Molecular signal rollback method and system based on cutting enzyme assistance

Abstract

The invention relates to the technical field of molecular calculation and DNA nanotechnology, in particular to a molecular signal rollback method and system based on cutting enzyme assistance. According to the method, the nicking enzyme is introduced as a rollback factor, and the problem that a traditional molecular sequential circuit enters an irreversible inhibition state due to abnormal input is solved. When a target DNA substrate is combined and inactivated by an inhibition signal, a nicking enzyme capable of specifically recognizing the inhibition compound is added, the inhibition signal is dissociated through a cutting effect, so that the substrate is recovered to an initial active state, then an activation signal can be responded again, output is triggered, and rollback from an abnormal silence state to a normal functional state is realized. The invention further provides a specified rollback module capable of performing fixed-point recovery on the specific abnormal path under multiple instruction input. The reliability and controllability of a molecular information processing system are remarkably enhanced, and a foundation is laid for constructing a complex and robust molecular calculation and diagnosis system.

Description

Technical Field

[0001] This invention relates to the fields of molecular computing and DNA nanotechnology, specifically to a molecular signal rollback method and system based on nicking enzyme assistance. Background Technology

[0002] Molecular sequential circuits, as one of the core technologies in molecular computing, are built based on DNA strand substitution reactions. They can process input signals according to a preset logical sequence and show broad application prospects in cutting-edge fields such as biosensing, intelligent drug delivery, and molecular information processing. Their functionality relies heavily on the ordered interactions between molecular components such as DNA strands, which strictly adhere to the design logic, as well as controllable state transition processes. They are a key carrier for realizing complex molecular-level computations and signal transduction.

[0003] However, traditional molecular sequential circuits have inherent flaws, with state transitions often being unidirectional and irreversible. The reaction paths of these circuits are typically unidirectional or conditionally triggered, with state transitions relying on the specific binding of the input signal to a pre-defined path. Once a specific reaction is initiated, the system enters a fixed next-level state and cannot autonomously backtrack. The most prominent problem is that when the inhibitory signal arrives at the reaction system before the activation signal and binds to the key DNA substrate, a structurally stable and inert intermediate complex is formed. This complex's closed structure blocks the recognition and binding of subsequent activation signals, causing the system to be permanently locked in a "shutdown" or quiescent state, completely losing its ability to respond to the original design function. This irreversible characteristic becomes the core bottleneck restricting performance improvement.

[0004] This inherent limitation of unresettable states severely restricts the application of molecular sequential circuits in dynamic control scenarios. In practical applications such as simulating the dynamic feedback of complex signaling pathways in biological systems and constructing multi-round cyclic detection systems, the system must possess the ability to recover from an abnormal state to its initial responsive state. Traditional technologies cannot meet this core requirement. In computer science, "rollback" technology has been maturely applied to abnormal state repair, effectively reversing the effects of abnormal instructions by restoring the system to a previous correct checkpoint. However, at the molecular scale, how to construct a precise, controllable, efficient, and stable mechanism to actively restore a molecular system "locked" by abnormal input to its initial triggerable state remains an unsolved technical challenge, urgently requiring the development of novel molecular control strategies to overcome this bottleneck. Summary of the Invention

[0005] The purpose of this invention is to propose a molecular signal rollback method and system based on nicking enzyme assistance. By introducing a nicking enzyme with highly specific sequence recognition and single-chain cleavage function as a rollback factor, molecular circuits that have been deactivated due to abnormal inhibitory signal input can be effectively restored to their initial functional state, thereby achieving the "cancellation" of abnormal operations and the autonomous recovery of system state at the molecular level.

