An in situ detection method for DNA molecular reaction networks

Abstract

This invention provides an in-situ detection method for DNA molecular reaction networks, comprising: providing a reaction system including erroneous nodes; adding a diagnostic probe to the target node, reading the fluorescence signal and comparing it with a fluorescence intensity threshold to determine the state of the target node; the diagnostic probe includes an amplifier module and a fluorescent reporter molecule module, the amplifier module receiving the node signal chain of the target node and converting it into an amplified signal, and the fluorescent reporter molecule module converting the amplified signal into a fluorescence response; removing the fluorescent reporter molecule module of the diagnostic probe through biotin-streptavidin interaction and a magnetic field; repeating the addition and removal steps until the location of the erroneous node is accurately determined. This invention, by adding a diagnostic probe targeting a single node and combining it with a magnetic field-assisted fluorescent reporter molecule removal mechanism, achieves in-situ, stepwise, and site-specific fault detection of nodes at all levels of a DNA molecular reaction network.

Description

Technical Field

[0001] This invention relates to the field of DNA molecular reaction networks and DNA computing, specifically to an in-situ detection method for DNA molecular reaction networks, used to achieve in-situ fault detection of DNA molecular network reactions. Background Technology

[0002] DNA molecular reaction networks, with their high parallel computing power, programmable base sequences and structures, and biocompatibility, exhibit unique advantages over traditional silicon-based circuits in specific scenarios, and are expected to become the core carrier of next-generation molecular information processing. With the rapid development of DNA computing, information processing systems based on DNA molecular reaction networks have been gradually applied to complex logic operations, intelligent diagnostics, and molecular regulation.

[0003] Common DNA molecular reaction networks (MRNNs) consist of DNA strands, nucleases, ribozymes, and small molecule regulatory elements, including functional modules such as DNA logic gates, DNA amplifiers, threshold chains, and fuel chains. Each module achieves signal transmission and logical operations through base pairing and strand substitution reactions. However, the reliability of DNA MRNNs faces significant challenges in practical operation. Because DNA MRNNs rely on base pairing and strand substitution reactions for signal transmission and logical operations, the kinetic uncertainties caused by instantaneous intermolecular binding and dissociation, the accumulation of background leakage reactions, and the signal attenuation caused by interlayer transport all gradually accumulate in the multi-layer reaction chain and eventually translate into considerable computational errors. These problems not only limit the scalability of the circuit but also pose challenges to the stability and reproducibility of complex DNA MRNNs in practical applications.

[0004] Currently, fault detection in DNA molecular reaction networks mainly relies on fluorescence intensity reading or stepwise reconstruction methods. Specifically, fluorescence intensity reading involves applying fluorescent reporter molecules labeled with fluorescent and quenching groups to the output of the DNA molecular reaction network, using fluorescence kinetics to monitor changes in fluorescence signals in real time, thus determining the output result of the DNA molecular reaction network. However, this method can only read the terminal output and cannot analyze the reaction states of intermediate levels. When the circuit output is abnormal, it only tells us that there is an output error, but cannot determine which logic gate or signal transmission stage the fault occurred in. Stepwise reconstruction verification, on the other hand, starts by re-preparing and mixing the DNA logic gates, and combines this with fluorescence reading to detect and verify the correctness of each level, thereby locating the fault in the overall circuit. While this method can achieve fault location, it requires preparing all components of the sub-reaction network separately for each logic gate, a cumbersome process that increases experimental costs.

[0005] In summary, the existing methods have the following drawbacks: 1) Lack of fault location function: Traditional methods can usually only obtain the final output signal of the overall system, and cannot accurately locate the specific DNA reaction network node where the fault occurred.

[0006] 2) Low detection efficiency. In order to achieve fault location, the step-by-step reconstruction verification method requires the preparation of multiple sub-reaction networks and step-by-step testing, which is cumbersome and time-consuming, and cannot meet the rapid diagnosis needs of complex circuits.

[0007] 3) Inability to detect in situ, meaning it is difficult to identify and pinpoint faults within the circuit without re-fabricating the existing DNA reaction network components.

[0008] 4) Difficulty in repairing faulty reaction networks. Due to the lack of analytical capabilities regarding the internal state of the circuit, it is necessary to test individual sub-reaction networks step by step, making it impossible to establish a systematic fault diagnosis method for complex DNA reaction networks and thus preventing immediate repair of the reaction networks.

[0009] Therefore, developing an in-situ detection method for fault nodes in DNA molecular reaction networks is of great significance for improving the reliability, scalability, and engineering application capabilities of DNA computing systems. Summary of the Invention

[0010] The purpose of this invention is to provide an in-situ diagnostic method for DNA molecular reaction network faults, so as to realize in-situ, step-by-step and fixed-point fault detection of nodes at all levels of the DNA molecular reaction network.

[0011] To achieve the above objectives, the present invention provides an in-situ detection method for DNA molecular reaction networks, comprising: Step S1: Provide a reaction system, which includes the DNA molecule reaction network to be tested and the input signal of the DNA molecule reaction network, and includes error nodes; Step S2: Using the undetected child node as the target node, add the diagnostic probe of the target node to the reaction system, read the fluorescence signal and compare it with the fluorescence intensity threshold to determine the status of the target node; The diagnostic probe includes an amplifier module and a fluorescent reporter molecule module. The amplifier module of the diagnostic probe is used to receive the node signal chain released by the target node in the DNA molecular reaction network and convert it into an amplified signal. The fluorescent reporter molecule module converts the amplified signal output by the amplifier module into a fluorescent response through a chain substitution reaction. Step S3: Remove the fluorescent reporter molecule module of the diagnostic probe by biotin-streptavidin interaction and magnetic field; Step S4: Repeat steps S2 and S3 until the location of the erroneous node can be located based on the status of all detected target nodes. At this point, output the location of the erroneous node.

