the cleavage of multiple substrate molecules. This strategy was important in that it allowed investigators to (i) apply biochemical methods for enzymatic analysis, (ii) conduct experiments to identify essential structural elements of the ribozyme-substrate complex, and (iii) develop engineered ribozymes that have been used for biomedical applications, including preventing the replication of pathogenic viruses, and the study of the function of individual genes. Reaction chemistry In common with several other ribozymes and protein ribonucleases, the cleavage reaction of the hairpin ribozyme generates RNA fragments with termini consisting of a 2',3'-cyclic phosphate and a 5'-hydroxyl group. The ligation reaction appears to be a simple reversal of cleavage, i.e. covalent joining of RNA fragments ending with a 2',3'-cyclic phosphate and a 5'-hydroxyl group to generate the ordinary 3'-5' phosphodiester linkage used in both RNA and DNA. Studies of this reaction in multiple ribozymes have served to establish that the reaction chemistry (catalytic mechanism) is an endogenous property of the RNA molecule itself and is not mediated by metal ions, as is true for some protein enzymes and some other ribozymes. Moreover, cleavage activity is still observed when Mg2+ is replaced by [Co(NH3)6]3+. Co3+ binds NH3 so tightly in solution that NH3 does not dissociate to any appreciable extent, and therefore does not become protonated. This suggests there is no metal-catalyzed proton transfer or direct coordination to the RNA, but instead metals are only required for folding. Furthermore, in crystal structures of a ribozyme-inhibitor complex and a transition state mimic, it was shown that the three-dimensional architecture splays apart A-1 and G+1, positioning the 2'-OH of A-1 for an in-line nucleophilic attack on the scissile phosphate linkage. Additionally, G8, A38, and A9 have been suggested to play roles in the catalysis by deprotonating the 2'-OH of A-1, stabilizing the developing negative charge of the pentacoordinate phosphate oxygens, and protonating the 5'-O leaving group of G+1. Structure The minimal hairpin ribozyme-substrate complex folds into a secondary structure that includes two domains, each consisting of two short base paired helices separated by an internal loop. Domain A (helix 1 – loop A – helix 2) contains the substrate and the primary substrate-recognition region of the ribozyme. Domain B (helix 3 – loop B – helix 4) is larger and contains the primary catalytic determinants of the ribozyme. The two domains are covalently joined via a phosphodiester linkage that connects helix 2 to helix 3. These