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DOI: 10.1039/D4CC03579H (Communication) Chem. Commun., 2024, 60, 10536-10539

DNA domino circuits based on a hairpin exonuclease assistance signal transmission architecture for temporal logic operations†

Xun Zhang a, Yao Yao a, Xin Liu a, Xiaokang Zhang a, Shuang Cui a, Bin Wang b and Qiang Zhang *a
aSchool of Computer Science and Technology, Dalian University of Technology, Dalian 116024, China. E-mail: madao@mail.dlut.edu.cn; zhangq@dlut.edu.cn
bKey Laboratory of Advanced Design and Intelligent Computing, Dalian University, Dalian 116622, China

Received 18th July 2024 , Accepted 27th August 2024

First published on 28th August 2024

Abstract

DNA circuits are important fundamental tools for performing temporal logic operations with complex structures, but they lack sequence orthogonality. Here, we developed a simple and orthogonal hairpin exonuclease assistance signal (H-EAST) architecture to construct DNA domino circuits with time-delay characteristics and temporal logic operations, which has potential applications in biomolecular computing.

Organisms typically maintain their survival using a series of energy dissipation systems. These often include significant features such as intracellular division and movement,1,2 and signal transmission.3,4 Such energy dissipation systems are characterized by reaction processes involving molecular spatiotemporal information under non-equilibrium conditions. Cells execute various biological activities through dissipative reactions in chemical reaction networks (CRNs),5,6 including aptamer formation,7 transcription,8 and translation.9 These dissipative reactions, through the kinetics of chemical reactions, allow the timing behaviour of some natural systems to be mimicked and can thus be used to construct precise time-controlled CRNs in vitro,10–12 which is of great significance to biomimetic and synthetic biology research. DNA computing, as a novel computing paradigm, benefits from the predictable base-pairing of DNA materials, programmable reactive substrates, and good biocompatibility. It is frequently used to design molecular biological circuits to meet the processing needs of molecular events in CRNs that have been applied in the contexts of DNA origami,13,14 DNA tiles,15,16 and nanoclusters and particles.17 To enhance the generality and ease of operation of DNA circuits, it is necessary not only to provide multiple biological marker input–output signal transmission interfaces from the perspective of biological computing, but also to adjust the temporal characteristics of related reaction network molecular events through programming.18–20

Dynamic DNA reaction networks involving biological enzymes provide a powerful tool for time-controlled functionalities,21–23 which can be used to develop nucleic acid-based dissipative systems. These can facilitate applications such as biological detection, nanostructure assembly, and molecular logic operations. In particular, nucleic acid exonucleases do not require specific sequence requirements or complex nanostructures to construct DNA circuits.24–28 This maximizes their scalability over a wide range of applications. However, in the process of molecular signal propagation in most molecular reaction systems, there are two major limitations. First, during the reaction process, by-products are generated. Owing to the lack of a catalytic effect, the issue of signal attenuation often becomes more severe as cascade levels increase.29–31 Second, a significant portion of the sequences carried by the input and output DNA strands are related. This requires stricter design criteria for DNA reaction substrates to prevent interference between different reaction levels. For example, three-base encoding can be used to avoid the formation of abnormal secondary structures, which also represents a challenge in traditional toehold mediated strand displacement (TSMD) reactions.32–34

This study introduces the H-EAST architecture, inspired by DNA-based time-controlled reactions. It duplicates a domino effect for signal transmission, multi-level feedback, and catalytic amplification within CRNs. By modifying the sequence length, reaction nodes, and substrate concentration, a temporal OR gate for a DNA circuit is developed, showcasing the efficiency of H-EAST in handling molecular information with temporal aspects. H-EAST is poised to advance the study of CRNs with time-processing abilities, fostering biomolecular computing technology.

In this study, we introduce the H-EAST architecture, which utilizes the EXO lambda enzyme to initiate hydrolysis at the 5′ phosphate group, enabling the production of a complementary DNA strand (Fig. 1A). Based on the traditional EAST architecture, we developed the H-EAST system, demonstrated with the Sub1-2 reaction example. The architecture features a hairpin with a toehold that, when opened by In1, exposes the 5′ phosphate group and allows EXO lambda to hydrolyse and recycle In1 (Fig. 1B). This enables the reuse of In1 to amplify the molecular signalling effect. The hydrolysis rate exhibits a fast–slow–fast pattern (Fig. S1, ESI†), resulting in sequence orthogonality and a catalytic effect without by-products. To quantify the signalling intensity, we modified Sub1-2 with 5′-FAM and 3′-BHQ1 to create Sub1-2F, which was tested via electrophoresis and FRET experiments. Even at a 0.1× input concentration, continuous recycling of Sub1-2F allows for catalytic activity, with In1 acting as a catalyst for Out2 production at concentrations much higher than 0.1× (Fig. 1C and D). The yield of Out2 is shown in Fig. S2 (ESI†) and the optimal reaction pH and temperature are shown in Fig. S3 (ESI†).35,36