[0012] The amplifier module includes complementary paired amplifier release chains and amplifier complement chains; The fluorescent reporter module is a fluorescently labeled-quenched double-stranded DNA reporter structure, comprising complementary fluorescent reporter strands and quenched strands.

[0013] The amplifier complementary chain includes a node signal binding domain and a fuel chain binding domain. The node signal binding domain refers to the extended single-chain segment of the amplifier complementary chain outside the complementary pairing region of the amplifier release chain, and the fuel chain binding domain refers to the extended single-chain segment of the amplifier complementary chain outside the complementary pairing region of the fuel chain.

[0014] The node signal binding domain is 5 nucleotides long; the molar ratio of the fuel chain to the amplifier module is 5:1; and the fuel chain binding domain is 4 nucleotides long.

[0015] The fluorescent reporter chain is modified with biotin, enabling the fluorescent reporter molecule module to specifically bind to streptavidin-modified magnetic beads, thereby achieving in-situ removal using magnetic field separation.

[0016] Step S1 specifically includes: Step S11: Provide a reaction system, the reaction system including a DNA molecule reaction network to be tested and a terminal fluorescent reporter molecule for generating a terminal fluorescent signal of the DNA molecule reaction network, the DNA molecule reaction network including multiple computing nodes; Step S12: Add an input signal to the DNA molecular reaction network to cause the terminal fluorescent reporter molecule to generate a terminal fluorescent signal. Read the terminal fluorescent signal to determine whether the DNA molecular reaction network includes erroneous nodes. Step S13: If it is determined that there is an erroneous node, proceed to step S2; before proceeding to step S2, the following steps are also included: removing the terminal fluorescent reporter molecule by biotin-streptavidin interaction and magnetic field.

[0017] In step S2, the rule for determining the state of the target node is as follows: If the intensity of the fluorescence signal exceeds the positive determination threshold under the condition that the expected output is negative, the output is determined to be positive, and thus it is determined to be a false positive fault. If the intensity of the fluorescence signal is lower than the negative determination threshold under the expected positive output condition, the output is determined to be negative, and thus it is determined to be a false negative failure. Otherwise, it is considered normal.

[0018] For each child node, the corresponding sub-reaction network includes a high-signal channel component and a low-signal channel component, and the diagnostic probes include high-signal channel diagnostic probes and low-signal channel diagnostic probes.

[0019] For the same target node, the fluorescent molecules modified by the diagnostic probes of the high signal channel and the low signal channel are different and are added to the reaction system at the same time; when performing threshold comparison, the spectra of the two fluorescent molecules do not overlap, thereby realizing the simultaneous comparison of the high signal channel and the low signal channel with the threshold of fluorescence intensity to determine the state of the target node. Alternatively, for the same target node, diagnostic probes with high signal channels and low signal channels are added to the reaction system sequentially to compare the high signal channels and low signal channels with the fluorescence intensity thresholds respectively, in order to determine the state of the target node.

[0020] The in-situ detection method for DNA molecular reaction networks further includes step S5: replacing the sub-reaction network corresponding to the erroneous node in the reaction system to achieve in-situ repair of the DNA molecular reaction network.

[0021] The method of this invention adds a diagnostic probe targeting a single node in situ to the DNA molecular reaction network that has obtained erroneous output, and removes the output fluorescent reporter molecule by combining a magnetic field-assisted fluorescent reporter molecule removal mechanism. This enables in situ, stepwise and site-specific fault detection of nodes at each level of the DNA molecular reaction network. It eliminates the need to prepare a separate sub-reaction network for each level for fault diagnosis and can also be used to repair the molecular reaction network. Attached Figure Description

[0022] Figure 1 This is a flowchart of the in-situ detection method for DNA molecular reaction networks according to the present invention.

[0023] Figure 2 This is a schematic diagram of the structure of the DNA molecular reaction network and the location for introducing diagnostic probes.

[0024] Figure 3A and Figure 3B This is a schematic diagram of fluorescence signals for different fault types according to the rules for determining the state of the target node. Figure 3A The fluorescence signal indicating a false positive failure was shown. Figure 3B The fluorescence signal of the false negative failure is shown.

[0025] Figures 4A-4D This diagram illustrates the working principle and optimized parameters of the diagnostic probe. Figure 4A This is a schematic diagram illustrating the installation location of the diagnostic probe and the detection reaction principle, where the diagnostic probe is introduced into... Figure 2 Between sub-reaction network 1 and sub-reaction network 2 shown; Figure 4BThis is a diagram showing the dynamic optimization results of the amplifier binding domain length; Figure 4C This is a graph showing the effect of the fuel chain to amplifier molar ratio on probe kinetics; Figure 4D This is a graph showing the effect of the fuel chain binding domain length on probe performance.

[0026] Figure 5 This is a schematic diagram of the fluorescent reporter strand removal process in the in-situ detection method for DNA molecular reaction networks according to the present invention.

[0027] Figure 6A This is a schematic diagram of the process for stepwise in-situ fault localization in a DNA molecular reaction network with false positive faults, involving sub-node detection and magnetic bead-assisted reporter molecule removal.

[0028] Figure 6B This is a diagram illustrating the detection output results of each sub-node and the principle of step-by-step fault localization in a DNA molecular reaction network with false positive faults.

[0029] Figure 7A This is a schematic diagram of the process for stepwise in-situ fault localization in a DNA molecular reaction network with false negative faults, involving sub-node detection and magnetic bead-assisted reporter molecule removal.

[0030] Figure 7B This is a diagram illustrating the detection output results of each sub-node and the principle of step-by-step fault localization in a DNA molecular reaction network with false negative faults.

