The field of RNA structural biology tries to understand the three-dimensional folds of RNA molecules and how these structures contribute to their biological function. Structural studies of often flexible RNA molecules can be difficult, and so there remain many challenges and opportunities for study. The relative paucity of RNA structural information limits machine learning techniques from accurately predicting RNA structures, so more RNA structures still must be solved with direct structural evidence.

The work reported here begins by presenting the expansion and characterization of a suite of portable RNA epitopes that can be recognized by antibody fragment (Fab) chaperones, proven to be useful in high-resolution x-ray crystallographic studies of RNA molecules. In addition to the previously reported loops that bind the BL3-6 and HAVx Fabs, new portable RNA loops are selected to bind to the BRG and HCV2 Fabs and the 3D structures of these new RNA-Fab complexes are determined. The suite of tools is comprised of RNA loops with distinct sequences and cognate Fabs that bind to their loops in distinct spatial arrangements with high (low to mid nM) affinity. These loops can be used to screen for crystallization conditions that may form using differing lattice contacts.

Furthermore, this work reports the use of one of these loops to be more broadly generalizable as a structural biology chaperone through the cryo-electron microscopy (cryo-EM) study of a novel RNA motif. Using one of these portable Fab chaperones, the structure of the newly described hovlinc ribozyme present in a human lncRNA transcript is determined to a resolution of 4.1 Å. The Fab chaperone more than doubles the size of the RNA motif alone, helping in the cryo-EM analysis of the small RNA given the intrinsic limitations of performing cryo-EM on small particles. The global fold of the structured RNA motif is determined unambiguously, consisting of two parallel stacks of helices held together by the double pseudoknot interactions of the RNA.

The structural information revealed about the hovlinc ribozyme is used to more carefully explore the determinants for the ribozyme’s self-cleaving activity in spatial proximity to the cut site. Through a high throughput sequencing experiment and extensive synthetic atomic perturbations around the active site, many of the active site features necessary for endonucleolytic activity are explored. A guanine is identified that is predicted to serve as the general base in the catalytic mechanism, and the overall active site architecture is hypothesized to have structural similarities to the pistol ribozyme’s active site. However, hovlinc does not appear to employ general acid catalysis in the same way as the pistol ribozyme’s mechanism. These results showcase a unique opportunity to expand on the utility of an RNA structural biology toolset and to study the activity and novel folding motif of a human ribozyme from a lncRNA.