image file: d4cc03579h-f1.tif
Fig. 1 H-EAST reaction mechanism. (A) Reaction diagram of the lambda exonuclease-assisted signal transmission (EAST) architecture. (B) Reaction diagram of the hairpin lambda exonuclease-assisted signal transmission (H-EAST) architecture. (C) Electrophoretic gel stained with stains-all (left) using a FAM-BHQ1-modified Sub1-2F reaction substrate with H-EAST architecture and exposed to UV irradiation (right). Lane 1: 20 pmol Sub1-2F; Lane 2: 20 pmol Out2; Lane 3: 20 pmol In1; Lane 4: 20 pmol Sub1-2F + 20 U EXO lambda; Lanes 5–8: 20 pmol Sub1-2F + 20 U EXO lambda with 20 pmol (1×), 10 pmol (0.5×), 5 pmol (0.2×), 2 pmol (0.1×) In1, 20 μL reaction volume. (D) FRET experiment of the H-EAST architecture with different concentrations of In1 with 20 U EXO lambda in a 50 μL reaction volume. The interval between each detection cycle of the FRET experiment was 2 min.

To explore the multi-level positive feedback catalytic effects produced by the interaction and stacking of different circuit modules in the H-EAST architecture during complex molecular reaction processes, we designed a cross-catalytic reaction circuit with feedback characteristics. As shown in Fig. 2A, the TSub1-2 reaction substrate can be triggered by TIn1 in the cross-catalytic reaction. Under the hydrolysis of EXO lambda, it then releases the Out2 signal. At the same time, the Out2 signal is also the TIn2 signal, which can be used to activate the Sub2-1 reaction substrate and further release the Out1 signal, thus forming a signal catalytic loop from node 1 to node 2. We modified FAM–BHQ1 on the reaction substrates TSub1-2 and TSub2-1 for real-time fluorescence detection. We controlled [TSub1-2]0 = [TSub2-1F]0 = 400 nM in 50 μL reaction systems and added 0.2–1× of [TIn1]0 and [TIn2]0. The real-time fluorescence changes were detected in the presence of 20 U of EXO lambda. The specific fluorescence data are further detailed in Fig. S4 (ESI†). The fluorescence values after a 3 h reaction are shown in Fig. 2B. Similarly, by controlling [TSub1-2F]0 = [TSub2-1]0 = 400 nM, we were able to investigate the effects of varying concentrations of TIn1 and TIn2 on the reaction substrate TSub1-2 (Fig. 2C). We observed that both TIn1 and TIn2 exhibited catalytic effects on the reaction to varying degrees, maintaining low leakage rates. This validated the feasibility of using the H-EAST architecture to construct DNA circuits with feedback characteristics. Owing to the influence of DNA sequences on the reaction rates of EXO lambda hydrolysis and strand displacement reactions, certain differences in the intensities of the output signals persisted, even though the two substrates for the reactions had the same structures and sequence base numbers. In the ternary cross-cascade catalytic reaction, an additional signal node is added and an enclosed circular information catalytic loop is constructed through the reaction substrates TSub1-2, TSub2-3, and TSub3-1. By inputting any one of the DNA strands TIn1, TIn2, and TIn3, the other two DNA strand signals can be catalytically released in a clockwise sequence (Fig. S5, ESI†).


image file: d4cc03579h-f2.tif
Fig. 2 (A) Schematic diagram of the cross-catalytic reaction. (B) FRET experiment for the cross-catalytic reaction with input strands. (C) Detecting the fluorescence output of nodes 1 and 2 after 3 h to the input strand concentrations at 0–1× with 20 U EXO lambda.

Benefiting from the orthogonal characteristics of the H-EAST architecture, there are no predefined sequence constraints between the input and output strands. This allows for the realization of various programmable parallel pathways, such as implementing fan-out/fan-in operations and cascaded networks. As shown in Fig. 3A, we first constructed a three-output fan-out DNA circuit containing three reaction substrates (Sub0-1, Sub0-2 and Sub0-3), all of which can recognize In0 and trigger strand displacement reactions. Under the effect of EXO lambda, three different corresponding outputs (Out1, Out2, and Out3) are released. We also added reporter1, reporter2, and reporter3, which are modified by FAM–BHQ1, ROX–BHQ2, and Cy5–BHQ3, respectively, in order to detect the output signal intensity corresponding to the different pathways (Fig. 3B). Particularly, the positions of the input and output sequences can be reversed in the reaction substrates, resulting in Sub1-0, Sub2-0, and Sub 3-0, which recognize and receive signals from In1, In2 and In3, and release the Out0 signal with the EXO lambda (Fig. 3C). Using this property, we constructed a three-input fan-in DNA circuit, where any one of the three different DNA inputs can trigger the reaction and yield Out0 (Fig. 3D). Fig. S6 (ESI†) shows the real-time fluorescence curve for this application.


image file: d4cc03579h-f3.tif
Fig. 3 Schematic diagram of the DNA circuit illustrating (A) fan-out and (B) fan-in characteristics. (C) FRET experiment validating the fan-out circuits: in a 50 μL reaction system, adjusting the concentrations of [Sub0-1]0, [Sub0-2]0, [Sub0-3]0, [Reporter1]0, [Reporter2]0, and [Reporter3]0 to 400 nM each. We added In0 at concentrations of 0–1200 nM, conducted the reaction in an environment with 20 U EXO lambda for 4 h, and measured the fluorescence intensity values of different channels. (D) FRET experiment validating fan-in circuits.