[0031] Figure 8A This is a schematic diagram of the process for stepwise in-situ fault localization in a fan-in architecture DNA molecular reaction network, involving sub-node detection and magnetic bead-assisted reporter molecule removal.

[0032] Figure 8B This is a diagram illustrating the detection output results of each sub-node and the principle of step-by-step fault location in a fan-in architecture DNA molecular reaction network.

[0033] Figure 9A This is a flowchart illustrating the stepwise in-situ fault localization process for sub-node detection and magnetic bead-assisted reporter molecule removal in a DNA molecular reaction network calculated using square root.

[0034] Figure 9B This is a diagram showing the detection output results of each sub-node and the principle analysis of step-by-step fault location in a DNA molecular reaction network calculated by square root.

[0035] Figure 10A This is a flowchart illustrating the in-situ diagnosis and repair of false-negative DNA molecular reaction networks using the in-situ detection method for DNA molecular reaction networks of this invention.

[0036] Figure 10BThis is a diagram showing the detection output results of each sub-node in the in situ diagnosis and in situ repair of the DNA molecular reaction network with negative faults.

[0037] Figure 10C This is the output dynamics curve of output terminal 2 after a normal logic gate is added in situ to repair the DNA molecular reaction network with a false negative fault. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto. The pharmaceuticals and reagents used in the embodiments can be purchased from chemical pharmaceutical companies or prepared by methods disclosed in the art.

[0039] like Figure 4A As shown, the in-situ detection method for DNA molecular reaction networks of the present invention is based on a diagnostic probe introduced into the target node between sub-reaction networks, including an amplifier module 10 and a fluorescent reporter molecule module 20. The amplifier module 10 of the diagnostic probe receives the node signal chain released by the target node in the DNA molecular reaction network and converts it into an amplified signal to improve signal accessibility and partially compensate for signal attenuation in deep cascades. The fluorescent reporter molecule module 20 employs a double-stranded DNA molecule structure labeled with a fluorescent group and a quencher group, and converts the amplified signal output from the amplifier module into a fluorescent response through a strand substitution reaction.

[0040] The amplifier module 10 is a double-stranded DNA structure, including complementary amplifier release strands S2 and S5 and amplifier complementary strands S1 and S4.

[0041] The amplifier complementary chain includes a node signal binding domain and a fuel chain binding domain. The node signal binding domain refers to an extended single-chain segment outside the complementary pairing region of the amplifier complementary chain and the amplifier release chain. This extended single-chain segment is used to specifically identify and bind to the node signal chain released by the target node when the amplifier release chain binds to the amplifier complementary chain, triggering a chain replacement reaction to release the amplifier release chain. The fuel chain binding domain refers to an extended single-chain segment outside the complementary pairing region of the amplifier complementary chain and the fuel chain. This extended single-chain segment is used to undergo a chain replacement reaction with the fuel chain when the node signal chain binds to the amplifier complementary chain, releasing the node signal chain and enabling the cyclic operation of the amplifier module.

[0042] The amplifier release chain is paired with the amplifier complementary chain. Under the sequential triggering of the node signal chain and the fuel chain, the amplifier release chain is dissociated and released, and transmitted as an amplified signal to the fluorescent reporter molecule module.

[0043] The fluorescent reporter module 20 is a fluorescently labeled-quenched double-stranded DNA reporter structure, including complementary fluorescent reporter strands S7 and S9 and quenched strands S8 and S10.

[0044] When the amplifier releases the reporter chain, the fluorescent reporter module dissociates the reporter chain from the quencher chain via a chain substitution reaction, generating a detectable fluorescent signal, thereby converting the molecular signal into a fluorescent readout signal. One end of the reporter chain is modified with a fluorophore, and one end of the quencher chain is modified with a quencher group. Thus, the quencher group and the fluorophore, in a double-stranded pairing state, form a fluorescence resonance energy transfer, placing the fluorescent signal in a quenched state and allowing the fluorophore to emit a fluorescent signal in a dissociated state.

[0045] The fluorescent reporter chain is modified with biotin, enabling it to specifically bind to streptavidin-modified magnetic beads, and then remove them in situ using a magnetic field. Specifically, the fluorescent reporter chain is captured in situ through the biotin-streptavidin interaction, and then magnetically removed under an external magnetic field. This design allows the same reaction system to be repeatedly accessed in multiple readouts, avoiding signal crosstalk caused by the accumulation of fluorescent labels in the system. This provides a basis for signal reset for multi-round iterative detection, supports multi-level detection, eliminates the need for step-by-step preparation of sub-reaction networks, does not damage the original reaction system and environment, and achieves high detection efficiency.

[0046] In this embodiment, the 5' end of the fluorescent reporter chain is modified with biotin and the 3' end with a fluorescent group; the 5' end of the quencher chain is modified with a quencher group.

[0047] The key parameters of the diagnostic probe were optimized, including: 1) amplifier binding domain length (T act ): Optimized to 5 bases, this probe length provides the fastest response speed (e.g. Figure 4B As shown in the figure), the fluorescence kinetics of the tuning probe were measured using amplifier binding domain lengths of 4, 5, 6, and 7 nucleotides, respectively. The results showed that the design with a 5-nucleotide length had the fastest response speed. 2) Fuel chain concentration: The molar ratio of fuel chain to amplifier module was optimized to 5:1. At this molar ratio, the reaction rate reached saturation and non-specific interactions (such as...) could be avoided. Figure 4C (As shown). Increasing the fuel chain concentration accelerates the chain displacement reaction until a saturation region is reached; when the fuel chain concentration is 5 times excessive, a saturation region is reached, and further increases in concentration offer limited performance improvement. 3) Fuel chain binding domain length (T) amp ): Optimized to 4 bases, this length ensures the reaction rate while reducing the sequence length (e.g. Figure 4D (As shown).

