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Advances in the DNA nanotechnology have enabled the fabrication of DNA-based hydrogels with precisely controlled structures and tunable mechanical and biological properties. Compared to DNA hydrogel, preparation of RNA-based hydrogel remains challenging due to the inherent instability of naked RNA. To overcome these limitations, we fabricated a DNA-RNA hybrid hydrogel via stepwise dual enzymatic polymerization. Multimeric short hairpin RNAs (shRNAs) were hybridized with functional DNA aptamers for targeting and mechanical properties of the hydrogel. The obtained DNA-RNA hybrid hydrogel was ultrasoft, robust, and injectable hence reconfigurable into any confined structures. As a model system, the hydrogel was able to mimic microtubule structures under physiological conditions and designed to release the functional small interfering RNA (siRNA)-aptamer complex (SAC) sequentially. In addition, we encoded restriction enzyme-responsive sites in DNA-RNA hybrid hydrogel to boost the release of SAC. This novel strategy provides an excellent platform for systematic RNA delivery through double-controlled release, SAC release from hydrogel, and subsequent release of siRNA from the SAC, which has promising potential in RNA therapy.

Control of the release properties of drugs has been considered a key factor in the development of drug delivery systems (DDSs). However, drug delivery has limitations including cytotoxicity, low loading efficiency, and burst release. To overcome these challenges, nano or micro-particles have been suggested as carrier systems to deliver chemical drugs. Herein, nano-sized DNA particles (DNAp) were manufactured to deliver netropsin, which is known to bind to DNA minor grooves. The rationally designed particles with exposed rich minor grooves were prepared by DNAp synthesis via rolling circle amplification (RCA). DNAp could load large quantities of netropsin in its minor grooves. An analytical method was also developed for the quantification of netropsin binding to DNAp by UV&ndash, visible spectrometry. Moreover, controlled release of netropsin was achieved by forming a layer of Ca2+ on the DNAp (CaDNAp). As a proof of concept, the sustained release of netropsin by CaDNAp highlights the potential of the DNAp-based delivery approach.

A novel fabrication method for RNA particles (RPs) was developed based on enzymatic polymerization, and the size of the RPs was controlled intentionally by adjusting the RNA polymerase concentration for a variety of potential applications.