In vitro selection of a novel nuclease-resistant RNA phosphodiesterase

Background: Ribonucleotide-based enzymes (ribozymes) that cleave pathologic al RNAs are being developed as therapeutic agents. Chemical modification of the hammerhead ribozyme has produced nuclease-resistant catalysts that cle ave targeted mRNAs in cell culture and exhibit antitumor activity in animal s. Unfortunately, stabilizing modifications usually reduce the catalytic ra te in vitro. An alternative to rationally designed chemical modifications o f existing ribozymes is to identify novel motifs through in vitro selection of nuclease-stable sequence space. This approach is desirable because the catalysts can be optimized to function under simulated physiological condit ions. Results: Utilizing in vitro selection, we have identified a nuclease-stable phosphodiesterase that demonstrated optimal activity at simulated physiolo gical conditions. The initial library of 10(14) unique molecules contained 40 randomized nucleotides with all pyrimidines in a nuclease-stabilized 2'- deoxy-2'-amino format. The selection required trans-cleaving activity and b ase-pairing specificity towards a resin-bound RNA substrate. Initial select ive pressure was permissive, with a 30 min reaction time and 25 mM Mg2+. St ringency of selection pressure was gradually increased until final conditio ns of 1 mM Mg2+ and less than 1 min reaction times were achieved. The resul ting 61-mer catalyst required the 2'-amino substitutions at selected pyrimi dine positions and was stable in human serum (half-life of 16 h). Conclusions: We demonstrated that it is possible to identify completely nov el, nuclease-resistant ribozymes capable of trans-cleaving target RNAs at p hysiologically relevant Mg2+ concentrations. The new ribozyme motif has min imal substrate requirements, allowing for a wide range of potential RNA tar gets.