OLIGONUCLEOTIDE FACILITATORS MAY INHIBIT OR ACTIVATE A HAMMERHEAD RIBOZYME

Facilitators are oligonucleotides capable of affecting hammerhead ribo zyme activity by interacting with the substrate at the termini of the ribozyme, Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effec ts of 9mer and lamer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated, A ki netic model was developed which allows determination of the apparent d issociation constant of the ribozyme-substrate complex from single tur nover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding t o an additional stabilization energy of Delta Delta G = 1.7 kcal/mol w ith 3'-end facilitators, The cleavage rate constant was increased by 3 '-end facilitators and decreased by 5'-end facilitators, Values for k( m) were slightly lowered by all facilitators and k(cat) was increased by 3'-end facilitators and decreased by 5'-end facilitators in our sys tem. Generally the facilitator effects increased with the length of th e facilitators and RNA provided greater effects than DNA of the same s equence. Results suggest facilitator influences on several steps of th e hammerhead reaction, substrate association, cleavage and dissociatio n of products, Moreover, these effects are dependent in different mann ers on ribozyme and substrate concentration, This leads to the conclus ion that there is a concentration dependence whether activation or inh ibition is caused by facilitators. Conclusions are drawn with regard t o the design of hammerhead ribozyme facilitator sytems.