Cleaving Folded RNA with DNAzyme Agents

Abstract

Cleavage of biological mRNA by DNAzymes (Dz) has been proposed as a variation of oligonucleotide gene therapy (OGT). The design of Dz‐based OGT agents includes computational prediction of two RNA‐binding arms with low affinity (melting temperatures (Tm) close to the reaction temperature of 37oC) to avoid product inhibition and maintain high specificity. However, RNA cleavage might be limited by the RNA binding step especially if the RNA is folded in secondary structures. This calls for the need of two high‐affinity RNA‐binding arms. In this study, we optimized 10‐23 Dz‐based OGT agents for cleavage of three RNA targets with different folding energies under multiple turnover conditions in 2 mM Mg2+ at 37°C. Unexpectedly, one optimized Dz had each RNA‐binding arms with a Tm ≥ 50°C, without suffering from product inhibition or low selectivity. This phenomenon was explained by the folding of the RNA cleavage products into stable secondary structures. This result suggests that Dz with long (high affinity) RNA‐binding arms should not be excluded from the candidate pool for OGT agents. Rather, analysis of the cleavage products’ folding should be included in Dz selection algorithms. The Dz optimization workflow should include testing with folded rather than linear RNA substrates.

Multivalent DNAzyme agents for cleaving folded RNA

“…This binding step might be a limiting stage for suppression of natural mRNA in vivo . We, therefore, strongly advocate for using long model RNA substrates in optimization of Dz agents, as was expressed by us earlier ( 36 ) and supported recently by Zhang et al. ( 46 ).…”

“…It was shown earlier that the increase in the affinity of RNA-binding arms in Dz agents increases the cleavage efficiency of folded RNA substrates ( 11–14 ). However, none of the prior studies with one recent exception ( 36 ) have optimized the structures of Dz agents for the cleavage of folded RNA. We reasoned here that a fair comparison of the cleavage efficiency and selectivity of the bivalent agents should be done with optimal rather than suboptimal Dz of the traditional design.…”

“…We, therefore, targeted RNA-60 (folding Δ G of −6.30 kcal/mol, Figure 2C ) of a sequence unrelated to RNA-58. Cleaving agents 60_Dz1 and 60_Dz2 were designed according to the conventional rules ( 11 , 12 , 36 ) and then were linked together to form 60_BDD1, 2 and 3 constructions.…”

Cleaving Folded RNA by Multifunctional DNAzyme Nanomachines

“…We compared the parameters with that of Dz of conventional design optimized for the same RNA targets earlier. [17] Folded RNA Can Be Efficiently Cleaved by Long-Armed Dz of Conventional Design if the Cleavage Products are Folded in Stable Secondary Structures RNA-1 had the most stable global and local secondary structures of the three substrates used (Figure 1). Surprisingly, DNM approach did not improve the rate of RNA-1 cleavage.…”

“…We, therefore, concluded that fragments of biological RNA that produce folded RNA fragments might be considered as promising targets for Dz agents. [17] In this case, however, Dz of conventional designs need to be equipped by long RNA binding arms, which may jeopardize their specificity when exposed to human transcriptome. DNM approach, therefore, may become a strategy to achieve efficient and selective cleavage in such challenging cases.…”