Herein,we present a DNA circuit programmed for the deliveryofCpG oligodeoxynucleotides (CpG ODNs) with the pharmacological immunostimulationfunction. The circuit employs a complementary DNA (cDNA) strand todeactivate the biological function of CpG ODNs via hybridization,while T7 exonuclease mediates the activation by hydrolyzing the cDNAand releasing the CpG ODN as an active moiety. We investigated theinfluence of several factors on the kinetic profile and temporal behaviorof the circuit. These include the design of the cDNA strand, the concentrationof the DNA duplex, and the concentration of T7 exonuclease. The DNAcircuit's in vitro activation resulted in toll-like receptor9 stimulation in the HEK-engineered cell line, as well as tumor necrosisfactor-alpha release by J774A.1 macrophages. By programming the DNAcircuit to control the release of the CpG ODN, we achieved an alteredpharmacological profile with acute and potent immunostimulation, incomparison to a system without controlled CpG ODN release, which exhibiteda slow and delayed response. Our findings demonstrate the potentialof DNA circuits in controlling the pharmacological activity of DNAstrands for controlled drug delivery.
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