Cleavage of Structured RNAs Is Accelerated by High Affinity DNAzyme Agents

DNAzymes (Dz) have been suggested as sequence-specific agents for cleaving RNA for therapeutic purposes. This concept paper discusses the challenges of Dz 10-23 design to effciently cleave folded RNA substrates. Dz with traditionally designed RNA binding arms (T_m~37 °C) have low affinity to the folded RNA substrates, which limits the overall cleavage rate. The RNA cleavage can be facilitated using Dz with high-affinity arms. However, this strategy is efficient only for cleaving RNA into folded RNA products. The unfolded products inhibit multiple substrate turnover. In a more general approach, Dz should be equipped with additional RNA binding arms to achieve tight RNA binding. This can be accomplished by bivalent and multivalent Dz constructs that have multiple catalytic cores. In all cases, high selectivity toward single nucleotide variations can be achieved in addition to multiple turnovers. The presence of RNase H, which plays a role in the antisense effect of oligonucleotide gene therapy agents, stabilizes the Dz:RNA complex and reduces its selectivity but significantly increases RNA cleavage efficiency. This work proposes changes in the algorithms of Dz design, which can help in constructing potent Dz agents for RNA inhibition both in cell cultures and in vivo. The concept article is supplemented with a quiz, which tests knowledge of the main concepts discussed in this work.