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Expert laboratory scientists describe in detail their methodologies for constructing ribozymes designed to elucidate the role of specific genes as key routes to the development of novel therapies for a wide variety of diseases. The authors review the many sites targeted with ribozymes in various diseases and provide specific accounts of the practical techniques required for the proper use of ribozymes in these systems. Their cutting-edge protocols demonstrate how to achieve ribozyme expression in distinct cellular systems, the preparation and use of high-efficiency ribozyme DNA or RNA delivery, and the studies required to prove the efficacy of ribozyme-mediated inhibition. Therapeutic Applications of Ribozymes contains all the technical support needed to make ribozyme technology a widely used and significant methodology.
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Small Efficient Hammerhead Ribozymes Philip Hendry, Trevor J. Lockett, and Maxine J. McCall
1. Introduction The hammerhead ribozyme was discovered as a self-cleavmg RNA molecule in certain plant vtroids and satellite RNAs (1). Shortly after its conserved features were defined (2,3), the hammerhead was shown to be able to act as a true enzyme, cleaving multiple substrates m a bimolecular reaction (4). The self-cleaving hammerhead can be divided in a number of ways into two, or even three, separate strands (45). The most useful form has almost all of the conserved nucleotides on the ribozyme strand, leaving minimal sequence requirements in the substrate strand. To be cleavable the substrate must possessthe sequence 5’ UH (H is C, U, or A), where cleavage occurs to the 3’ stde of H (6) (Fig. 1). This particular configuration has been the paradigm for hammerhead ribozyme design since 1988. Here we describe variations on this basic design, with the constant theme being to minimize the size of the ribozyme. The advantages of minimizing the