[0006] According to a first aspect of the embodiments of this disclosure, a molecular signal rollback method based on nicking enzyme assistance is provided, comprising the following steps: A double-stranded DNA substrate Sub1 containing a nicking enzyme-specific recognition sequence is provided. The substrate is capable of responding to an activation signal AS1 and triggering the generation of an output signal, while also being able to bind to an inhibition signal IS1 and enter an inhibition state. When the inhibitory signal IS1 arrives in the reaction system before the activation signal AS1, IS1 specifically binds to the double-stranded DNA substrate Sub1 to form the inactive intermediate product Intermediate3. This intermediate product can block the molecular reaction pathway with the relay signal S1 and enter a quiescent state. A nicking enzyme is introduced into the quiescent reaction system. The nicking enzyme can specifically recognize and cleave the inactive intermediate product Intermediate3, causing the inhibitory signal IS1 to dissociate from the intermediate product, thereby restoring the inactive intermediate product Intermediate3 to the initially active double-stranded DNA substrate Sub1. An activation signal AS1 is provided to the reactivated double-stranded DNA substrate Sub1. The activation signal AS1 triggers the restart of the molecular timing circuit reaction path, prompting the generation of an output signal and realizing the signal rollback from the quiescent state to the activated state.

[0007] In one embodiment, the activation signal AS1 triggers a strand displacement reaction in the double-stranded DNA substrate Sub1, thereby releasing single-stranded DNA U1; the single-stranded DNA U1 can specifically bind to and react with the fluorescently labeled reporter complex RE to generate a detectable output signal.

[0008] In one embodiment, a specific recognition sequence of a nicking enzyme is embedded in the domain of the inhibition signal IS1; when the inhibition signal IS1 binds to the double-stranded DNA substrate Sub1 to form an inactive intermediate product, the specific recognition sequence is exposed and recognized and cleaved by the corresponding nicking enzyme.

[0009] In one embodiment, the length of the binding domain d2 of the inhibition signal IS1 and the double-stranded DNA substrate Sub1 is 2 to 14 bases; in the domain of the inhibition signal IS1, the length of the spacer sequence between the region containing the nicking enzyme's specific recognition sequence and the binding domain d2 is not less than 2 bases.

[0010] In one embodiment, after the nicking enzyme cleaves the inactive intermediate, it exposes a foothold of 2 to 6 bases in length on the double-stranded DNA substrate Sub1.

[0011] In one embodiment, the concentrations of each component in the reaction system must meet the following conditions: the concentration of relay signal S1 is 1-2 times the concentration of substrate Sub1; the concentration of inhibition signal IS1 is 1-2 times the concentration of double-stranded DNA substrate Sub1; the concentration of activation signal AS1 is 1 to 4 times the concentration of double-stranded DNA substrate Sub1, wherein a 4-fold concentration is selected when triggering rollback to achieve complete rollback; and the concentration of nicking enzyme used is 1 to 3 units (U) per reaction.

[0012] In one embodiment, the reaction time between the inhibition signal IS1 and the double-stranded DNA substrate Sub1 is 0-20 minutes, the interval between enzyme digestion recovery and rollback activation is 0-20 minutes, and the activation signal AS1 is added at the same time as the nicking enzyme is introduced.

[0013] In one embodiment, the cleaving enzyme is selected from at least one of Nt.BbvCI, Nb.BtsI, and functional analogs having the same recognition and cleavage specificity.

[0014] According to a second aspect of the present disclosure, a molecular signal rollback system based on nicking enzyme assistance is provided, comprising: The substrate preparation module provides a double-stranded DNA substrate Sub1 containing a nicking enzyme-specific recognition sequence. The substrate has the function of responding to the activation signal AS1 and triggering the generation of an output signal, and can also bind to the inhibition signal IS1 and enter an inhibition state. The inhibition trigger module, when the inhibition signal IS1 arrives in the reaction system before the activation signal AS1, IS1 specifically binds to the double-stranded DNA substrate Sub1 to form an inactive intermediate product Intermediate3. This intermediate product can block the molecular reaction pathway with the relay signal S1 and enter a quiescent state. The enzyme digestion recovery module introduces a nicking enzyme into the quiescent reaction system. The nicking enzyme can specifically recognize and cleave the inactive intermediate product Intermediate3, causing the inhibition signal IS1 to dissociate from the intermediate product, thereby restoring the inactive intermediate product Intermediate3 to the initially active double-stranded DNA substrate Sub1. The rollback activation module provides an activation signal AS1 to the reactivated double-stranded DNA substrate Sub1. The activation signal AS1 triggers the restart of the molecular timing circuit reaction path, prompting the generation of an output signal and realizing the signal rollback from the quiescent state to the activated state.

