Signal processing method based on exonuclease-driven modular DNA reaction networks

Abstract

The application belongs to the field of molecular information processing, and discloses a signal processing method based on exonuclease-driven modular DNA reaction network. The application is based on a simple core unit, and by combining other functional units, four modular DNA reaction networks including signal selection, signal time-adjustable self-resetting, multi-input and output signal amplification, and signal delay continuous adjustment are established, and various signal processing tasks are realized. The method can simplify the design process of the DNA reaction network, improve the reusability and scalability of the network, and is expected to bring new design ideas and application methods for the fields of biological computing, nanomachines, biosensing and the like.

Description

Technical Field

[0001] This invention belongs to the field of molecular information processing, specifically relating to a signal processing method based on a modular DNA reaction network driven by exonuclease. Background Technology

[0002] DNA reaction networks are widely used to construct nanoscale machines and complex computational circuits to perform specific molecular information processing tasks. To date, DNA reaction networks are used to construct logic gate circuits through calculations such as AND, OR, NOT, XOR, and NAND, and by combining these basic functions, more complex digital functions are realized. Furthermore, DNA reaction networks can also simulate the information processing processes of chemical reaction networks, finding wide application in molecular detection and disease diagnosis (DNA-Based Dynamic Reaction Networks. Trends in Biochemical Sciences, 2018, 43(7), 547-560). DNA reaction networks have been proven to have the ability to process information at the nanoscale. However, realizing diverse signal processing tasks using DNA reaction networks often requires large amounts of structurally complex DNA substrates, making modular design of DNA reaction networks difficult, thus increasing network complexity and making it difficult to build large-scale integrated reaction networks (Information processing using an integrated DNA reaction network. Nanoscale, 2021, 13(11), 5706-5713.). Summary of the Invention

[0003] The purpose of this invention is to provide a method for performing multiple signal processing tasks based on a simple molecular structure. It uses a simple core unit as a foundation and, by combining other functional units, establishes four modular DNA reaction networks: signal selection, adjustable signal time self-resetting, multi-input / output signal amplification, and continuously adjustable signal delay. This enables the implementation of various signal processing tasks. The reusability of the core unit significantly reduces the workload of DNA sequence design and simplifies the design process of DNA reaction networks. This lays a solid foundation for building more powerful and complex DNA networks and provides a promising tool for processing molecular information.

[0004] The technical solution of this invention:

[0005] A signal processing method based on a nuclease-driven modular DNA reaction network includes the following steps:

[0006] Step 1: Construction and operation of the signal selection module

[0007] The substrate for the signal selection module consists of a core unit and two reporters. The core unit is a symmetrical structure formed by annealing two 5' phosphate-modified DNA strands and two unphosphate-modified DNA strands. The two 5' phosphate-modified DNA strands hybridize to form two 3' overhangs, which then hybridize with the two unphosphate-modified DNA strands to form a core unit with two 5' overhangs. When an input is added to the core unit, the two input strands can select the two 5' overhangs of the core unit respectively, and different output strands are obtained through sequential DNA strand substitution reactions and enzymatic digestion by lambda exonuclease (λExo), ensuring the scalability of subsequent modular network design. The two reporters are used to receive the two unphosphate-modified output strands produced by the core unit. One reporter consists of two complementary strands modified with a ROX fluorophore and a BHQ2 quencher, while the other reporter consists of two complementary strands modified with a FAM fluorophore and a BHQ1 quencher. Based on the two different output strands received, the two reporters split their fluorescent and quenching groups, thus producing two different fluorescence signals. This signal selection module can generate two different fluorescence signals based on two different input chains, thus achieving signal selection functionality.

[0008] Step 2: Construction and Operation of the Signal Self-Reset Module

[0009] The signal self-reset module consists of a core unit and a self-reset unit. The core unit is the same as that of the signal selection module. The self-reset unit comprises three DNA strands: one strand is modified with a ROX fluorescent group at its 3' end, and another strand is modified with a BHQ2 quenching group at its 5' end. Both of these DNA strands hybridize with the unmodified third DNA strand. During operation, the core unit generates an output strand, which then undergoes a DNA strand substitution reaction with the self-reset unit, inducing the release of the signal strand and resulting in enhanced fluorescence. At this point, the self-reset unit forms a hydrolyzable substrate that can interact with λExo. After hydrolysis, the signal strand rehybridizes with the hydrolyzed substrate, completing the re-quenching of fluorescence and achieving the signal self-reset function.