[0048] like Figure 1As shown, an in-situ detection method for DNA molecular reaction networks according to the present invention includes the following steps: Step S1: Provide a reaction system, which includes the DNA molecule reaction network to be tested and the input signal of the DNA molecule reaction network, and includes error nodes; Specifically, step S1 includes: Step S11: Provide a reaction system, the reaction system including a DNA molecule reaction network to be tested and a terminal fluorescent reporter molecule for generating a terminal fluorescent signal of the DNA molecule reaction network, the DNA molecule reaction network including multiple computing nodes; The DNA molecular reaction network may include erroneous nodes, such as intermediate breakpoints, that cause errors in the computational results.

[0049] Step S12: Add an input signal to the DNA molecular reaction network to cause the terminal fluorescent reporter molecule to generate a terminal fluorescent signal. Read the terminal fluorescent signal to determine whether the DNA molecular reaction network includes erroneous nodes. like Figure 4A and Figure 4B As shown, each DNA molecular reaction network includes multiple sub-reaction networks and signal transmission lines connecting different sub-reaction networks. The DNA molecular reaction network adopts a dual-track logic architecture, that is, each sub-reaction network and signal transmission line includes a high-signal channel component and a low-signal channel component. The combination of the output signals of the high-signal channel and the low-signal channel represents the logic operation result. Therefore, the read terminal fluorescence signal includes the terminal fluorescence signals of the high-signal channel and the low-signal channel. The logic operation result is determined based on these two terminal fluorescence signals and compared with the expected logic operation result of the DNA molecular reaction network to confirm whether the logic operation result is correct.

[0050] In this embodiment, as Figure 2 and Figure 4A As shown, the DNA molecule reaction network includes sub-reaction network 1, sub-reaction network 2, sub-reaction network 3 and corresponding signal transmission lines; the yellow parts of sub-reaction network 1 and sub-reaction network 2 are high signal channel components, and the blue parts are low signal channel components; the red parts of the signal transmission lines are high signal channel components, and the green parts are low signal channel components.

[0051] Since sub-reaction network 1 is an OR gate, it performs dual-track logic operations based on the following principle: when the high-signal channel input chain in... H When triggered and involved in the reaction, the high-signal channel is activated, thereby outputting the high-signal channel output chain out. H As the result of the logic operation output of the high-signal channel, it corresponds to logic "1"; when the first and second low-signal channel input chains in1L in2 L When triggered and involved in the reaction, the low-signal channel is activated, thereby outputting the low-signal channel output chain (out). L The result of the logic operation on the low-signal channel output corresponds to logic "0". After the operation is completed, only one channel generates a valid output chain (i.e., only the high-signal channel output chain "out" is output). H or low signal channel output chain out L ), and serve as the input signal for downstream sub-reaction networks or the node signal identified by diagnostic probes, with the high-signal channel output chain out. H Only the high-signal channel of the downstream sub-reaction network can be triggered; the low-signal channel output chain is out. L It can only trigger the low-signal channels of downstream sub-reaction networks. High and low signal channels operate mutually exclusively to avoid signal crosstalk and ensure stable computation results.

[0052] Sub-reaction network 2 is an AND gate. Its high-signal channel operates in a similar manner to the low-signal channel of sub-reaction network 1, and its low-signal channel operates in a similar manner to the high-signal channel of sub-reaction network 2.

[0053] In other embodiments, the number and specific type of each sub-reaction network can vary as needed, meaning the specific structure of the DNA molecule reaction network is arbitrary.

[0054] The signal transmission line is set between two connected sub-reaction networks. It consists of a threshold module and a signal transmission amplifier. The threshold module is used to absorb some leakage signals generated by the previous sub-reaction network. The signal transmission amplifier is used for signal amplification and signal conversion, amplifying the signal generated by the previous sub-reaction network to the same concentration and using it as the input signal for the next sub-reaction network.

[0055] Step S13: If it is determined that an erroneous node is included, proceed to step S2.

[0056] If the logical operation result is incorrect, it is determined that an erroneous node is included, and step S2 needs to be executed; otherwise, if the logical operation result is correct, it is determined that there is no erroneous node, and the process ends.

[0057] Additionally, optionally, prior to performing step S2, the process may include removing the terminal fluorescent reporter molecule via biotin-streptavidin interaction and a magnetic field.

[0058] Figure 5 This is a schematic diagram of the fluorescent reporter strand removal process in the in-situ detection method for DNA molecular reaction networks according to the present invention. Figure 5As shown, in this embodiment, the terminal fluorescent reporter molecule is modified with biotin. By adding streptavidin-modified magnetic beads to the reaction system, the magnetic beads capture the biotin-modified terminal fluorescent reporter molecule through biotin-streptavidin interaction. Under the action of an external magnetic field, the magnetic beads containing the captured terminal fluorescent reporter molecule are separated and removed from the reaction system, restoring the fluorescence signal to the baseline level. This design allows the same reaction system to be repeatedly accessed in multiple readouts, avoiding signal crosstalk caused by the accumulation of fluorescent labels in the system from multiple rounds. This provides a basis for signal reset for multi-round iterative detection, supports multi-level detection, eliminates the need for step-by-step preparation of sub-reaction networks, does not damage the original reaction system and reaction environment, and has high detection efficiency.

