Kinetic and binding analysis of the catalytic involvement of ribose moieties of a trans-acting δ ribozyme

We have identified ribose 2'-hydroxyl groups (2'-OHs) that are critical for the activity of a trans-cleaving S ribozyme derived from the antigenomic strand of the hepatitis 5 virus. Initially, an RNA-DNA mixed ribozyme composed of 26 deoxyribo- (specifically the nucleotides forming the P2 stem and the P4 stem-loop) and 31 ribonucleotides (those forming the catalytic center) was engineered. This mixed ribozyme catalyzed the cleavage of a small substrate with kinetic parameters virtually identical to those of the all-RNA ribozyme. The further substitution of deoxyribose for ribose residues permitted us to investigate the contribution of all 2'-OHs to catalysis. Determination of the kinetic parameters for the cleavage reaction of the resulting ribozymes revealed (i) 10 2'-OH groups appear to be important in supporting the formation of several hydrogen bonds within the catalytic core, 00 none of the important 2'-OHs seem to coordinate a magnesium cation, and (iii) 1 of the tested RNA-DNA mixed polymers appeared to stabilize the ribozyme-substrate transition-state complex, resulting in an improvement over the all-RNA counterpart. The contribution of the 2'-OHs to the catalytic mechanism is discussed, and differences with the crystal structure of a genomic 5 self-cleaved product are explained. Clearly, the 2'-OHs are essential components of the network of interactions involved in the formation of the catalytic center of the 5 ribozyme.