OLIGONUCLEOTIDES 15:303–309 (2005) © Mary Ann Liebert, Inc. Brief Communication Hairpin Ribozyme-Catalyzed Ligation in Water-Alcohol Solutions ALEXANDER V. VLASSOV, 1,2 BRIAN H. JOHNSTON, 1,3 and SERGEI A. KAZAKOV 1 ABSTRACT The hairpin ribozyme (HPR) is a naturally existing RNA that catalyzes site-specific RNA cleavage and ligation. At 37°C and in the presence of divalent metal ions (M 2 ), the HPR efficiently cleaves RNA substrates in trans. Here, we show that the HPR can catalyze efficient M 2 -independent liga- tion in trans in aqueous solutions containing any of several alcohols, including methanol, ethanol, and isopropanol, and millimolar concentrations of monovalent cations. Ligation proceeds most effi- ciently in 60% isopropanol at 37°C, whereas the reverse (cleavage) reaction is negligible under these conditions. We suggest that dehydration of the RNA is the key factor promoting HPR activity in wa- ter-alcohol solutions. Alcohol-induced ribozyme ligation may have practical applications. INTRODUCTION T HE HAIRPIN RIBOZYME (HPR) is a small catalytic RNA derived from the minus strand of the tobacco ringspot virus satellite RNA, which also harbors a hammerhead ri- bozyme in its plus strand (Buzayan et al., 1986a,b, 1988; van Tol et al., 1990). The presumed biological function of its catalytic activity is to generate unit-length genomes from products of rolling circle replication through site-spe- cific self-cleavage and circularization (Buzayan et al., 1995; Chay et al., 1997). The catalytic core of the ri- bozyme catalyzes both cleavage and ligation of RNA sub- strates, with cleavage yielding 5'-hydroxyl and 2',3'- cyclic phosphate termini. The equilibrium between cleavage and ligation depends on salt concentration, tem- perature, and whether the reaction is cis or trans. Under physiological conditions (37°C and the presence of Mg 2+ ), the HPR efficiently cleaves substrates in trans, and the re- sulting products readily dissociate from the ribozyme. In contrast, when RNA-RNA interactions are stabilized by reduced temperature or increased salt concentration, re- lease of cleavage products is slowed, and the equilibrium shifts toward ligation (Nesbitt et al., 1999; Fedor, 2000). The hairpin ribozyme consist of two structural do- mains, each containing an internal loop and two short he- lices. The active site comprises multiple conserved func- tional groups located at and around the interface of the two domains in the docked, active enzyme-substrate complex (Rupert and Ferre-D’Amare, 2001; Hampel, 1998; Burke, 2001; Strobel and Ryder, 2001). Detailed investigations of the HPR reaction have shown that diva- lent metal ions (M 2+ ) have a structural but not catalytic role, as they can be substituted by cobalt (III) hexam- mine, spermidine, or high concentrations of monovalent cations, such as Na + (Nesbitt et al., 1997; Nesbitt et al., 1999; Chowrira et al., 1993; Young et al., 1997; Hampel and Cowan, 1997; Earnshaw and Gait, 1998; Murray et al., 1998). Crystallographic studies have provided no ev- 1 SomaGenics, Inc., Santa Cruz, CA 95060. 2 Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia. 3 Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305. 303