[0059] Step S2: Using the undetected child node as the target node, add the diagnostic probe of the target node to the reaction system, read the fluorescence signal and compare it with the fluorescence intensity threshold to determine the status of the target node; Therefore, by introducing diagnostic probes into the sub-nodes between sub-reaction networks, direct detection of intermediate states can be achieved, thereby distinguishing faulty modules. For example... Figure 2 and Figure 4A As shown, for example, diagnostic probes can be introduced into Figure 2 The sub-nodes between sub-reaction network 1 and sub-reaction network 2 are shown to enable state monitoring of the target node at the output end of sub-reaction network 1.

[0060] In this embodiment, a fluorescence detection device is used to read the intensity of the fluorescence signal, record the fluorescence dynamics curve of the target node, and compare the intensity of the fluorescence signal with a fluorescence intensity threshold to determine whether the target node is in a normal or faulty state.

[0061] The fluorescence intensity threshold refers to the positive and negative determination thresholds for the expected output fluorescence intensity of a normal DNA molecule reaction network. In this embodiment, the positive determination threshold is 0.6 and the negative determination threshold is 0.4. When the fluorescence intensity is below the negative determination threshold of 0.4, the output is determined to be 0 (i.e., negative), and when it is above the positive determination threshold of 0.6, the output is determined to be 1 (i.e., positive). Figure 3A and Figure 3B The rules for judging the status of the target node are as follows: if the intensity of the fluorescence signal exceeds the positive judgment threshold (i.e., the output is judged to be positive) under the condition that the expected output is negative, it is judged to be a false positive fault; if the intensity of the fluorescence signal is lower than the negative judgment threshold (i.e., the output is judged to be negative) under the condition that the expected output is positive, it is judged to be a false negative fault; otherwise, it is judged to be normal.

[0062] For example, in an OR logic circuit, given the input combination 11, the expected output is 1, but the actual output is lower than 0.4, which is a false negative output. Similarly, in an OR logic circuit, given the input combination 00, the theoretical output is 0, but the actual output is lower than 0.6, which is a false positive output.

[0063] In this embodiment, for each sub-node, the corresponding sub-reaction network includes a high-signal channel component and a low-signal channel component. Correspondingly, the diagnostic probes include high-signal channel diagnostic probes and low-signal channel diagnostic probes. In this embodiment, for the same target node, the high-signal channel diagnostic probes and low-signal channel diagnostic probes are modified with different fluorescent molecules and are added to the reaction system simultaneously. For example, the high-signal channel diagnostic probe is a modified TET fluorescent molecule, excited at 510 nm and emitting at 540 nm; the low-signal channel diagnostic probe is a modified Cy5 fluorescent molecule, excited at 640 nm and emitting at 670 nm. When threshold comparison is performed using a microplate reader, the spectra of the two fluorescent molecules do not overlap, thereby enabling simultaneous comparison of the high-signal channel and low-signal channel with the fluorescence intensity threshold to determine the state of the target node.

[0064] In other embodiments, for the same target node, diagnostic probes with high signal channels and diagnostic probes with low signal channels are added to the reaction system in sequence to compare the high signal channels and low signal channels with the fluorescence intensity thresholds respectively, so as to determine the state of the target node.

[0065] Step S3: Remove the fluorescent reporter molecule module of the diagnostic probe by biotin-streptavidin interaction and magnetic field; As described above, the fluorescent reporter chain of the fluorescent reporter molecule module is modified with biotin, enabling the fluorescent reporter chain to specifically bind to streptavidin-modified magnetic beads, and then remove them in situ using magnetic field separation. In step S3, streptavidin-modified magnetic beads are added to the reaction system, and the magnetic beads capture the biotin-modified fluorescent reporter chain through biotin-streptavidin interaction. Under the action of an external magnetic field, the magnetic beads containing the captured fluorescent reporter chain are separated and removed from the reaction system, restoring the fluorescence signal to the baseline level. This design allows the same reaction system to be repeatedly accessed in multiple readouts, avoiding signal crosstalk caused by the accumulation of fluorescent labels in the system from multiple rounds, thus providing a signal reset basis for multi-round iterative detection, supporting multi-level detection, eliminating the need for step-by-step preparation of sub-reaction networks, not damaging the original reaction system and reaction environment, and achieving high detection efficiency.

[0066] In this process, the fluorescence intensity of the reaction system was measured before the streptavidin-modified magnetic beads were added, and this was used as the first fluorescence intensity. After the streptavidin-modified magnetic beads were added, an external magnetic field was applied to separate and remove the magnetic beads that captured the fluorescent reporter molecules from the reaction system, leaving the solution system. The fluorescence intensity of the reaction system was then measured, and this was used as the second fluorescence intensity. The first fluorescence intensity was normalized, and if the second fluorescence intensity was lower than 0.4, it was considered to have recovered to the baseline level.

[0067] Step S4: Repeat steps S2 and S3 until the location of the erroneous node can be located based on the status of all detected target nodes. At this point, output the location of the erroneous node.

[0068] In this embodiment, when repeating step S2, undetected child nodes are selected sequentially level by level according to the connection relationship of the sub-reaction networks to serve as target nodes. Specifically, after detecting the child nodes at the output end of the current level's sub-reaction network, a diagnostic probe is added to the child nodes at the output end of the previous level's sub-reaction network. The previous level's sub-reaction network is a sub-reaction network connected to the input end of the current level's sub-reaction network to determine the status of the target node in the next round. Thus, based on the detection results of each level's sub-reaction network, the fault range is eliminated level by level: if the status of the current level's child node is normal, the faulty node is located in the downstream module of the child node. If the status of the current level's child node is faulty, the faulty node is located in the current level's child node or its upstream module.

[0069] Step S5: Replace the sub-reaction network corresponding to the erroneous node in the reaction system to achieve in-situ repair of the DNA molecular reaction network.

[0070] Specifically, when the error node is a false negative fault, a sub-reaction network corresponding to the error node is added to achieve in-situ repair of the DNA molecular reaction network.

