Selection and analysis of RNA-cleaving deoxyribozymes and ribozymes: Application toward understanding enzyme structure and function.

摘要

Ribozymes and deoxyribozymes created by the process of in vitro selection allow researchers not only to study the capabilities of the nucleic acids themselves but also to probe some of the fundamental catalytic principles applicable to all enzymes. To explore the dynamic structures of RNA, allosteric hammerhead ribozymes were created that are activated more than 3,000 fold by theophylline binding. In addition, a variant allosteric ribozyme was evolved that uses subtle conformational changes to recognize 3-methylxanthine more readily than theophylline.; To explore the ability of DNA sequences to form catalytic structures, RNA-cleaving deoxyribozymes were selected in a complex, physiologically relevant buffer. At least six new active classes were identified in this population. Four classes were dissected to yield trans-cleaving motifs, the most common of which has a pseudo first order rate constant of ∼0.1 min−1 under cell-like conditions. These studies of allosteric ribozymes and RNA-cleaving deoxyribozymes provide additional evidence of the functional potential of nucleic acids. In a separate study, it was demonstrated that a self-cleaving deoxyribozyme sequence can be expressed in bacterial cells using a retroelement. In the future, RNA-cleaving deoxyribozymes might be similarly expressed in cells.; The RNA-cleaving deoxyribozymes were also examined under ideal reaction conditions along with several other RNA-cleaving deoxyribozymes and ribozymes. Of the 14 classes of nucleic acid enzymes studied, half attain a maximum rate constant of ∼1 min−1, compared to ∼80,000 min −1 for ribonuclease A. This commonly encountered ribozyme “speed limit” coincides with the theoretical maximum rate enhancement for complete activation of the 2′-hydroxyl nucleophile and optimum positioning of the atoms for nucleophilic displacement.; A review of the literature on the cleavage of RNA and its analogs reveals that in addition to nucleophile activation and geometric positioning, RNA transesterification is also be catalyzed by neutralizing overall negative charge and by assisting leaving group departure. The rate enhancements derived from non-enzymatic catalysis suggest that the ability of this reaction to be subjected to catalysis exceeds physical limits such as diffusion. This in turn supports the idea that, by similarly promoting the same chemical events, nucleic acid enzymes could match the speed of protein enzymes.