RESUMEN
Signal amplification schemes that do not rely on protein enzymes show great potential in areas as abstruse as DNA computation and as applied as point-of-care molecular diagnostics. Toehold-mediated strand displacement, a programmable form of dynamic DNA hybridization, can be used to design powerful amplification cascades that can achieve polynomial or exponential amplification of input signals. However, experimental implementation of such amplification cascades has been severely hindered by circuit leakage due to catalyst-independent side reactions. In this study, we systematically analyzed the origins, characteristics, and outcomes of circuit leakage in amplification cascades and devised unique methods to obtain high-quality DNA circuits that exhibit minimal leakage. We successfully implemented a two-layer cascade that yielded 7,000-fold signal amplification and a two-stage, four-layer cascade that yielded upward of 600,000-fold signal amplification. Implementation of these unique methods and design principles should greatly empower molecular programming in general and DNA-based molecular diagnostics in particular.