Controlled therapeutic nucleic acid delivery: from smart DNA- circuits to out-of-equilibrium systems Aman Ishaqat 1,2 , Xiaofeng Zhang 2 , Qing Liu 3 , Lifei Zheng 3 , Andreas Herrmann 1,2 1 DWI-Leibniz Institute for Interactive Materials, Forckenbeckstr. 50, 52074 Aachen, Germany. 2 Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Worringerweg 2, 52074 Aachen, Germany. 3 Wenzhou Institute, University of Chinese Academy of Sciences,325001, Wenzhou, China. DNA circuits are artificial nanodevices composed of short DNA strands, designed to perform various computational and signal processing tasks 1 . These circuits have potential applications in nanotechnology and biotechnology and can be used to regulate drug delivery by enabling kinetic control over drug release 2-5 . In this work, an enzyme-based DNA circuit was designed for delivering pharmacologically active CpG oligodeoxynucleotides (ODNs) that stimulate the immune system 6,7 . Our circuit employs a complementary DNA strand (cDNA) to temporarily deactivate the biological function of CpG ODNs via hybridization. T7 exonuclease then activates the circuit by hydrolyzing the cDNA, releasing active CpG ODN. We investigated the influence of several factors on the kinetic profile and temporal behaviour of the circuit. These include the design of the cDNA strand and the concentration of the DNA duplex and T7 exonuclease. The DNA-circuit’s in-vitro activation resulted in toll-like receptor 9 (TLR9) stimulation in HEK-engineered cell line, as well as tumor necrosis factor- alpha (TNF-α) release by J774A.1 macrophages. By programming the DNA-circuit to control the release of CpG ODN, we achieved an altered pharmacological profile with acute and potent immunostimulation, in comparison to a system without controlled CpG ODN release, which exhibited a slow and delayed response. The programmable DNA-circuit can be transformed into a dissipative system that operates out-of-equilibrium 8 . The repetitive addition of the cDNA strand results in initiating the deactivation of the CpG ODN and results in a renewable DNA-circuit coupled with energy dissipation. We achieved multiple loading/release cycles Our findings demonstrate the potential of DNA-circuits in controlling the pharmacological activity of DNA strands for controlled drug delivery. The renewable DNA-circuits can be programmed 9 to stimulate the target receptors multiple times with potential autonomous control. The DNA circuit serves as a delivery platform, leading to improved stability of the CpG ODN and kinetic control of its release. References 1. Scalise, D. & Schulman, R. Annu. Rev. Biomed. Eng. 21, 469-493 (2019) 2. Yang, X. et al. ACS Nano 10, 2324-2330 (2016). 3. Chen, J. et al. Small 18, 2108008 (2022).
4. Douglas, S. M et al. Science 335, 831-834 (2012). 5. Rangel, A. E. et al. Adv. Mater. 32, 2003704 (2020). 6. Klinman, D. M. Nat. Rev. Immunol. 4, 249-259 (2004). 7. Takeshita, F. et al. J. Immunol. 167, 3555-3558 (2001). 8. Del Grosso, E. et al. Nat. Chem. 2022, 14 , 600-613.Garg, S. et al. Small 14, 1801470 (2018).
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