Facilitation of RNA Enzyme Activity in the Molecular Crowding Media of Cosolutes

Abstract

Short RNA sequences exhibiting the activity of a target RNA cleavage are promising for cellular gene regulation and biosensor research, but the reaction media different from an aqueous solution may cause unanticipated molecular interactions and properties. In this study, we investigated the molecular crowding effects arising from steric crowding and altered solvent properties on the hammerhead ribozyme activity using water-soluble neutral cosolutes. Poly(ethylene glycol) (PEG) and other cosolutes at 20 wt % increased the RNA hydrolysis rate by a factor of 2.0-6.6 at 10 mM MgCl(2) and much more at lower MgCl(2) concentrations. Remarkably, although the cosolutes decreased the stability of the ribozyme stem helices, the thermal inactivation temperature of the ribozyme was significantly raised, resulting in a higher reaction rate, up to 270 times at 50 degrees C. More significantly, PEG decreased the metal ion concentration to perform the reaction even with a limiting Mg(2+) or Na(+) concentration, facilitated the catalytic turnover, and activated a catalytically less active ribozyme sequence. These observations agreed that the cosolutes acted as an osmolyte stabilizing the water-release reaction of the RNA tertiary folding but destabilizing the water-uptake reaction of Watson-Crick base pairing. The opposite cosolute effect on the stabilities of RNA secondary and tertiary structures, which is fundamentally different from a protein folding, suggests how RNA stabilizes a tertiary structure and enhances the catalytic activity in molecular crowding media.

Catalytic Activities of Ribozymes and DNAzymes in Water and Mixed Aqueous Media

“…The hammerhead ribozyme, ligase ribozyme, and substrate RNAs having a 5 -triphosphate group were prepared by in vitro transcription using T7 RNA polymerase and purified by polyacrylamide gel electrophoresis, as described previously [34]. Other RNAs, DNAs, and a DNA-RNA chimeric strand, purified by high performance liquid chromatography, were purchased from Hokkaido System Science (Sapporo, Japan) or Fasmac (Atsugi, Japan).…”

“…A simple interpretation of this correlation is that the transition state is electrostatically stabilized in less-polar media. However, our previous study identified an apparent increase in Mg 2+ -binding affinity to HH(S) in mixed solutions with a low dielectric constant [34,35]. The substrate cleavage catalyzed by the hammerhead ribozyme requires the involvement of several Mg 2+ ions, and at least one Mg 2+ ion binds to the site with highly negative electrostatic potential located near the scissile phosphate of the substrate [49].…”

“…Both ribozymes exhibited high RNA cleavage activity under single-turnover condition in the presence of 10 mM MgCl 2 , but the kinetic traces associated with substrate cleavage differed for the two systems. The time course of substrate cleavage by HH(S) was closely approximated by a single-exponential function [34], whereas HH(L) showed a biphasic kinetic behavior, whereby~10% of the substrate was cleaved at a fast rate (within less than a few seconds) and~70% of it was cleaved at a slow rate (taking a few tens of minutes), so that 80% substrate conversion was achieved after completion of the slow phase of the reaction (Figure 1b). This observation is consistent with a previous report that an extended ribozyme having a similar sequence to HH(L) showed a biphasic kinetic behavior [32].…”

“…We have previously reported the enhancement of the rate of cleavage catalyzed by HH(S) in mixed solutions containing PEG8000 or PEG200 [34,35]. When HH(L) was investigated, the effects of PEG on the fast and slow phases of the reaction were different: the amplitude of the fast phase conversion increased 9.8-fold with PEG8000 and 5.3-fold with PEG200, although the rates were too fast for the rate constants to be determined.…”

Characterization of Metal Ion-Nucleic Acid Interactions in Solution

“…investigated the stability and catalytic activity of a minimal hammerhead ribozyme with PEG8000 as co-solute [333,334]. The authors find that large neutral co-solutes stabilize the tertiary structure and enhance catalytic rates especially at low Mg 2+ concentrations.…”