In biological systems, energy dissipation controls reaction durations. Using Sub0-1 as an example, In0 strand introduction opens the hairpin loop, forming a double-strand, single-strand, double-strand structure. EXO lambda hydrolyses with a fast–slow–fast pattern, which can be delayed by adding thymine bases near the loop (Fig. 4A). We tested this by creating substrates H0-1, H0-/3T/-1, and H0-/6T/-1 with 0, 3, and 6 nt of thymine, respectively, and measured the delay effect using FRET (T1/2 parameter). Additionally, extension of reaction times can be achieved by increasing the number of nodes in substrates and reducing their concentration, in line with the domino effect principle (Fig. 4B and C).


image file: d4cc03579h-f4.tif
Fig. 4 (A) Reaction system containing 400 nM of each substrate [H0-1]0, [H0-/3T/-1]0, [H0-/6T/-1]0, and 200 nM [Reporter1]0. (B) By increasing the number of reaction nodes and adjusting the concentrations of the reaction substrates to [Sub2-3]0 = 400 nM, [Sub1-2]0 = [Sub2-3]0 = 400 nM, and [Sub0-1]0 = [Sub1-2]0 = [Sub2-3]0 = 400 nM, as well as controlling [Reporter3]0 = 200 nM, 400 nM each of In2, In1, and In0 were introduced. (C) By removing the 5′ phosphate group from the Sub1-2 substrate to reduce the concentration of the substrates corresponding to the nodes. [Sub1-2]0 was controlled at 400, 200, and 80 nM, [Sub2-3]0 = 400 nM, [Reporter3]0 = 200 nM, and 400 nM of In1 was added to the reaction system. Real-time fluorescence curves and T1/2 were obtained under the effect of 20 U of EXO lambda. For all FRET experiments, each detection cycle interval was 2 min.

Based on the mechanism above, the temporal OR gate is proposed in Fig. 5A, where the inputs In1 and In3 can react with the substrates Sub1-2 and Sub3-4, respectively, to produce Output2 and Output4, respectively. However, unlike a regular OR gate, the two output signals respond differently as a function of the timing characteristics of the input signals In1 and In3. Therefore, this temporal OR logic gate can offer more computational properties for handling temporal characteristics. The resulting truth table is shown in Fig. 5B.


image file: d4cc03579h-f5.tif
Fig. 5 (A) Schematic of the reaction mechanism for the temporal OR logic gate. (B) Truth table for the temporal OR logic gate. (C) Controlling the concentration [Sub1-2]0 = [Sub3-4]0 = 400 nM, [Reporter2-4]0 = 200 nM, and with inputs [In1]0 = [In3]0 = 200 nM, under the effect of 20 U EXO lambda, fluorescence outputs from the FAM and ROX channels were detected. (D) Adjusting the concentration of the input signal In3 from 1× to 0.2×, with a delay time ranging between 0 and 70 cycle periods, and measuring the final fluorescence signal after 4 h.

We can detect the temporal characteristics of the temporal OR logic gate by controlling the concentrations of In1, In3, Sub1-2, and Sub3-4. The real-time fluorescence data are shown in Fig. S7 (ESI†). It can be observed that when the reaction system does not add any input strand, the reaction system produces no output corresponding to the “0” output in the truth table. However, when In1 or In3 is added alone, the fluorescence values of FAM and ROX increase significantly, thus yielding the “1” output in the truth table (Fig. 5C). Next, we added In1 and In3 to the reaction system and recorded the corresponding times t1 and t3 to test their effects on the reaction system. In Fig. 5D, adding both substrates simultaneously results in a lower Output2 yield compared to Output4, likely due to sequence-specific EXO lambda hydrolysis and DNA strand displacement rates. When In1 is added first with a controlled delay of 20–140 min, the Output4 yield decreases and Output2 increases with increasing delay. In biochemical networks, temporal signal handling can be challenging, and altering DNA sequences to regulate reaction rates is costly. Our previously described method of adjusting reactant concentration can dynamically control delay times. By decreasing [In1]0 from 400 nM to 80 nM, Output4 formation can be inhibited.

In this study, inspired by the domino effect, we developed the H-EAST architecture, which used the unique hydrolytic properties of EXO lambda to introduce a delayed triggering strategy and construct a series of DNA domino circuits with signal transduction, catalysis, feedforward, and feedback outputs. We also provided several methods for handling nanoscale molecular temporal events for this architecture, which has novel sequence orthogonality and no waste by-products. Successfully mimicking the delay time characteristics of DNA domino circuits to construct a temporal OR logic gate could provide assistance for the development of biochemical sensing, molecular detection, and molecular robotics technologies in the nanotechnology field.

Data availability

The experimental data in this article are provided in the ESI.†

Conflicts of interest

There are no conflicts to declare.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4cc03579h
This journal is © The Royal Society of Chemistry 2024