OLIGONUCLEOTIDE FACILITATORS ENABLE A HAMMERHEAD RIBOZYME TO CLEAVE LONG RNA SUBSTRATES WITH MULTIPLE-TURNOVER ACTIVITY

Trans-acting hammerhead ribozymes are usually efficient in cleaving sh ort RNA model substrates under both single-turnover and multiple-turno ver conditions. In contrast, when long RNAs are the substrates, the cl eavage efficiency of these ribozymes decreases, including a loss of mu ltiple-turnover activity in many cases. Since target substrates for po tential therapeutical purposes are mostly long RNAs, a multiple-turnov er cleavage of long RNAs would essentially increase the efficiency of hammerhead ribozymes. Therefore, we explored if oligonucleotide facili tators, capable of enhancing multiple-turnover activity with short sub strates, can also affect or cause multiple turnover with long substrat es. We examined the effects of 12-base and 24-base oligonucleotide fac ilitators on the multiple-turnover activity with substrates of differe nt length containing 39-, 452- and 942-base sequences of the human tis sue factor (HTF) mRNA. In the absence of facilitator, the ribozyme cle aved only the 39-base substrate with multiple-turnover activity, but n ot the long 452-base and 942-base substrates. However, facilitator add ition enabled the ribozyme to cleave even the 452-base and the 942-bas e substrates with multiple-turnover activity. All facilitators tested showed a remarkable activating effect with the long substrates. The da ta demonstrate that a hammerhead ribozyme which, by itself, can only a ct as a single-turnover catalyst with long substrates, can be switched by facilitators into a multiple-turnover catalyst. Thus, the inactiva tion of long target RNAs in multiple-turnover reactions may be achieve d by addition of oligonucleotide facilitators.