[0015] In one embodiment, a designated rollback module under multiple command inputs is also included. This module provides at least two different double-stranded DNA substrates, each of which contains a corresponding preset molecular reaction pathway and a specific recognition sequence of a dedicated nicking enzyme. Each substrate also has a corresponding specific inhibition signal, a specific activation signal, a specific nicking enzyme, and a dedicated reporter complex. When one or more of the specific inhibition signals are input into the reaction system, the corresponding molecular reaction pathway is blocked. By introducing a specific nicking enzyme that matches the input specific inhibition signal, the abnormal state caused by the inhibition signal can be specifically rolled back, restoring the corresponding molecular reaction pathway without interfering with other molecular reaction pathways that have not received an inhibition signal. The reporter complex corresponding to each substrate independently monitors the signal output of each molecular reaction pathway.

[0016] The advantages of the above technical solutions adopted in this invention compared with the prior art are as follows: 1. This invention introduces the concept of "rollback" into the field of molecular circuits for the first time. By utilizing the specific cleavage function of nicking enzymes, the irreversible inhibition state of traditional molecular sequential circuits is transformed into a reversible dynamic control process, enabling the system to autonomously recover from abnormal silent states, thus greatly improving the fault tolerance and reliability of molecular information processing systems.

[0017] 2. The rollback process of this invention is precisely controlled by an exogenously added nicking enzyme and activation signal. Its specificity is strictly limited by a preset DNA sequence, exhibiting excellent programmability. Furthermore, by optimizing parameters such as reaction time and concentration, the rollback efficiency and rate can be finely controlled to meet the needs of different application scenarios.

[0018] 3. The designated rollback module designed in this invention can accurately locate and restore specific abnormal paths in complex molecular networks with multiple inputs and multiple pathways, avoiding the waste of resources caused by global reset, and laying the core technical foundation for building complex and robust molecular computing and diagnostic systems.

[0019] 4. The rollback mechanism of this invention can be used as an independent functional module and can be seamlessly integrated with various existing DNA logic gates, timing circuits and sensing systems, significantly improving its overall performance and applicability without changing the core architecture of the original system. Attached Figure Description

[0020] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments of this application and their descriptions are used to explain this application and do not constitute an undue limitation of this application.

[0021] Figure 1 This is a schematic diagram illustrating the specific working principle of the rollback mechanism of the present invention; Figure 2The activation and inhibition functional diagrams were verified by fluorescence experiments, showing that AS1 can activate two circuits and IS1 can inhibit two circuits. Figure 3 The results of the rollback real-time fluorescence experiment are shown for different d2 lengths (2, 4, 6, 8, 10, 12, 14 nt). Figure 4 The graph shows the effect of different addition intervals (0, 10, 20 min) between the nicking enzyme and AS1 on the rollback effect after 10 minutes of IS1 treatment. Figure 5 A detailed schematic diagram illustrating the working principle of the specified rollback module; Figure 6 The experimental results are shown in the figure, which shows the results of rolling back two exceptions in the specified rollback module. Detailed Implementation

[0022] The present disclosure will be further described below with reference to the accompanying drawings and embodiments.

[0023] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] Example 1: This embodiment provides a molecular signal rollback method based on nicking enzyme assistance, including the following steps: S1. Provides a double-stranded DNA substrate Sub1 containing a nicking enzyme-specific recognition sequence, wherein the substrate has the function of responding to an activation signal AS1 and triggering the generation of an output signal, and can also bind to an inhibition signal IS1 and enter an inhibition state; like Figure 1As shown, the core components of the system include: a double-stranded substrate Sub1, formed by annealing three complementary DNA strands; an inhibitory signal IS1, a single-stranded DNA with the following components from its 5' to 3' ends: a domain e* complementary to the Sub1 anchor site e (with an embedded Nt.BbvCI recognition sequence), a short spacer sequence (e.g., 2nt), and a domain d2 complementary to the Sub1 anchor site d2* (e.g., 10nt); an activation signal AS1 and a relay signal S1, designed according to a standard DNA strand substitution circuit; a reporter complex RE, a double-stranded DNA, with one strand modified with a fluorescent group (FAM) at its 3' end and a quencher group (BHQ) modified at its 5' end; and a nicking enzyme, Nt.BbvCI.

