Entropically Driven Enhancement of Cleavage Activity of a DNA-Armed Hammerhead Ribozyme:  Mechanism of Action of Hammerhead Ribozymes

Recent X-ray structures of hammerhead ribozymes demanded rearrangement of the scissile phosphate backbone into a conformation suitable for in-line attack by the 2‘-oxygen in the transition state or prior to it. As part of our efforts to detect the putative conformational change and also to characterize the transition state structure of a hammerhead ribozyme-catalyzed reaction, kinetic and thermodynamic parameters of a chimeric RNA/DNA ribozyme were compared with the corresponding values of an all-RNA ribozyme. Kinetic analysis revealed that (i) DNA portions enhanced k cat in the entire pH region between 6 and 9, and at all temperatures between 0 and 60 °C; (ii) the origin of the acceleration in k cat by the chimeric DNA-armed ribozyme was accomplished by two distinctly different phenomena: at the temperatures below 25 °C, the enhancement originated from the acceleration of dissociation of the products from the reaction complex ( k diss ), whereas at temperatures 25−45 °C, the DNA-portions enhanced the chemical cleavage ( k cleav ) of the ribozyme-catalyzed reaction; (iii) under any reaction conditions used in this study, a conformational change in the ribozyme·substrate complex did not become the rate-limiting step; and (iv) importantly, the enhancement of cleavage activity by the chimeric DNA-armed ribozyme was driven entropically, a result that cannot simply be explained by the recently proposed “fraying model” with open and closed ribozyme·substrate helixes (Hertel et al. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8497−8502).