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.

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