[0026] S2. When the inhibitory signal IS1 arrives in the reaction system before the activation signal AS1, IS1 specifically binds to the double-stranded DNA substrate Sub1 to form an inactive intermediate product Intermediate3. This intermediate product can block the molecular reaction pathway with the relay signal S1 and enter a quiescent state. Normal activation pathway: Upon addition of AS1, its domain e binds to the basement e of Sub1, triggering branching migration and generating Intermediate1 and waste W1. Subsequently, S1 binds to the basement c of Intermediate1, displacing and releasing single-stranded DNA U1 and waste W3. U1 then reacts with the reporter complex RE, displacing the output strand, causing FAM to separate from BHQ and generating a fluorescent signal.

[0027] Abnormal suppression: When the suppression signal IS1 arrives before the activation signal AS1, it combines with the base points e and d2* of Sub1 to form the inactive intermediate product Intermediate3, and the system enters a quiescent state.

[0028] S3. Introduce a cleaving enzyme into the quiescent reaction system, the cleaving enzyme being able to specifically recognize and cleave the inactive intermediate product Intermediate3, causing the inhibitory signal IS1 to dissociate from the intermediate product, thereby restoring the inactive intermediate product Intermediate3 to the initially active double-stranded DNA substrate Sub1. Reversal Path: At this point, because IS1 contains a specific recognition sequence for Nt.BbvCI, the addition of Nt.BbvCI allows the enzyme to precisely recognize and cleave this sequence in Intermediate3. Cleavage causes the IS1 chain to dissociate from the complex, simultaneously restoring Intermediate3 to its initial active substrate, Sub1. Adding AS1 again then triggers the fluorescence signal output along the normal activation pathway described above, successfully achieving "signal reversal."

[0029] S4. An activation signal AS1 is provided to the reactivated double-stranded DNA substrate Sub1. The activation signal AS1 triggers the restart of the molecular timing circuit reaction path, prompting the generation of an output signal and realizing the signal rollback from the quiescent state to the activated state.

[0030] like Figure 2 As shown, the results indicate that the circuit of the present invention not only possesses the same activation / inhibition function as the conventional circuit, but its uniqueness lies in the fact that after inhibition, the signal can be restored through "enzyme digestion + reactivation", while the conventional circuit will permanently fail.

[0031] Gel electrophoresis experiment: PAGE results directly confirm that the output signal band only reappears under the process of "IS1 inhibition → enzyme treatment → AS1 addition" (lane i), while simple inhibition followed by a second addition of AS1 is ineffective (lane h), which strongly proves the effectiveness of the rollback mechanism at the molecular level.

[0032] By adjusting the length of the binding domain d2 between the inhibitory signal IS1 and the double-stranded DNA substrate Sub1, and / or adjusting the length of the spacer sequence between the nicking enzyme recognition sequence and the binding domain d2 in the inhibitory signal IS1, the stability of the inhibitory signal binding and the accessibility of the rollback factor were optimized. The kinetics of subsequent strand displacement reactions were optimized by testing the length of the foothold exposed after nicking enzyme cleavage. The optimal working concentrations of the relay signal S1, the activation signal AS1, the inhibitory signal IS1, and the nicking enzyme were determined through testing. The timing precision of the rollback operation was optimized by testing the time required for the inhibitory signal IS1 to reach its maximum inhibitory effect and the interval between the addition of the activation signal AS1 after nicking enzyme action. Specifically: The structural optimization for rollback effectiveness in this embodiment: Optimization of domain d2 length: To balance complex stability and post-cleavage dissociation efficiency, d2 lengths ranging from 2 to 14 nt were tested. Figure 3 As shown, the rollback signal is significant when d2 is 6, 8, or 10nt. Further testing revealed that the background leakage is high when d2=6nt, and the rollback curve is unstable when d2=8nt. Therefore, a d2 length of 10nt is preferred, which can achieve efficient dissociation and rollback while ensuring stable bonding.

[0033] Optimization of the interval length between e* and d2: The effects of intervals of 0, 2, 4, and 6nt were tested. Rollback failed when the interval was 0nt, while the signal was good for intervals of 2-6nt. For the sake of simplifying the sequence, an interval length of 2nt is preferred.

