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

Shu-ichi Nakano, Masao Horita, Miku Kobayashi, Naoki Sugimoto
2017 Catalysts  
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
more » ... 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. Catalysts 2017, 7, 355 2 of 14 process to take place [14] [15] [16] [17] [18] [19] . Studies using simple aqueous solutions have provided detailed information regarding the catalytic activity of many types of ribozymes and DNAzymes and the metal ion conditions necessary for catalysis. However, the environment inside living cells and that in the area proximal to the surface of biosensors, where nucleic acid enzymes exert their therapeutic and diagnostic activity, are quite different from the aqueous solutions commonly used for in vitro evaluation of catalytic activity. In these particular cases, nucleic acid interactions occur under sterically-restricted conditions that alter the diffusion, dynamics, and effective concentration of molecules. Moreover, intracellular water and that in the vicinity of the surface of a biosensor have different physical properties (e.g., dielectric constant and water activity) from pure water. The characteristics of these environments are known to affect the thermodynamics and kinetics of nucleic acid interactions [20] . The molecular environment of an aqueous solution is also substantially affected by addition of organic solvents. Solvents like ethanol, methanol, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide (DMF) have been employed to improve oligonucleotide function in the polymerase chain reaction [21, 22] , molecular beacon assays [23], DNA strand exchanges [24] , and to assist DNAzyme functionalization [25] [26] [27] [28] . The presence of organic solvents changes the physical properties of a solution, and the organic molecules themselves may interact with nucleic acids. Understanding the effects of the molecular environment on the activity of nucleic acid enzymes is helpful for the use of the enzymes in practical applications. Aqueous solutions containing water-soluble organic compounds at a concentration of a few to several tenths of a percent have been used to investigate how environmental factors influence nucleic acid behavior. These types of studies have shown that the solution composition influences base-pair interaction energy, RNA folding, hydration status, and nucleic acid-metal ion interactions [20, 29] . Polyethylene glycol (PEG) is a commonly used reagent in these studies. This polymer is water-soluble, and highly purified PEGs of different molecular weights are commercially available. Notably, low-molecular-weight PEGs exist as liquids and high-molecular-weight PEGs as solids at room temperature. Mixed solutions, PEG-containing solutions in many cases, have been reported to increase the rate of RNA cleavage by ribozymes, such as the group I intron ribozyme, human delta virus-like ribozyme, hairpin ribozyme, leadzyme, and hammerhead ribozyme (see the references in [30] ). Several reports also exist on the catalytic activity of DNAzymes; in these cases, the reaction rates in mixed solutions containing ethanol or other organic solvents were higher than those in the solution containing no organic solvents [26] [27] [28] . These studies suggest the possibility of using mixed solutions to enhance the activity of nucleic acid enzymes. However, the effects of solution composition on nucleic acid behavior have been explained by a variety of different environmental factors: excluded volume interactions (molecular crowding environment), reduced water activity (dehydrating environment), and reduced dielectric constant (stronger electrostatic interaction environment) [31] . This uncertainty is partly the result of the limited use of mixed solutions to discuss the major environmental factor in these studies. In addition, most of the reported studies evaluated the catalytic activity under single-turnover conditions. In contrast to the case of protein enzymes, the turnover of nucleic acid enzymes is intrinsically difficult to achieve, because of the stable base-pairing interactions that are established with substrate strands and because of the slow kinetics of Watson-Crick base-pair opening. Methods for enhancing the turnover number would be useful to increase the effectiveness of technologies based on the catalytic activity of nucleic acid enzymes. The hypothesis can be made that organic solvents that reduce the strength of base-pairing interactions facilitate the release of reaction products and thus increase the catalytic turnover of the reactions catalyzed by ribozymes and DNAzymes. Here, we investigated the activities of several types of RNA-cleaving and RNA-ligating nucleic acid enzymes under single-and multiple-turnover conditions, using 21 different mixed aqueous solutions containing PEGs with an average molecular weight ranging from 2 × 10 4 to 2 × 10 2 , ethylene glycol derivatives (ethylene glycol (EG), glycerol (Glyc), 1,3-propanediol (PDO), 2-methoxyethanol (MME), and 1,2-dimethoxyethane (DME)), small primary alcohols (methanol (MeOH), ethanol (EtOH), and 1-propanol (PrOH)), amide compounds (urea, formamide (FA), DMF, and acetamide (AcAm)), aprotic compounds (acetonitrile (AcCN), DMSO,
doi:10.3390/catal7120355 fatcat:5qqaddowizgz3ag3ktshu3rz2m