Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA

Abstract

Despite decades of effort, gene therapy( GT) has failed to deliver clinically significant anticancer treatment, owingi np art to lows electivity,l ow efficiency,a nd poor accessibility of folded RNAt argets.H erein, we propose to solve these common problems of GT agents by using aD NA nanotechnology approach. We designed ad eoxyribozymebased DNAm achine that can i) recognizet he sequence of ac ancer biomarker with high selectivity,i i) tightly bind as tructured fragment of ah ousekeeping gene mRNA, and iii)cleave it with efficiency greater than that of at raditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapya pproaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave ahousekeeping gene mRNAa fter being activated by ac ancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNAm achine could become ap rototype platform for anew type of anticancer GT agent.

Deoxyribozyme‐Based DNA Machines for Cancer Therapy

“…Such DNA constructions can be easily assembled by annealing the constituent DNA strands (e.g., T1, T2, and T3 in Scheme ). We found that the DNA machine can recognize both the marker sequence and targeted RNA fragment with high selectivity under near physiological conditions . The fundamental principles of superior selectivity of multicomponent in comparison with monolith hybridization probes was comprehensively discussed earlier .…”

“…Another factor contributing to decreased RCDZ efficiency is folded RNA structure, which can significantly reduce accessibility of targeted sequences . Indeed, when 8–17 was used to cleave a long folded RNA substrate, its activity dropped more than 100 times in comparison with cleavage rate of a 19‐nt RNA substrate .…”

“…The DNA machine cleaved folded RNA substrate about 16 times more efficiently than a traditionally designed RCDZ agent. This effect was shown to be achieved by the presence of additional RNA binding arms (arms 1 and 4 in Scheme ) …”

“…Unfortunately, we did not achieve depletion of the targeted housekeeping gene in cell cultures using this design presumably due to poor endosomal escape of the DNA nanomachine in cell cytoplasm. DNA‐machines were efficiently delivered in cells, but the targeted gene expression remained practically unchanged . Most of RCDZ constructs were retained in endosomes during lipofectamine‐assisted delivery.…”

Orthogonal Activation of RNA‐Cleaving DNAzymes in Live Cells by Reactive Oxygen Species

“…[7] Not only do these RNA-cleaving DNAzymes specifically recognize their target RNAs ubstrates through Watson-Crick base pairing,they are also highly specific with regards to the metalion cofactors,thus making them very useful tools in chemical biology for sensing metal ions [8] and regulating gene expression. [10] To activate DNAzyme activities by external stimuli, caged DNAzymes containing light-responsive modifications have been developed for light-dependent control of intracellular metal-ion biosensing or gene regulation. [10] To activate DNAzyme activities by external stimuli, caged DNAzymes containing light-responsive modifications have been developed for light-dependent control of intracellular metal-ion biosensing or gene regulation.…”