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

Abstract

Catalytic nucleic acids are regarded as potential therapeutic agents and biosensors. The catalytic activities of nucleic acid enzymes are usually investigated in dilute aqueous solutions, although the physical properties of the reaction environment inside living cells and that in the area proximal to the surface of biosensors in which they operate are quite different from those of pure water. The effect of the molecular environment is also an important focus of research aimed at improving and expanding nucleic acid function by addition of organic solvents to aqueous solutions. In this study, the catalytic activities of RNA and DNA enzymes (hammerhead ribozyme, 17E DNAzyme, R3C ribozyme, and 9DB1 DNAzyme) were investigated using 21 different mixed aqueous solutions comprising organic compounds. Kinetic measurements indicated that these enzymes can display enhanced catalytic activity in mixed solutions with respect to the solution containing no organic additives. Correlation analyses revealed that the turnover rate of the reaction catalyzed by hammerhead ribozyme increased in a medium with a lower dielectric constant than water, and the turnover rate of the reaction catalyzed by 17E DNAzyme increased in conditions that increased the strength of DNA interactions. On the other hand, R3C ribozyme and 9DB1 DNAzyme displayed no significant turnover activity, but their single-turnover rates increased in many mixed solutions. Our data provide insight into the activity of catalytic nucleic acids under various conditions that are applicable to the medical and technology fields, such as in living cells and in biosensors.

Functional Nucleic Acids Under Unusual Conditions

“…[82] In another study, Nakano et al investigated the catalytic activities of two variants (17E(S) and 17E(L)) of the 8-17 DNAzyme (Figure 6B) in organic co-solvents. [83] The investigated aqueous solutions had different dielectric constants, water activities, and viscosities from the mixing of different components, which included PEGs, ethylene glycol derivatives, small primary alcohols (methanol, ethanol and 1-propanol), amide compounds, aprotic compounds, and dextran. In the presence of 10 mM Mg 2 + , most of the organic co-solvents inhibited or inactivated the cleaving reaction of the DNAzymes.…”

“…Furthermore, there exists a positive correlation between the catalytic rate increment and the T m increase of the DNAzymes in the mixed solutions. [83] The improvement of the DNAzyme's thermal stability may induce an increase in the catalytic activity, when present in organic solvents/co-solvents.…”

CRISPR Cpf1 proteins: structure, function and implications for genome editing

“…The catalytic activities of RNA enzymes (ribozymes) are beneficial in the expansion of medical and biotechnological tools. Specifically, the hammerhead ribozyme catalyzed the site-specific cleavage of the phosphodiester bond of an RNA substrate [71]. Therefore, crRNAs ribozyme cassettes, which were driven by different promoters, were utilized in genome editing.…”

Bulky cations greatly increase the turnover of a native hammerhead ribozyme

“…25 Furthermore, cationic additives that stabilize RNA-RNA interactions are expected to disfavor the dissociation of cleaved products, making the rate of multiple-turnover cleavage slow down. There have been reports of several-to several tens-fold enhancement in the turnover of hammerhead ribozymes using RNA-binding proteins, 26 oligonucleotide facilitators, 27,28 locked nucleic acid nucleotides, 29 poly(ethylene glycol) (PEG), 30,31 and aqueous droplet-based microuidics. 32 In this report, we show further improvements of the turnover rate of a long hammerhead ribozyme construct by the use of tetraalkylammonium ions, with increases of greater than a hundredfold at optimal salt concentrations.…”