Myers, J. M. | bioRxiv Science

bioRxiv2022

Log-order improved in trans hammerhead ribozyme turnover rates: reevaluating therapeutic space for small catalytic RNAs

We discovered an enhanced functionality hammerhead ribozyme (EhhRz), designed to act in trans against human rod opsin (RHO) mRNA, with turnover activity >300 nM min-1 under substrate-excess conditions and physiological Mg2+ levels (1 mM). We developed a real-time moderate-throughput fluorescence quantitative hhRz kinetic assay, which is linear with substrate and product moles and supported by gel-based measures. The EhhRz targets a CUC{downarrow} cleavage site in a substrate with no predicted secondary/tertiary structure and demonstrates classic Michaelis-Menten turnover behavior when the substrate is in 10-fold excess (Vmax/Km up to 1.60 x 108 min-1 M-1), which is comparable to RNase A. EhhRzs show cooperative titration with a Kd of 0.73 {+/-} 0.02 mM at cellular Mg2+ concentrations and a Hill coefficient of 1.73 {+/-} 0.07. The upstream EhhRz antisense flank (bound to a downstream substrate flank) interacts with stem-loop II, and examinations of different variants revealed that a U7 residue in the downstream flank of the substrate is not essential for enhanced activity. Under single-turnover conditions with substrate pre-annealed to enzyme, reaction rates exceeded 1,000 min-1. These findings show that RNA catalysis approaches the efficiency of the ribosome and suggests EhhRz in trans is a druggable nucleic acid therapeutic.

pharmacology and toxicology↗

bioRxiv2020

Contributions by N-terminal Domains to NMDA Receptor Currents

To investigate the role of the N-terminal domains (NTDs) in NMDA receptor signaling we used kinetic analyses of one-channel currents and compared the reaction mechanism of recombinant wild-type GluN1/GluN2A and GluN1/GluN2B receptors with those observed for NDT-lacking receptors. We found that truncated receptors maintained the fundamental gating mechanism characteristic of NMDA receptors, which includes a multi-state activation sequence, desensitization steps, and mode transitions. This result establishes that none of the functionally-defined NMDA receptor activation events require the NTD. Notably, receptors that lacked the entire NTD layer retained isoform-specific kinetics. Together with previous reports, these results demonstrate that the entire gating machinery of NMDA receptors resides within a core domain that contains the ligand-binding and the channel-forming transmembrane domains, whereas the NTD and C-terminal layers serve modulatory functions, exclusively.

biophysics↗