Abstract
Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge-loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge-loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.