Tandem‐Controlled Dynamic DNA Assembly Enables Temporally‐Selective Orthogonal Regulation of cGAS–STING Stimulation

Abstract Despite advances in the controlled reconfiguration of DNA structures for biological applications, the dearth of strategies that allow for orthogonal regulation of immune pathways remains a challenge. Here, we report for the first time an endogenous and exogenous tandem‐regulated DNA assembly strategy that enables orthogonally controlled stimulation of the cyclic GMP‐AMP synthase (cGAS)‐stimulator of interferon genes (STING) pathway. A DNA motif containing two palindromic sequences is engineered with an abasic site (AP)‐connected blocking sequence to inhibit its self‐assembly function, while apurinic/apyrimidinic endonuclease 1 (APE1)‐triggered enzymatic cleavage of the AP site enables the reconfiguration and self‐assembly of DNA motif into long double‐stranded structures, thus realizing allosteric activation of the catalytic activity of cGAS to produce 2’3’‐cyclic‐GMP‐AMP for STING stimulation. Importantly, we demonstrate that APE1‐regulated DNA assembly allows for cell‐selective activation of cGAS–STING signaling. Furthermore, by re‐engineering the DNA motif with a photocleavable group, enzyme‐triggered DNA assembly allows the cGAS–STING stimulation to operate (switched “ON”), whereas light‐mediated fragmentation of the double‐stranded DNA enables termination of such stimulation (switched “OFF”), thereby achieving orthogonal control over immune regulation. This work highlights an endogenous and exogenous tandem regulated strategy to modulate the cGAS–STING pathway in an orthogonally controlled manner.