Minimizing Leakage in Stacked Strand Exchange Amplification Circuits

Signal amplification is ubiquitous in biology and engineering. Protein enzymes, such as DNA polymerases, can routinely achieve >10⁶-fold signal increase, making them powerful tools for signal enhancement. Considerable signal amplification can also be achieved using nonenzymatic, cascaded nucleic acid strand exchange reactions. However, the practical application of such kinetically trapped circuits has so far proven difficult due to uncatalyzed leakage of the cascade. We now demonstrate that strategically positioned mismatches between circuit components can reduce unprogrammed hybridization reactions and therefore greatly diminish leakage. In consequence, we were able to synthesize a three-layer catalytic hairpin assembly cascade that could operate in a single tube and that yielded 3.7 × 10⁴-fold signal amplification in only 4 h, a greatly improved performance relative to previous cascades. This advance should facilitate the implementation of nonenzymatic signal amplification in molecular diagnostics, as well as inform the design of a wide variety of increasingly intricate nucleic acid computation circuits.