Engineering high-robustness DNA molecular circuits by utilizing nucleases

摘要

Toehold-mediated strand displacement (TMSD) as an important player in DNA nanotechnology has been widely utilized for engineering non-enzymatic molecular circuits. However, these circuits suffer from uncontrollable leakage and unsatisfactory response speed. We utilized site-specific and sequence-independent nucleases to engineer high- robustness DNA molecular circuits. First, we found that the kinetics of the APE1-catalyzed reaction is highly dependent on substrate stability, allowing for the elimination of asymptotic leakage of DNA split circuits. Second, we obtained strict substrate preference of lambda exonuclease (lambda(exo)) by optimizing the reaction conditions. Robust single-layer and cascade gates with leak resistance were established by using lambda exo. Owing to the remarkably fast kinetics of these nucleases, all the circuits yield a high speed of computation. Compared to TMSD-based approaches, nuclease-powered circuits render advanced features such as leakage resistance, hundreds of times higher speed, and simplified structures, representing a class of promising artificial molecule systems.