[0034] Optimization of the exposed site length after enzyme digestion: The effectiveness of exposing 2, 4, and 6 nt single-stranded regions as new sites was tested. The reaction was difficult to initiate at 2 nt, while the fluorescence signals were strong and similar at 4 nt and 6 nt. To allow for a wider reaction kinetic window, a site length of 6 nt was preferred.

[0035] The concentrations of each component were optimized in this embodiment: Optimization of relay signal S1 concentration: Fluorescence and electrophoresis experiments showed that the reaction reached saturation and the output signal was strongest when the S1 concentration reached twice the substrate Sub1 concentration (2×). Therefore, the optimal working concentration of S1 is 2×.

[0036] Maximum output concentration of activation signal AS1: AS1 reaches maximum output saturation at a concentration of 2×. This condition has also been verified in other substrate systems.

[0037] Optimization of IS1 concentration for suppression: Under fixed conditions, the interference of different IS1 concentrations on rollback was tested. The results showed that the higher the IS1 concentration, the worse the rollback effect. To ensure effective suppression while minimizing interference with rollback, an optimal IS1 working concentration of 1× (equivalent to Sub1 concentration) was selected.

[0038] The threshold concentration at which the activation signal AS1 triggers complete rollback: the final signal level after rollback increases with increasing AS1 concentration. When the AS1 concentration reaches 4×, rollback is complete, meaning the fluorescence intensity recovers to the maximum output level.

[0039] In this embodiment, the rollback rate and time parameters are optimized: Optimization of nicking enzyme (rollback factor) concentration: The rollback kinetics were tested at enzyme concentrations of 1U, 2U, and 3U. The results showed that the higher the enzyme concentration, the faster the initial rollback rate, with 2U and 3U having similar effects. Considering both efficiency and economy, the optimal nicking enzyme dosage was 2U.

[0040] Determination of the inhibition signal duration: To ensure that IS1 exerts its inhibitory effect, the effects of adding AS1 after incubation with the substrate for different times (0, 2, 5, 10, 15 min) were tested. It was found that the inhibition was most thorough after 10 minutes of incubation, with AS1 barely activating the system. Therefore, the optimal inhibition time was determined to be 10 minutes.

[0041] Determination of the interval between enzyme addition and AS1 addition during the rollback operation: After 10 minutes of IS1 inhibition, the effect of different intervals (0, 10, 20 min) between the addition of nicking enzyme and AS1 was tested. Figure 4 As shown, the results indicate that the interval affects the rollback initiation rate but not the final rollback extent. To simplify the procedure, it is preferable to add the nicking enzyme and AS1 simultaneously (0 minutes interval) after adding IS1 and reacting for 10 minutes.

[0042] Example 2: This embodiment provides a molecular signal rollback system based on nicking enzyme assistance, including: The substrate preparation module provides a double-stranded DNA substrate Sub1 containing a nicking enzyme-specific recognition sequence. The substrate has the function of responding to the activation signal AS1 and triggering the generation of an output signal, and can also bind to the inhibition signal IS1 and enter an inhibition state. The inhibition trigger module, when the inhibition signal IS1 arrives in the reaction system before the activation signal AS1, IS1 specifically binds to the double-stranded DNA substrate Sub1 to form an inactive intermediate product Intermediate3. This intermediate product can block the molecular reaction pathway with the relay signal S1 and enter a quiescent state. The enzyme digestion recovery module introduces a nicking enzyme into the quiescent reaction system. The nicking enzyme can specifically recognize and cleave the inactive intermediate product Intermediate3, causing the inhibition signal IS1 to dissociate from the intermediate product, thereby restoring the inactive intermediate product Intermediate3 to the initially active double-stranded DNA substrate Sub1. The rollback activation module provides an activation signal AS1 to the reactivated double-stranded DNA substrate Sub1. The activation signal AS1 triggers the restart of the molecular timing circuit reaction path, prompting the generation of an output signal and realizing the signal rollback from the quiescent state to the activated state.

