PMC

(A) Scheme for the ‘YES’ gate using the DNAzyme-based regulating strategy. (B) The abstract graph of the YES gate. The purple and green circles represent the two catalysis amplifications as entropy-driven and enzymatic mechanisms, respectively. (C) Gel analysis of the YES gate reaction using a 12% PAGE. Lane 1, DNAzyme 1 consisting of strands B and A; lane 2, the previous complex B; lane 3, the previous B complex with addition of strand A; lane 4, the B complex and A of the gate strands after adding catalyst H1. (D) Time-dependent fluorescent intensity changes in the presence of (1) input strand H1 or (2) no input. The time interval is 6 min. All data represent the average of three replicates. Error bars represent one standard deviation from triplicate analyses.

(A) Illustration of a two-level cascading circuit YES gate. The first-level YES gate consists of the duplex complex D, single strand C’, catalyst H4 and input DNAzyme-3. The second-level YES gate is composed of the duplex complex B*, single strand A, fluorescent reporter BrA and catalyst H1. The output DNAzyme-2a in the first-level gate triggers the downstream reaction as an input to induce a significant fluorescence increase. (B) Illustration of three DNAzyme catalytic reactions (DNAzyme-3, DNAzyme-2a and DNAzyme-1) and two entropy-driven reactions (catalysts H4 and H1) in the two-level cascading circuit. (C) Time-dependent fluorescence changes with different inputs. The time interval is 6 min. Curves (1), (2) and (3) demonstrate the effect of the addition of (1) DNAzyme-3 without catalysts H1 and H4, (2) with catalysts H1 and H4 in the absence of DNAzyme-3, (3) in the presence of DNAzyme-3 and both catalysts, respectively. All data represent the average of three replicates. Error bars represent one standard deviation from triplicate analyses.

(A) Illustration of a self-catalysis circuit. With the help of catalyst H1, DNAzyme-2 is used as an input to trigger the whole circuit, generating two products: DNAzyme-2b and DNAzyme-4. DNAzyme-2b has the same enzyme cleavage site as the input DNAzyme-2 and can therefore cut strand B*-CrD to initiate the self-catalysis reaction. (B) Illustration of a control experiment. DNAzyme-2 is used as an input to trigger the whole circuit, generating two products: DNAzyme-5 and DNAzyme-4. DNAzyme-5 is a waste product. (C) Abstract graph of three DNAzyme catalytic reactions (DNAzyme-2, DNAzyme-2b and DNAzyme-4) and one entropy-driven reaction (catalyst H1) in the self-catalysis circuit and control experiment. (D) Analysis of the self-catalysis circuit products using a 12% PAGE gel. Lanes 1, products of the duplex substrate B*; lane 2, the product of DNAzyme-4; lanes 3–6, a series of different DNAzyme-2 concentrations added to the gate solution with strands A0 and A2 as 0.05, 0.15, 0.45 and 1.35 μM, respectively; lanes 7–10, a series of different DNAzyme-2 concentrations added to the gate solution with strands A1 and A2 as 0.05, 0.15, 0.45 and 1.35 μM, respectively. Other DNA components were used at fixed concentrations 0.6 μM of B’/B*-CrD, 0.15 μM of catalyst H1 and 0.1 μM of ssDNA (A0, A1 and A2). (E) Fluorescence signal analysis. The columns 1–6 correspond to the relative fluorescence increase percentage (△F) before and after self-catalyzing at 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 μM, respectively.

A cascading ‘YES’ logic gate based on the DNAzyme-based regulating strategy. (A) Illustration of a cascading YES gate. The loop region of strand B*-CrD has a TrAGG (purple arrow), which is similar to the pre-catalysts H2. The fluorophore FAM and the quencher BHQ are functionalized at either end of strand BrA. DNAzyme-1 can cleave the reporter strand BrA to trigger fluorescent signals. (B) The abstract graph of two enzymatic catalytic processes (DNAzyme-2 and DNAzyme-1) and one entropy-driven amplification process (catalyst strand H2*) in the cascading ‘YES’ logic reaction. (C) Analysis of YES-gate products using a 12% PAGE gel: ‘+’ denotes addition of the strand and ‘−’ denotes absence of the strand. Lane 1, the products of DNAzyme-1; lane 2, the product of the duplex substrate B*; lane 3, the gate stands consist of strand A and the duplex substrate; lane 4, the gate strands and one hairpin pre-catalyst H2; lane 5, the product after adding DNAzyme-2. (D) Time-dependent fluorescence changes according to different inputs. The time interval is 6 min. Curve (1) reflects the reaction with the addition of DNAzyme-2 and curve (2) is the case with no input. All data represent the average of three replicates. Error bars represent one standard deviation from triplicate analyses.

Regulations of the OR gate using DNAzymes-2 and -3. (A) Schematic illustration of the logic gate. The loop region of strand B4 has two TrAGGs (purple arrow, loop site 1; pink arrow, loop site 2), which are similar to catalysts H2 (purple arrow) and H3 (pink arrow), respectively. The fluorophore FAM and quencher BHQ are functionalized at either end of strand CrE. DNAzyme-1 can cleave the reporter strand CrE to trigger fluorescent signals. (B) Abstract graph of three DNAzyme catalytic reactions (DNAzyme-3, -2 and -4) and one entropy-driven reaction (catalysts H2 or H3) in the OR gate. (C) Time-dependent fluorescence changes with different inputs. The time interval is 6 min. The curves (1), (2), (3) and (4) reflect the reactions with the addition of DNAzymes-2 and -3, DNAzyme-2, DNAzyme-3 and no input, respectively. All data represent the average of three replicates. Error bars represent one standard deviation from triplicate analyses. (D) Analysis of OR gate products using a 12% PAGE gel. ‘+’ denotes addition of the strand. Lanes 1, the products of the complex E; lane 2, the product of DNAzyme-4; lane 3, gate stands consisting of strand A2 and the duplex complex; lane 4, gate strands and one hairpin catalyst H2; lane 5, gate strands and one hairpin catalyst H3; lanes 6–8, in the presence of DNAzyme-3, DNAzyme-2 and both, respectively.