Comparison of Antisense Oligonucleotides, DNAzymes, and Their Bivalent Forms in RNAse H Dependent Cleavage of Folded RNA

Abstract

Objective: Antisense oligonucleotide (ASO) and DNAzyme (Dz) agents have been suggested for suppression specific mRNA in vivo. It was reported that Dz agents are more selective in recognition their targets than ASO. However, Dz failed to produce therapeutically significant drugs due to their low efficiency. Here we compared the performance of the two types of agents in cleavage a folded RNA fragment in reconstituted system containing RNase H. Methods: Thermodynamic parameters and predicted 2D-structure of the RNA fragments were obtained using RNAFold application in UNAFold web server. To perform the experiments with RNA cleavage by enzymes we used commercial Mg₂⁺-containing reaction 10X RNAse H Buffer (200 mM Tris-HCl (pH 8.3), 150 mM DTT, 1 M KCl, 45 mM MgCl₂). Results of RNA cleavage were visualized with 20% denaturing PAGE (AA : BA (29 : 1), 7 M Urea, 1× TBE) running 150 min at 80 V. Results and Discussion: Individual ASO agents were ~3­–6 times more active in RNA cleaving than the equivalent Dz agents. Both agents demonstrated low selectivity toward RNA cleavage. Combining two Dz complementary to the abutting position of the RNA target bivalent (BDD) agent improved RNA cleavage to the level of the most active ASO agent. Conclusions: Comparing the obtained data with published earlier for RNase H—free system suggests that RNase H stabilizes the Dz:RNA complex and reduces its selectivity but significantly increase RNA cleavage efficiency. The contribution of RNase H effects on the performance of Dz agents in cell culture and in vivo should be taken in account.