[0043] Preferably, the system also includes a designated rollback module under multiple instruction inputs. This module provides at least two different double-stranded DNA substrates, each containing a corresponding preset molecular reaction pathway and a specific recognition sequence of a dedicated nicking enzyme. Each substrate also has a corresponding specific inhibition signal, specific activation signal, specific nicking enzyme, and dedicated reporter complex. When one or more of the aforementioned specific inhibition signals are input into the reaction system, the corresponding molecular reaction pathway is blocked. By introducing a specific nicking enzyme that matches the input specific inhibition signal, the abnormal state caused by the inhibition signal can be specifically rolled back, restoring the corresponding molecular reaction pathway without interfering with other molecular reaction pathways that have not received an inhibition signal. The reporter complex corresponding to each substrate independently monitors the signal output of each molecular reaction pathway.

[0044] like Figure 5As shown, the module comprises two independent reaction systems: System 1 (substrate Sub1, signal AS1 / IS1, enzyme Nt.BbvCI) and System 2 (substrate Sub2, signal AS2 / IS2, enzyme Nb.BtsI). The two systems are distinguished and monitored using different fluorescently labeled reporter complexes (such as FAM and ROX). The core principle is the high specificity of the two nicking enzymes (Nt.BbvCI and Nb.BtsI) and their recognition sequences, ensuring accurate identification and targeted recovery of abnormal pathways.

[0045] Single-path anomaly, specified rollback: When AS1 and the anomaly signal IS2 are input, only system 1 (FAM channel) has an output, while system 2 is suppressed by IS2. At this time, if Nb.BtsI and AS2 specifically targeting IS2 are added, system 2 is restored, and the system generates dual signal (FAM+ROX) outputs, both of which are restored to their maximum strength, realizing "specified rollback" only for the path affected by IS2.

[0046] If both paths fail, roll back separately: For example Figure 6 As shown, when IS1 and IS2 are input simultaneously, both channels are suppressed. By simultaneously adding Nt.BbvCI and Nb.BtsI along with their corresponding activation signals, the two channels can be restored separately, achieving parallel and non-interfering specified rollback.

[0047] Tests were conducted under different input combinations (AS1+IS2, AS2+IS1, IS1+IS2). Experimental results show that this specified rollback module can accurately identify the suppressed channel and restore the channel individually by adding the corresponding specific nicking enzyme, without affecting the state of other channels. This fully verifies its accurate anomaly handling and state recovery capabilities in a multi-instruction environment.

[0048] In summary, this invention establishes an efficient, reliable, and controllable nicking enzyme-assisted molecular signal rollback mechanism through precise molecular design and multi-parameter system optimization. This mechanism significantly improves the fault tolerance and environmental adaptability of molecular timing circuits, providing a novel technical tool for developing smarter and more robust molecular computing systems and biosensing platforms.

[0049] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

[0050] While the specific embodiments of this disclosure have been described above in conjunction with the accompanying drawings, this is not intended to limit the scope of protection of this disclosure. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of this disclosure are still within the scope of protection of this disclosure.

Claims

1. A molecular signal rollback method based on nicking enzyme assistance, characterized in that, Includes the following steps: A double-stranded DNA substrate Sub1 containing a nicking enzyme-specific recognition sequence is provided. The substrate is capable of responding to an activation signal AS1 and triggering the generation of an output signal, while also being able to bind to an inhibition signal IS1 and enter an inhibition state. When the inhibitory signal IS1 arrives in the reaction system before the activation signal AS1, IS1 specifically binds to the double-stranded DNA substrate Sub1 to form the inactive intermediate product Intermediate3. This intermediate product can block the molecular reaction pathway with the relay signal S1 and enter a quiescent state. A nicking enzyme is introduced into the quiescent reaction system. The nicking enzyme can specifically recognize and cleave the inactive intermediate product Intermediate3, causing the inhibitory signal IS1 to dissociate from the intermediate product, thereby restoring the inactive intermediate product Intermediate3 to the initially active double-stranded DNA substrate Sub1. An activation signal AS1 is provided to the reactivated double-stranded DNA substrate Sub1. The activation signal AS1 triggers the restart of the molecular timing circuit reaction path, prompting the generation of an output signal and realizing the signal rollback from the quiescent state to the activated state.

2. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, The activation signal AS1 triggers a strand displacement reaction in the double-stranded DNA substrate Sub1, thereby releasing the single-stranded DNA U1; The single-stranded DNA U1 can specifically bind to and react with the fluorescently labeled reporter complex RE to generate a detectable output signal.

3. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, The inhibitory signal IS1 contains a specific recognition sequence of the nicking enzyme embedded in its structural domain. When the inhibitory signal IS1 binds to the double-stranded DNA substrate Sub1 to form an inactive intermediate product, this specific recognition sequence is exposed and recognized and cleaved by the corresponding nicking enzyme.

4. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, The length of the binding domain d2 between the inhibition signal IS1 and the double-stranded DNA substrate Sub1 is 2 to 14 bases; in the domain of the inhibition signal IS1, the length of the spacer sequence between the region containing the nicking enzyme's specific recognition sequence and the binding domain d2 is not less than 2 bases.

5. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, After the nicking enzyme cleaves the inactive intermediate, it exposes a foothold of 2 to 6 bases in length on the double-stranded DNA substrate Sub1.

6. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, The concentrations of each component in the reaction system must meet the following conditions: the concentration of relay signal S1 is 1-2 times the concentration of substrate Sub1; the concentration of inhibition signal IS1 is 1-2 times the concentration of double-stranded DNA substrate Sub1; the concentration of activation signal AS1 is 1 to 4 times the concentration of double-stranded DNA substrate Sub1, of which 4 times the concentration is used to trigger rollback to achieve complete rollback; the concentration of nicking enzyme is 1 to 3 units (U) per reaction.

7. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, The reaction time between the inhibition signal IS1 and the double-stranded DNA substrate Sub1 is 0-20 minutes, the interval between enzyme digestion recovery and rollback activation is 0-20 minutes, and the activation signal AS1 is added at the same time as the nicking enzyme is introduced.

8. The molecular signal rollback method based on nicking enzyme assistance according to claim 1, characterized in that, The cleaving enzyme is selected from at least one of Nt.BbvCI, Nb.BtsI, and their functional analogs with the same recognition and cleavage specificity.

9. A molecular signal rollback system based on nicking enzyme assistance, characterized in that, include: The substrate preparation module provides a double-stranded DNA substrate Sub1 containing a nicking enzyme-specific recognition sequence. The substrate has the function of responding to the activation signal AS1 and triggering the generation of an output signal, and can also bind to the inhibition signal IS1 and enter an inhibition state. The inhibition trigger module, when the inhibition signal IS1 arrives in the reaction system before the activation signal AS1, IS1 specifically binds to the double-stranded DNA substrate Sub1 to form an inactive intermediate product Intermediate3. This intermediate product can block the molecular reaction pathway with the relay signal S1 and enter a quiescent state. The enzyme digestion recovery module introduces a nicking enzyme into the quiescent reaction system. The nicking enzyme can specifically recognize and cleave the inactive intermediate product Intermediate3, causing the inhibition signal IS1 to dissociate from the intermediate product, thereby restoring the inactive intermediate product Intermediate3 to the initially active double-stranded DNA substrate Sub1. The rollback activation module provides an activation signal AS1 to the reactivated double-stranded DNA substrate Sub1. The activation signal AS1 triggers the restart of the molecular timing circuit reaction path, prompting the generation of an output signal and realizing the signal rollback from the quiescent state to the activated state.

10. The molecular signal rollback system based on nicking enzyme assistance according to claim 9, characterized in that, It also includes a designated rollback module under multiple command inputs. This module provides at least two different double-stranded DNA substrates. Each substrate contains a corresponding preset molecular reaction pathway and a specific recognition sequence of a dedicated nicking enzyme. Each substrate also has a corresponding specific inhibition signal, specific activation signal, specific nicking enzyme, and dedicated reporter complex. When one or more of the aforementioned specific inhibition signals are input into the reaction system, the corresponding molecular reaction pathway is blocked. By introducing a specific nicking enzyme that matches the input specific inhibition signal, the abnormal state caused by the inhibition signal can be specifically rolled back, restoring the corresponding molecular reaction pathway without interfering with other molecular reaction pathways that have not received an inhibition signal. The reporter complex corresponding to each substrate independently monitors the signal output of each molecular reaction pathway.

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