Guanine‐Locked DNA Circuit Drives Homogeneous Coupling of Catalytic Hairpin Assembly and dCTP‐Free Rolling Circle Amplification

Both enzyme-free DNA circuits and polymerase-mediated amplification represent powerful and versatile platforms for nucleic acid signal transduction. However, DNA polymerases' intrinsic polymerizing/exonucleolytic activities cause nonspecific extension/degradation of hairpins, restricting the combination of these two reactions to stepwise operations with limited efficiency. Herein, we report a guanine-locked DNA circuit resisting polymerase-mediated side reactions, enabling homogeneous coupling of catalytic hairpin assembly and dCTP-free rolling circle amplification. Modified onto magnetic nanoparticles for optomagnetic biosensing, this platform unifies target recognition, amplification, and real-time transduction in a one-pot isothermal format, achieving a detection limit of 9 aM with a wide dynamic range in 1 h for Botrytis cinerea DNA. The biosensor discriminates Botrytis species and correlates strongly with qPCR (r = -0.986) for infected rose petal samples. This work resolves the incompatibility between hairpin cascades and DNA polymerases, providing a generalizable strategy for crosstalk-free homogeneous nucleic acid circuits.