herein referred to as N15HH, as in canonical hammerhead ribozymes, consists of a conserved catalytic core supported by three stem-loop structures. The minimal stem-loop II structure of N15HH consists of a putative two-base stem and a 5’-UAA-3’ triplet as a short loop. In natural instances of this class of ribozymes, a kissing-loop interaction between loop II and loop I, or loop II and a bulge in stem I can enhance catalytic activity. Such interactions cannot occur in N15HH, thus raising the question how the active form of this ribozyme is stabilized. Through extensive in vitro mutational analyses, we show that the minimal stem-loop II structure supports fast self-cleavage rates in N15HH (K_obs ~10 min^-1 in 10mM MgCl₂). To further investigate, we carried out de novo and homology-based structural modeling and in silico simulations to obtain models that can account for the high catalytic rates of this ribozyme. Cluster analysis of microseconds Molecular Dynamics simulations resulted in the identification of two najor structures: A first structure displaying a direct loop II-stem I interaction (between the 2’OH of a guanosine in stem I and the NH₂ at position six of the middle adenine in loop II), and a second structure displaying a novel interaction between loop II and the catalytic core of the ribozyme. In the latter, the 2’OH of the middle loop II adenine hydrogen bonds with the nitrogen at position 3 of the second core uracil (or U⁷ in the 5’-CUGAU⁷GA-3’ core motif), which may mediate stabilization of its catalytically active conformation. To experimentally corroborate these predictions, ribozymes with deoxy-base substitutions at the interacting positions were synthesized to probe the proposed interactions. A significant decrease in catalytic activity was observed in the chimeric ribozymes, consistent with the two structures. Based on these results, we propose two distinct active conformations are co-occurring in N15HH. Altogether, our results indicate that despite the overall simplicity of hammerhead ribozyme structural framework, multiple tertiary site interactions can occur to prime the ribozyme for activity.