A multi-axial RNA joint with a large range of motion promotes sampling of an active ribozyme conformation.

Investigating the dynamics of structural elements in functional RNAs is important to better understand their mechanism and for engineering RNAs with novel functions. Previously, we performed rational engineering studies with the Varkud satellite (VS) ribozyme and switched its specificity toward non-natural hairpin substrates through modification of a critical kissing-loop interaction (KLI). We identified functional VS ribozyme variants with surrogate KLIs (ribosomal RNA L88/L22 and human immunodeficiency virus-1 TAR/TAR*), but they displayed ∼100-fold lower cleavage activity. Here, we characterized the dynamics of KLIs to correlate dynamic properties with function and improve the activity of designer ribozymes. Using temperature replica exchange molecular dynamics, we determined that the natural KLI in the VS ribozyme supports conformational sampling of its closed and active state, whereas the surrogate KLIs display more restricted motions. Based on in vitro selection, the cleavage activity of a VS ribozyme variant with the TAR/TAR* KLI could be markedly improved by partly destabilizing the KLI but increasing conformation sampling. We formulated a mechanistic model for substrate binding in which the KLI dynamics contribute to formation of the active site. Our model supports the modular nature of RNA in which subdomain structure and dynamics contribute to define the thermodynamics and kinetics relevant to RNA function.

Rational engineering of the Neurospora VS ribozyme to allow substrate recognition via different kissing-loop interactions.

The Neurospora VS ribozyme is a catalytic RNA that has the unique ability to specifically recognize and cleave a stem-loop substrate through formation of a highly stable kissing-loop interaction (KLI). In order to explore the engineering potential of the VS ribozyme to cleave alternate substrates, we substituted the wild-type KLI by other known KLIs using an innovative engineering method that combines rational and combinatorial approaches. A bioinformatic search of the protein data bank was initially performed to identify KLIs that are structurally similar to the one found in the VS ribozyme. Next, substrate/ribozyme (S/R) pairs that incorporate these alternative KLIs were kinetically and structurally characterized. Interestingly, several of the resulting S/R pairs allowed substrate cleavage with substantial catalytic efficiency, although with reduced activity compared to the reference S/R pair. Overall, this study describes an innovative approach for RNA engineering and establishes that the KLI of the trans VS ribozyme can be adapted to cleave other folded RNA substrates.

Helix-length compensation studies reveal the adaptability of the VS ribozyme architecture.

Compensatory mutations in RNA are generally regarded as those that maintain base pairing, and their identification forms the basis of phylogenetic predictions of RNA secondary structure. However, other types of compensatory mutations can provide higher-order structural and evolutionary information. Here, we present a helix-length compensation study for investigating structure-function relationships in RNA. The approach is demonstrated for stem-loop I and stem-loop V of the Neurospora VS ribozyme, which form a kissing-loop interaction important for substrate recognition. To rapidly characterize the substrate specificity (k(cat)/K(M)) of several substrate/ribozyme pairs, a procedure was established for simultaneous kinetic characterization of multiple substrates. Several active substrate/ribozyme pairs were identified, indicating the presence of limited substrate promiscuity for stem Ib variants and helix-length compensation between stems Ib and V. 3D models of the I/V interaction were generated that are compatible with the kinetic data. These models further illustrate the adaptability of the VS ribozyme architecture for substrate cleavage and provide global structural information on the I/V kissing-loop interaction. By exploring higher-order compensatory mutations in RNA our approach brings a deeper understanding of the adaptability of RNA structure, while opening new avenues for RNA research.

Role of SLV in SLI substrate recognition by the Neurospora VS ribozyme.

Substrate recognition by the VS ribozyme involves a magnesium-dependent loop/loop interaction between the SLI substrate and the SLV hairpin from the catalytic domain. Recent NMR studies of SLV demonstrated that magnesium ions stabilize a U-turn loop structure and trigger a conformational change for the extruded loop residue U700, suggesting a role for U700 in SLI recognition. Here, we kinetically characterized VS ribozyme mutants to evaluate the contribution of U700 and other SLV loop residues to SLI recognition. To help interpret the kinetic data, we structurally characterized the SLV mutants by NMR spectroscopy and generated a three-dimensional model of the SLI/SLV complex by homology modeling with MC-Sym. We demonstrated that the mutation of U700 by A, C, or G does not significantly affect ribozyme activity, whereas deletion of U700 dramatically impairs this activity. The U700 backbone is likely important for SLI recognition, but does not appear to be required for either the structural integrity of the SLV loop or for direct interactions with SLI. Thus, deletion of U700 may affect other aspects of SLI recognition, such as magnesium ion binding and SLV loop dynamics. As part of our NMR studies, we developed a convenient assay based on detection of unusual (31)P and (15)N N7 chemical shifts to probe the formation of U-turn structures in RNAs. Our model of the SLI/SLV complex, which is compatible with biochemical data, leads us to propose novel interactions at the loop I/loop V interface.