CAN HAMMERHEAD RIBOZYMES BE EFFICIENT TOOLS TO INACTIVATE GENE-FUNCTION

In order to improve hammerhead ribozyme efficiency and specificity, we have analyzed, both in vitro and in vivo, the activity of a series of ribozyme/substrate combinations that have the same target sequence bu t differ in the length of the ribozyme/substrate duplex or in their st ructure, i.e., the total length of the RNA. In vitro, we have found th at optimal kcat/Km (at 37 degrees C) is obtained when the ribozyme/sub strate duplex has a length of 12 bases, which according to the base co mposition represents a calculated free energy of binding of -16 kcal/m ol. We discuss the importance of this value for ribozyme specificity a nd present strategies that may improve it. Increasing the length of th e duplex from 14 to 17 bases (from -19 to -26 kcal/mol) produces a red uced ribozyme activity which is probably due to a slower rate of produ ct dissociation. In addition, inclusion of either the substrate or the ribozyme in a long transcript produces a reduction (10 fold) of the k cat/Km, probably because of a different accessibility of the target se quence. In vivo, the activity of the trans-acting ribozyme was extreme ly low and detected in only one case: with a ribozyme/substrate duplex length of 13 bases and with both ribozyme and substrate embedded in s hort RNAs expressed at a very high level. The similarity of the result s obtained in vitro and in vivo indicates that it is possible to use a n in vitro system to optimize ribozymes which are to be used in vivo. Satisfactory results were obtained in vivo only with cis-acting ribozy mes. Altogether these results suggest that the ribozyme/substrate hybr idization step is the limiting step in vivo and therefore it is not cl ear if ribozymes represent an improvement over antisense RNAs.