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Nature Protocols

Nature Protocols

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A bioswitchable delivery system for microRNA therapeutics based on a tetrahedral DNA nanostructure

Published:30 August 2024 DOI: 10.1038/s41596-024-01050-7 PMID: 39215132
Songhang Li, Taoran Tian, Tao Zhang, Yunfeng Lin, Xiaoxiao Cai

Abstract

As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use. This protocol extension details two approaches to creating a bioswitchable delivery system for microRNA therapeutics, based on a tetrahedral DNA nanostructure with a ribonuclease H-sensitive sequence as a bioswitchable apparatus for the controlled release of cargo.

Substances (4)

Materials
Procduct Name CAS Molecular Formula Supplier Price
Chlorpromazine Sulfone Hydrochloride 18683-73-3 C17H20Cl2N2O2S 12 suppliers Inquiry
Chlorpromazine Sulfone Hydrochloride 18683-73-3 C17H20Cl2N2O2S 12 suppliers Inquiry
Chlorpromazine Sulfone Hydrochloride 18683-73-3 C17H20Cl2N2O2S 12 suppliers Inquiry
Chlorpromazine Sulfone Hydrochloride 18683-73-3 C17H20Cl2N2O2S 12 suppliers Inquiry

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