Fig. 1.

Cascading of the CHA reaction. In the first-layer reaction C₀ catalyzes the formation of M₁:A₁ from substrate hairpins M₁ and A₁. (See Fig. S1 for the detailed pathway of this reaction.) The formation of M₁:A₁ exposes the 5* toehold domain of M₁, enabling M₁:A₁ to catalyze the formation of M₂:A₂ in the second-layer reaction. The formation of M₂:A₂ can be sensed by using a fluorescent reporter S₂. Hairpins of the same layer are shown in the same color. (Insets in gray) Additional reagents that are useful in testing individual CHA reactions (such as C₁ and S₂). Gray dashed arrows connect reactants and products, whereas red dashed arrows connect catalysts and catalyzed reactions. This two-layer cascade can be abbreviated using the presentation shown in the second row of Fig. 2A.