[0071] Example 1: Preparation of a diagnostic probe for in-situ detection of DNA molecular reaction networks

[0072] The sequences of the diagnostic probes are designed based on the sequences of the corresponding sub-reaction networks (such as logic gates). Each probe contains two modules: an amplifier module 10 and a fluorescent reporter molecule module 20. The amplifier module 10 receives the node signal strands released from the target node in the DNA molecular reaction network and converts them into amplified signals to improve signal accessibility and partially compensate for signal attenuation in deep cascades. The fluorescent reporter molecule module 20 uses a double-stranded DNA molecule structure labeled with a fluorescent group and a quencher group, converting the amplified signal output from the amplifier module into a fluorescent response through a strand substitution reaction. The amplifier module 10 includes complementary pairing amplifier releasing strands S2 and S5, and amplifier complementary strands S1 and S4, which are obtained through annealing. The fluorescent reporter molecule module 20 includes complementary pairing of fluorescent reporter strands S7 and S9, and quencher strands S8 and S10, which are obtained through annealing.

[0073] The buffers used for preparing the amplifier release strand, amplifier complementary strand, fluorescent reporter strand, and quencher strand were all 1×TE buffer containing 12.5 mM Mg²⁺. The annealing procedure was the same as that used for other double-stranded complexes. All DNA strands were synthesized by Sangon Biotech Co., Ltd. via the solid-phase phosphoridamide method. According to design requirements, specific strands of the fluorescent reporter molecule were modified. The fluorescent strand was double-labeled with biotin at the 5' end and a fluorescent group at the 3' end, and hybridized with the complementary quencher strand labeled with BHQ2 at the 5' end. The synthesized products were purified by high-performance liquid chromatography (HPLC) to remove short-chain impurities and byproducts generated during synthesis. The purified DNA strands were identified by electrospray ionization mass spectrometry (ESI-MS), confirming that the molecular weight was consistent with the theoretical value and the purity was ≥95%. Each purified DNA strand was dissolved in 1×TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to prepare a 100 μM stock solution, which was stored at -20°C for later use.

[0074] The amplifier release strand and amplifier complementary strand of the amplifier module of the DNA diagnostic probe were prepared at a molar ratio of 1:1.2, with corresponding concentrations of 10 μM for the amplifier release strand and 12 μM for its complementary strand. The quencher strand and fluorescent reporter strand of the fluorescent reporter molecule module of the DNA diagnostic probe were prepared at a molar ratio of 1.5:1, with corresponding concentrations of 15 μM for the quencher strand modified with the quencher group and 10 μM for the fluorescent reporter strand modified with biotin and a fluorescent group. The single strands were mixed in 1×TE buffer containing 12.5 mM MgCl2 to prepare the corresponding double-stranded DNA. The PCR program used for preparing the double-stranded DNA was: 95 °C for 2 minutes, followed by slow cooling to room temperature at a rate of 0.1 °C every 6 seconds.

[0075] Example 2: Removal of fluorescent reporter molecules assisted by magnetic beads

[0076] like Figure 5 As shown, firstly, the fluorescence intensity of the DNA molecular reaction network was measured at the endpoint of the reaction, with a fluorescent reporter molecule concentration of 500 nM. Then, at a mass ratio of biotin-modified fluorescent reporter DNA molecules to streptavidin (SA)-modified magnetic beads of appropriate amounts, a 1.5 mL microcentrifuge tube was transferred and pretreated according to commercially available magnetic bead treatment methods (such as Thermo Scientific). Next, the completed DNA molecular reaction network solution was added to the centrifuge tube containing the magnetic beads, and the tube was gently incubated at room temperature on a rotary mixer for 30 minutes to promote efficient binding. After incubation, the centrifuge tube was placed on a magnetic rack and allowed to stand for 5 minutes. The supernatant (containing the remaining logic gates in the DNA molecular reaction network and intermediate strands generated during the reaction) was collected for subsequent fluorescence measurement to quantify the residual reporter molecule signal in the supernatant.

[0077] Example 3: In-situ detection of DNA molecular reaction network faults

[0078] like Figure 6A As shown, a fault detection demonstration is performed on a dual-track, three-layer cascaded reaction network, introducing a false positive fault at gate 2. Under an input condition of 110, the following steps were performed sequentially: readout of the reaction network's endpoint signal, removal of the fluorescent reporter molecule, detection of sub-node 1, removal of the fluorescent reporter chain, and detection of sub-node 2. The terminal output is shown at out. H The channel is behaving abnormally; instead of the expected negative output, a false positive occurs, indicating an error in the terminal's (i.e., child node 3) output. L The channel remained correct. After removing the terminal reporter molecule, fluorescence returned to baseline levels, confirming effective reset of the endpoint readout (e.g., Figure 6B (As shown). In the same reaction mixture, the output node 2 of gate 2 is tested, and an abnormal signal is generated again, indicating that the output of node 2 is incorrect. After the second round of extraction and testing, the output node 1 of gate 1 exhibits the expected normal behavior (e.g., ...). Figure 6B As shown in the diagram, this indicates that sub-node 1 outputs correctly. This sequential pattern ultimately locates the faulty node at the output of gate 2, consistent with the preset defect location. These results collectively demonstrate that magnetically assisted reporter molecule extraction can effectively reset the readout layer while preserving sufficient circuitry functionality to support iterative fault localization. This reset mechanism enables stepwise fault localization directly within the original DNA circuitry mixture.

