Investigation of the pKa of the Nucleophilic O2' of the Hairpin Ribozyme.

Small ribozymes cleave their RNA phosphodiester backbone by catalyzing a transphosphorylation reaction wherein a specific O2' functions as the nucleophile. While deprotonation of this alcohol through its acidification would increase its nucleophilicity, little is known about the pKa of this O2' in small ribozymes, in part because high pKa's are not readily accessible experimentally. Herein, we turn to molecular dynamics to calculate the pKa of the nucleophilic O2' in the hairpin ribozyme and to study interactions within the active site that may impact its value. We estimate the pKa of the nucleophilic O2' in the wild-type hairpin ribozyme to be 18.5 ± 0.8, which is higher than the reference compound, and identify a correlation between proper positioning of the O2' for nucleophilic attack and elevation of its pKa. We find that monovalent ions may play a role in depression of the O2' pKa, while the exocyclic amine appears to be important for organizing the ribozyme active site. Overall, this study suggests that the pKa of the O2' is raised in the ground state and lowers during the course of the reaction owing to positioning and metal ion interactions.

Template-directed RNA polymerization and enhanced ribozyme catalysis inside membraneless compartments formed by coacervates.

Membraneless compartments, such as complex coacervates, have been hypothesized as plausible prebiotic micro-compartments due to their ability to sequester RNA; however, their compatibility with essential RNA World chemistries is unclear. We show that such compartments can enhance key prebiotically-relevant RNA chemistries. We demonstrate that template-directed RNA polymerization is sensitive to polycation identity, with polydiallyldimethylammonium chloride (PDAC) outperforming poly(allylamine), poly(lysine), and poly(arginine) in polycation/RNA coacervates. Differences in RNA diffusion rates between PDAC/RNA and oligoarginine/RNA coacervates imply distinct biophysical environments. Template-directed RNA polymerization is relatively insensitive to Mg2+ concentration when performed in PDAC/RNA coacervates as compared to buffer, even enabling partial rescue of the reaction in the absence of magnesium. Finally, we show enhanced activities of multiple nucleic acid enzymes including two ribozymes and a deoxyribozyme, underscoring the generality of this approach, in which functional nucleic acids like aptamers and ribozymes, and in some cases key cosolutes localize within the coacervate microenvironments.

Elucidation of Catalytic Strategies of Small Nucleolytic Ribozymes From Comparative Analysis of Active Sites.

A number of small, self-cleaving ribozyme classes have been identified including the hammerhead, hairpin, hepatitis delta virus (HDV), Varkud satellite (VS), glmS, twister, hatchet, pistol, and twister sister ribozymes. Within the active sites of these ribozymes, myriad functional groups contribute to catalysis. There has been extensive structure-function analysis of individual ribozymes, but the extent to which catalytic devices are shared across different ribozyme classes is unclear. As such, emergent catalytic principles for ribozymes may await discovery. Identification of conserved catalytic devices can deepen our understanding of RNA catalysis specifically and of enzymic catalysis generally. To probe similarities and differences amongst ribozyme classes, active sites from more than 80 high-resolution crystal structures of self-cleaving ribozymes were compared computationally. We identify commonalities amongst ribozyme classes pertaining to four classic catalytic devices: deprotonation of the 2'OH nucleophile (γ), neutralization of the non-bridging oxygens of the scissile phosphate (ß), neutralization of the O5' leaving group (δ), and in-line nucleophilic attack (α). In addition, we uncover conservation of two catalytic devices, each of which centers on the activation of the 2'OH nucleophile by a guanine: one to acidify the 2'OH by hydrogen bond donation to it (γ') and one to acidify the 2'OH by releasing it from non-productive interactions by competitive hydrogen bonding (γ''). Our findings reveal that the amidine functionalities of G, A, and C are especially important for these strategies, and help explain absence of U at ribozyme active sites. The identified γ' and γ'' catalytic strategies help unify the catalytic strategies shared amongst catalytic RNAs and may be important for large ribozymes, as well as protein enzymes that act on nucleic acids.