Fig. 5. Logic component layering allows more complex computation.
a, A two-input NOR gate is built by layering two RNA NOT gates. The NOT gates are regulated by either TetR or SmtB that sequester the unregulated InvadeR molecules from the DNA signal gate. b, Fluorescence activation is observed only in the absence of both inputs. c, A two-input IMPLY gate combines a DNA OR gate with an RNA NOT gate. In this specific example (ZnSO4 IMPLY tetracycline), the OR gate is regulated by TetR, and the NOT gate is regulated by SmtB, preventing signal generation in the presence of ZnSO4 only. d, Fluorescence activation is observed unless only ZnSO4 is added. Faster signal generation is observed from the tetracycline only input condition due to no mismatch between the OR gate output strand and the signal gate. e, A two-input NAND gate layers two unregulated DNA OR gates with two regulated RNA NOT gates. In this configuration, the presence of both inputs is required to hinder signal generation. Thermodynamic drivers (highlighted in red) are incorporated in the NOT gates to favor the interactions with their respective InvadeR strands. f, The expected NAND gate computation is observed. g, A two-input NIMPLY gate is built by combining the DNA AND gate and the RNA NOT gate. In this specific example, tetracycline-induced InvadeR and unregulated InvadeR are required for the AND gate activation. A SmtB-regulated NOT gate sequesters the unregulated InvadeR. h, Fluorescence activation is observed in the presence of tetracycline only. All data shown are n = 3 independent biological replicates each plotted as a line with raw fluorescence value standardized to MEF (μM fluorescein). Shading indicates the average of the replicates ± s.d. Domains with the same color share the same sequence except for the AND gate where the domain highlighted in orange is modified from the orange domain in the OR gate. All nucleic acid gates are drawn according to the secondary structures predicted using NUPACK at 37 °C30. The sequence of each domain and the concentrations of components in each gate can be found in Supplementary Data 4 and 5, respectively.