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  • Title: Efficient improvement of hammerhead ribozyme mediated cleavage of long substrates by oligonucleotide facilitators.
    Author: Jankowsky E, Schwenzer B.
    Journal: Biochemistry; 1996 Dec 03; 35(48):15313-21. PubMed ID: 8952482.
    Abstract:
    Hammerhead ribozymes were found to be not very efficient in cleaving long RNA substrates in trans. Oligonucleotide facilitators, capable of affecting hammerhead ribozymes by interacting with the substrate at the termini of the ribozyme, may improve this reaction. We determined in vitro the effects of 18 DNA and RNA oligonucleotide facilitators on three substrates containing 39, 452, and 942 nucleotides, respectively, by estimating the facilitator influences on association between ribozyme and substrate and on the cleavage step. The effects increase with the length of the substrates. With the 39mer substrate a maximal 4-fold enhancement of the ribozyme activity could be detected, the reaction with the 942mer substrate was accelerated up to 115-fold by facilitator addition. In long, structured substrates the facilitators have the potential to preform the substrate for the ribozyme attack. Due to this preforming effect, the rate of ribozyme-substrate association was increased as well as the rate of the cleavage step. 3'-End facilitators accelerate both of these rates, largely independent on the facilitator length. The rate of the cleavage step is raised as a result of a favorable activation energy gain by these facilitators. With all substrates, the 5'-end facilitators increase the association rate between ribozyme and substrate in dependence on their length. With the 39mer substrate the 5'-end facilitators decrease the rate of the cleavage step. With the long substrates 5'-end facilitators partially increase the rate of the cleavage step due to their preforming potential with these substrates. In some examples, combinations of several 5'-end and 3'-end facilitators provide an additional improvement over single facilitators in both the association between ribozyme and substrate and the cleavage step. Results suggest that even short facilitators may be efficient effectors enhancing hammerhead ribozyme mediated cleavage of long substrates.
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