Expanding the rule set of DNA circuitry with associative toehold activation

Abstract

Toehold-mediated strand displacement has proven extremely powerful in programming enzyme-free DNA circuits and DNA nanomachines. To achieve multistep, autonomous, and complex behaviors, toeholds must be initially inactivated by hybridizing to inhibitor strands or domains and then relieved from inactivation in a programmed, timed manner. Although powerful and reasonably robust, this strategy has several drawbacks that limit the architecture of DNA circuits. For example, the combination between toeholds and branch migration (BM) domains is 'hard wired' during DNA synthesis thus cannot be created or changed during the execution of DNA circuits. To solve this problem, I propose a strategy called 'associative toehold activation', where the toeholds and BM domains are connected via hybridization of auxiliary domains during the execution of DNA circuits. Bulged thymidines that stabilize DNA three-way junctions substantially accelerate strand displacement reactions in this scheme, allowing fast strand displacement initiated by reversible toehold binding. To demonstrate the versatility of the scheme, I show (1) run-time combination of toeholds and BM domains, (2) run-time recombination of toeholds and BM domains, which results in a novel operation 'toehold switching', and (3) design of a simple conformational self-replicator.

Figure 1

Scheme of dissociative and associative toehold activation. (a) Dissociative toehold activation. (b) General concept of associative toehold activation. (c) Hybridization-based associative toehold activation. (d) Toehold-mediated strand displacement across a 3-way junction. See text for the discussion on the intermediate Int1 and see Text S3 for discussions on rate constants k₁, k₋₁, and k₂. In all panels, toeholds (THs) are shown as red lines. Branch-migration domains (BMs) are shown as green lines. 5′ and 3′ termni are shown as squares and arrows, respectively. Auxiliary domains are shown as dashed back lines. Complementary domains are denoted with asterisks (*).

Figure 2

Kinetics of toehold-mediated strand displacement across a 3-way junction. (a) Scheme of the assay. The sequence near the 3-way junction of the invader strand is shown in the insets. For C2_x, the CTTG/Y motif (see Text S2) is shown in bold. T: TYE665; rQ: IowaBlack RQ. (b and c) Kinetics of toehold-mediated strand displacement when the toehold and BM domain are directly linked via direct hybridization (C1_x, b) or with two bulged thymidines (C2_x, c). Real-time fluorescence measurements are shown in blue (for C1₈ and C2₈), green (for C1₁₀ and C2₁₀), and red (for C1₁₄ and C2₁₄). The results of two independent measurements were shown in the darker and lighter variations of the same color. The average fluorescence values from the 2 measurements were used to fit the time course into a single-exponential equation (fitted time course shown as dotted lines). See Text S1 for the estimation of the second-order rate constants shown in (d). The concentrations of reaction components are listed in the insets. s.f.: structure-free. The sequences of oligonucleotides used in this study are shown in Figure S2.

Figure 3

A multiple-turnover reaction catalyzed by a strand whose toehold and BM domain are linked via a hairpin stem with two bulged thymidines. (a) Scheme of the catalytic reaction and the fluorescent reporter to monitor the progression of the reaction in real time. The sequence near the 3-way junction in the intermediate C-hp_x:M3 is shown in the insets. The CTTG/Y motif (see Text S2) is shown in bold. Note that the intermediates C-hp_x:M3 and C-hp_x:M3:A3 can also react with the fluorescent reporter in way similar to M3:A3. However these side reactions do not affect the determination of the rates and turnovers of the circuit. T: TYE665; rQ: IowaBlack RQ. (b) Real-time fluorescence readouts. The concentrations of reaction components are listed in the inset. The results of two independent measurements were shown in the darker and lighter variations of the same color. The kinetic data from the 2 measurements were averaged and used to fit into a double-exponential equation, as shown by dotted lines. (c) The initial rates of the reactions shown in a bar graph. s.f.: structure-free. The sequences of oligonucleotides used in this study are shown in Figure S3.

Figure 4

Scheme (a) and performance (b to d) of hybridization-based associative toehold activation. The substrates and reporter in these reactions are the same as those shown in Figure 3. (b) Comparison among reactions in the presence of 5 nM toehold-carrying strand C-TH alone, 5 nM BM domain-carrying strand C-BM alone, and 5 nM of the annealed product of the two strands. (c and d) Addition of 5 nM C-BM to a reaction mixture containing 5 nM C-TH (c), or vice versa (d). All reactions contained M3, A3, and S3-F:S3-Q, whose concentrations are listed in the inset of (a). The identity of each trace is labeled on the figures. The sequences of oligonucleotides used in this study are shown in Figure S3.

Figure 5

Toehold switching. (a) The scheme of toehold switching. The catalyst, duplex C-TH^A:C-BM₂, is converted into C-TH^B:C-BM₂ upon the addition of C-TH^B. C-TH^A:C-BM₂ and C-TH^B:C-BM₂ catalyze two hairpin assembly reactions shown as Circuit 1 and Circuit 2, respectively. The mechanism of catalysis is similar to that shown in Figure 3. Intermediates of the reactions are now shown. The products of the two reactions are monitored by fluorescent reporters S^A-F:S^A-Q and S^B-F:S^B-Q, simultaneously. F: FAM; fQ: IowaBlack FQ; T: TYE665; rQ: IowaBlack RQ. (b) Performance of toehold switching. The identity of each trace is labeled on the figure. Whenever present, the concentrations of reactants are listed in the inset. The sequences of oligonucleotides used in this study are shown in Figure S4.

Figure 6

A simple conformational self-replicator. (a) The scheme of the replicator and the sensor to monitor the progression of the reaction. For clarity, the names of DNA strands are written in the same color as strands themselves. Due to space limitations, when Top:BotA acts as a catalyst, only the part used to catalyze the reaction is shown in detail, as indicated in the inset. (b) Kinetics of self-replication. The identity of each trace is labeled on the figure. Whenever present, the concentrations of reaction components are listed in the inset. For self-replication reactions with (red traces) and without (blue traces) external catalyst (1 nM of Top:BotA), two independent measurements were carried out and shown in the darker and lighter variations of the same color. The sequences of oligonucleotides used in this study are shown in Figure S5.