Hammerhead Ribozymes Against Virus and Viroid RNAs Alberto Carbonell, Ricardo Flores, and Selma Gago Contents 1 A Historical Overview: Hammerhead Ribozymes in Their Natural Context ................................................................... 412 2 Manipulating Cis-Acting Hammerheads to Act in Trans ................................. 414 3 A Critical Issue: Colocalization of Ribozyme and Substrate ............................. 416 4 An Unanticipated Participant: Interactions Between Peripheral Loops of Natural Hammerheads Greatly Increase Their Self-Cleavage Activity ........................... 417 5 A New Generation of Trans-Acting Hammerheads Operating In Vitro and In Vivo at Physiological Concentrations of Magnesium ........ 419 6 Trans-Cleavage In Vitro of Short RNA Substrates by Discontinuous and Extended Hammerheads ........................................... 420 7 Trans-Cleavage In Vitro of a Highly Structured RNA by Discontinuous and Extended Hammerheads ........................................... 421 8 Trans-Cleavage In Vivo of a Viroid RNA by an Extended PLMVd-Derived Hammerhead ........................................... 422 9 Concluding Remarks and Outlooks ........................................................ 424 References ....................................................................................... 425 Abstract The hammerhead ribozyme, a small catalytic motif that promotes self- cleavage of the RNAs in which it is found naturally embedded, can be manipulated to recognize and cleave specifically in trans other RNAs in the presence of Mg 2+ . To be really effective, hammerheads need to operate at the low concentration of Mg 2+ existing in vivo. Evidence has been gathered along the last years showing that tertiary stabilizing motifs (TSMs), particularly interactions between peripheral loops, are critical for the catalytic activity of hammerheads at physiological levels of Mg 2+ . These TSMs, in two alternative formats, have been incorporated into a A. Carbonell • R. Flores (*) • S. Gago (*) Instituto de Biologı ´a Molecular y Celular de Plantas (UPV-CSIC), Universidad Polite ´cnica de Valencia, Avenida de los Naranjos, Valencia 46022, Spain e-mail: rflores@ibmcp.upv.es; selmag@ibmcp.upv.es V.A. Erdmann and J. Barciszewski (eds.), From Nucleic Acids Sequences to Molecular Medicine, RNA Technologies, DOI 10.1007/978-3-642-27426-8_16, # Springer-Verlag Berlin Heidelberg 2012 411