[0079] Example 4: In-situ detection of reaction networks for various types of faulty DNA molecules

[0080] The in-situ detection method for DNA molecular reaction networks of this invention is universally applicable to different failure modes and DNA molecular reaction network architectures. For example... Figure 7A and Figure 7B As shown, the in-situ detection method for DNA molecular reaction networks of the present invention is applied to the fault detection of DNA molecular reaction networks containing false negative faults. Its steps include reading out the fluorescence signal of the terminal, removing the fluorescent reporter molecule, and detecting sub-node 1. Among these steps, the high signal channel out... H The system should have output a positive fluorescence signal, but initially, the output was below the threshold of 0.4, resulting in a negative result and a false negative. Under input conditions where an output signal should have been generated, the endpoint fluorescence showed the expected output signal was missing. Sequential testing of child node 1 revealed that the upstream child node remained functional, while the downstream terminal node (i.e., child node 3) exhibited an erroneous response, thus pinpointing the fault to gate 3 corresponding to child node 3. This result demonstrates that this detection strategy can not only identify abnormal inputs but also detect the loss of transmitted signals in the original reaction mixture, i.e., it can identify false negative results.

[0081] In fan-in architecture DNA molecular reaction networks, anomalous endpoint behavior can stem from defects in any branch or downstream integrated logic gates, making localization solely through output readout particularly difficult. Therefore, we introduce node-specific diagnostic probes (such as...) at the two input branches and the converging downstream node. Figure 8A As shown), the sequence of the diagnostic probes is designed based on the sequence of the corresponding sub-reaction network. Step-by-step detection is displayed (e.g.) Figure 8B As shown in the diagram, one upstream branch exhibits the expected response, while another branch exhibits anomalous behavior, and this anomalous behavior is propagated to the integration stage. This result allows for the direct identification of defective branches without refactoring subcircuits, demonstrating that the method of this invention remains applicable even when multiple upstream paths contribute to the same output.

[0082] We then extend our analysis to more complex multi-layered architectures: square root-calculated DNA molecular reaction networks. For example... Figure 9A and Figure 9B As shown, despite increased circuit depth and topological complexity, this detection method can still progressively narrow down the fault location. By comparing the fluorescence responses of selected internal nodes, we can distinguish normally functioning modules from defective regions, thereby locating the pre-defined fault within the complete circuit. In summary, these results demonstrate that the proposed fault location framework is applicable to both false positive and false negative fault behaviors and remains effective in cascaded and branched DNA circuits. This universality supports its use as a general tool for multilayer DNA computational in situ analysis.

[0083] Example 5: In-situ detection and repair of faulty nodes for false negative faults

[0084] The method of this invention can achieve in-situ detection in various faulty circuits, and the identified faulty sites can be repaired by directly adding the correct components to the original reaction mixture. The repair principle is that the false-negative output behavior of the DNA molecular reaction network stems from the faulty module's inability to generate sufficient output signals in response to upstream inputs. Once the faulty module is successfully located, in principle, the DNA molecular reaction network should be repairable by simply replacing the faulty logic gates, without needing to reconstruct the entire circuit (e.g., ...). Figure 10A (As shown).

[0085] To demonstrate this repair strategy, a reaction network exhibiting a false negative output at output port 2 was constructed. With an input combination of 110, the endpoint report showed that output port 1 behaved normally, while the outH signal at output port 2 failed to reach the expected activation state. To locate the defect, the endpoint report molecule was first removed to reset the system, followed by breakpoint detection using debug probes. Gate 1 responded normally, ruling out insufficient upstream input or module failure. Detection of the parallel branch of gate 2 also showed the expected output, narrowing the defect to gate 4 (e.g., ...). Figure 10B (As shown).

[0086] After fault location was completed, a newly fabricated, functional AND gate was directly added to the system to replace the defective module in situ. Following this modular update, the outH signal at output port 2 resumed its expected upward trend and exceeded the threshold, while the outL signal maintained a low background level. The terminal output thus returned to the correct logic state (e.g., ...). Figure 10C (As shown). This repair model is naturally applicable to loss-of-function failures (i.e., false negative failures).

[0087] The method of this invention adds a diagnostic probe targeting a single node in situ to the DNA molecular reaction network that has obtained erroneous output, and removes the output fluorescent reporter molecule by combining a magnetic field-assisted fluorescent reporter molecule removal mechanism. This enables in situ, stepwise and site-specific fault detection of nodes at each level of the DNA molecular reaction network. It eliminates the need to prepare a separate sub-reaction network for each level for fault diagnosis and can also be used to repair the molecular reaction network.

[0088] Compared with the prior art, the present invention has the following characteristics: (1) In-situ detection without disrupting the original state of the reaction network. This invention achieves in-situ analysis of the state of intermediate nodes by directly adding diagnostic probes to the reaction system. Compared with the fault detection methods in the prior art that require reconstruction of sub-networks, this invention analyzes the internal state of the network step by step in situ without disturbing the original reaction mixture, thus preserving the integrity of the reaction network and the original reaction environment.

[0089] (2) Precisely locate the fault node. The present invention uses a modularly designed diagnostic probe that specifically identifies the chain signal released by the intermediate node through DNA sequence and converts it into fluorescent output. Through an iterative process of "adding probe - reading signal - removing probe - next level detection", the fault range is eliminated step by step, and the failure node is precisely located.

[0090] (3) Fault detection in multi-level networks does not require the preparation of sub-reaction networks at each level, thus preserving the original reaction system and environment and achieving high detection efficiency. This invention utilizes biotin-streptavidin interactions and magnetic field-assisted separation to achieve in-situ capture and removal of fluorescent reporter molecules, enabling the same reaction system solution to support multiple rounds of iterative detection. Compared with existing step-by-step reconstruction verification methods that require the separate preparation of sub-reaction network solutions for each logic gate, which is cumbersome and costly, this invention can complete multi-level detection in the original reaction mixture, significantly improving detection efficiency and reducing experimental costs.

[0091] (4) Supports systematic fault diagnosis and enables transferable understanding of failure mechanisms. The in-situ detection method of this invention transforms the single endpoint readout in the prior art into hierarchical local state readout through iterative detection steps, possessing the operability and scalability of cross-level iterative readout. Through standardized detection procedures, the operating state of each node within the DNA reaction network can be systematically analyzed, providing data support for the study of failure mechanisms in complex DNA reaction networks and contributing to the establishment of transferable reliability optimization methods.

[0092] (5) Possesses repairable and expandable capabilities. Based on the precise location of the failure node, this invention can also achieve in-situ modular repair of the fault: by directly replacing the located faulty module, the correct output function can be restored without reconstructing the entire reaction network. This feature transforms the DNA molecular reaction network from a "black box" that can only read the endpoint into a detectable, maintainable, and repairable molecular information system, laying an experimental foundation for the construction of future adaptive and self-repairing DNA computing systems.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. An in-situ detection method for DNA molecular reaction networks, characterized in that, include: Step S1: Provide a reaction system, which includes the DNA molecule reaction network to be tested and the input signal of the DNA molecule reaction network, and includes error nodes; Step S2: Using the undetected child node as the target node, add the diagnostic probe of the target node to the reaction system, read the fluorescence signal and compare it with the fluorescence intensity threshold to determine the status of the target node; The diagnostic probe includes an amplifier module and a fluorescent reporter molecule module. The amplifier module is used to receive the node signal chain released by the target node and convert it into an amplified signal. The fluorescent reporter molecule module converts the amplified signal into a fluorescent response through a chain substitution reaction. Step S3: Remove the fluorescent reporter molecule module of the diagnostic probe by biotin-streptavidin interaction and magnetic field; Step S4: Repeat steps S2 and S3 until the location of the erroneous node can be located based on the status of all detected target nodes. At this point, output the location of the erroneous node.

2. The in-situ detection method for DNA molecular reaction networks according to claim 1, characterized in that, The amplifier module includes complementary paired amplifier release chains and amplifier complement chains; The fluorescent reporter module is a fluorescently labeled-quenched double-stranded DNA reporter structure, comprising complementary fluorescent reporter strands and quencher strands.

3. The in-situ detection method for DNA molecular reaction networks according to claim 2, characterized in that, The amplifier complementary chain includes a node signal binding domain and a fuel chain binding domain. The node signal binding domain refers to the extended single-chain segment of the amplifier complementary chain outside the complementary pairing region of the amplifier release chain, and the fuel chain binding domain refers to the extended single-chain segment of the amplifier complementary chain outside the complementary pairing region of the fuel chain.

4. The in-situ detection method for DNA molecular reaction networks according to claim 3, characterized in that, The node signal binding domain is 5 nucleotides long; the molar ratio of the fuel chain to the amplifier module is 5:1; and the fuel chain binding domain is 4 nucleotides long.

5. The in-situ detection method for DNA molecular reaction networks according to claim 3, characterized in that, The fluorescent reporter chain is modified with biotin, enabling the fluorescent reporter molecule module to specifically bind to streptavidin-modified magnetic beads, thereby achieving in-situ removal using magnetic field separation.

6. The in-situ detection method for DNA molecular reaction networks according to claim 1, characterized in that, Step S1 specifically includes: Step S11: Provide a reaction system, the reaction system including a DNA molecule reaction network to be tested and a terminal fluorescent reporter molecule for generating a terminal fluorescent signal of the DNA molecule reaction network, the DNA molecule reaction network including multiple computing nodes; Step S12: Add an input signal to the DNA molecular reaction network to cause the terminal fluorescent reporter molecule to generate a terminal fluorescent signal. Read the terminal fluorescent signal to determine whether the DNA molecular reaction network includes erroneous nodes. Step S13: If it is determined that there is an erroneous node, proceed to step S2; before proceeding to step S2, the following steps are also included: removing the terminal fluorescent reporter molecule by biotin-streptavidin interaction and magnetic field.

7. The in-situ detection method for DNA molecular reaction networks according to claim 1, characterized in that, In step S2, the rule for determining the state of the target node is as follows: If the intensity of the fluorescence signal exceeds the positive determination threshold under the condition that the expected output is negative, the output is determined to be positive, and thus it is determined to be a false positive fault. If the intensity of the fluorescence signal is lower than the negative determination threshold under the expected positive output condition, the output is determined to be negative, and thus it is determined to be a false negative fault. Otherwise, it is considered normal.

8. The in-situ detection method for DNA molecular reaction networks according to claim 1, characterized in that, For each child node, the corresponding sub-reaction network includes a high-signal channel component and a low-signal channel component, and the diagnostic probes include high-signal channel diagnostic probes and low-signal channel diagnostic probes.

9. The in-situ detection method for DNA molecular reaction networks according to claim 1, characterized in that, For the same target node, the fluorescent molecules modified by the diagnostic probes of the high signal channel and the low signal channel are different and are added to the reaction system at the same time; when performing threshold comparison, the spectra of the two fluorescent molecules do not overlap, thereby realizing the simultaneous comparison of the high signal channel and the low signal channel with the threshold of fluorescence intensity to determine the state of the target node. Alternatively, for the same target node, diagnostic probes with high signal channels and low signal channels are added to the reaction system sequentially to compare the high signal channels and low signal channels with the fluorescence intensity thresholds respectively, in order to determine the state of the target node.

10. The in-situ detection method for DNA molecular reaction networks according to claim 1, characterized in that, It also includes step S5: replacing the sub-reaction network corresponding to the erroneous node in the reaction system to achieve in-situ repair of the DNA molecular reaction network.

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