[0010] Step 3: Construction and operation of the signal cross-catalysis module

[0011] The signal cross-catalysis module consists of two identical core units, two reporters for real-time monitoring of the cross-catalytic signal, and two threshold units. The core units are designed using the same method as the signal selection module, but the sequence design ensures that the input of one core unit is the output of the other. Furthermore, unlike the core units of the signal selection and signal self-resetting modules, one phosphate-modified output strand of the core unit in the signal cross-catalysis module is replaced with an unmodified strand to prevent phosphate-modified strands from being hydrolyzed by λExo over a long period. The two reporters are designed using the same method as the self-resetting unit in the signal self-resetting module, with fluorescent quenching groups modified using ROX and BHQ2, and FAM and BHQ1, respectively. The two threshold units lack the 3' end-modified fluorescent DNA strand compared to the reporter structure. During operation, regardless of which core unit is added to the substrate, a cross-catalytic closed loop is formed, continuously catalyzing the generation of two input strands and two output strands until all core units in the substrate are consumed. The signal cross-catalysis module not only amplifies the signal but also enables multiple input and output signals.

[0012] Step 4: Construction and operation of the signal delay module

[0013] A signal delay module was further implemented by utilizing the substrate of the signal cross-catalysis module. After the output is generated using the cross-catalysis module, the output preferentially hybridizes with the threshold unit because the output and threshold unit have a faster reaction rate, at which point no fluorescence signal enhancement is produced. If a small amount of output undergoes a chain displacement reaction with the reporter, which is slower than hybridization, the displaced signal will still hybridize with the threshold unit to form the reporter; this process also does not increase fluorescence intensity. Only when the threshold unit is completely consumed can the output undergo a chain displacement reaction with the reporter to induce fluorescence signal enhancement, thereby achieving delayed signal triggering. In the signal delay module, the signal delay time can be continuously adjusted by changing the concentration of the threshold unit.

[0014] The signal delay module relies entirely on the substrate of the signal cross-catalysis module; the specific difference lies in the concentration of the threshold units used. The threshold units in the signal cross-catalysis module are solely for generating a better signal-to-noise ratio, while the threshold units in the signal delay module are for achieving delayed signal triggering.

[0015] The beneficial effects of this invention are:

[0016] 1. By fully utilizing the high efficiency, persistence, and sequence independence of λExo, a signal processing core unit with input-output orthogonality was established to realize multi-input-output signal transmission mode.

[0017] 2. By combining the core unit and the self-reset unit, the signal self-reset function is realized. Furthermore, by changing the concentration of λExo, the time adjustability of the signal self-reset function is achieved, thereby improving the time controllability of the modular DNA reaction network.

[0018] 3. Based on the sequence independence of λExo and the orthogonality between the input and output of the core unit, two core units with the same structure are used, and the DNA sequence constituting the core unit is cleverly designed to realize the signal amplification function with multiple input and output characteristics.

[0019] 4. Based on the signal cross-catalysis module, the signal delay time can be continuously adjusted by controlling the concentration of the threshold unit, which provides greater flexibility and precision for modular DNA reaction networks.

[0020] 5. The core unit can be flexibly selected, adjusted, and combined with other functional units, providing scalability for modular DNA reaction network design. The reusability of the core unit greatly reduces the workload of DNA sequence design and simplifies the design process of DNA reaction networks.

[0021] 6. The modular design of the DNA reaction network improves its reusability, scalability, and ability to be flexibly customized for different applications. A proof-of-concept study was successfully conducted by implementing various information processing tasks.

[0022] This invention develops a signal processing method based on exonuclease-driven modular DNA reaction networks. By combining functional units, various modular DNA reaction networks can be constructed to meet different signal processing needs. This method simplifies the design of DNA reaction networks, improves their scalability, and provides design ideas for large-scale networks with multiple functions, potentially bringing new application methods to fields such as biocomputing, nanomachines, and biosensing. Attached Figure Description

[0023] Figure 1 This explains the working principle of the signal selection module.

[0024] Figure 2 The gel electrophoresis experiment was used to verify the core unit. In this experiment, A represents adding input 1 to perform gel electrophoresis analysis on the core unit, and B represents adding input 2 to perform gel electrophoresis analysis on the core unit.

[0025] Figure 3 For fluorescence verification of the signal selection module, A is to add input 1 to perform fluorescence analysis on signal 1, and B is to add input 2 to perform fluorescence analysis on signal 2.

[0026] Figure 4 This describes the working principle of the signal self-reset module.

[0027] Figure 5 Fluorescence verification of the signal self-reset module.

[0028] Figure 6 The fluorescence curves of the signal self-reset module at different enzyme concentrations were monitored.

[0029] Figure 7 This describes the working principle of the signal cross-catalysis module.

[0030] Figure 8 The fluorescence verification of the signal cross-catalysis module is shown in Figure 1. A represents the fluorescence curve monitoring of signal 1 at different input 1 concentrations, B represents the fluorescence curve monitoring of signal 2 at different input 1 concentrations, C represents the fluorescence curve monitoring of signal 1 at different input 2 concentrations, and D represents the fluorescence curve monitoring of signal 2 at different input 2 concentrations.

[0031] Figure 9 This explains the working principle of the signal delay module.

[0032] Figure 10 The fluorescence verification and analysis of the signal delay module are as follows: A represents the fluorescence verification of the signal delay module, and B represents the fluorescence analysis of the signal delay module. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. It should be understood that the described examples are only a part of the examples of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0034] In this invention, all substrates were obtained using the same polymerase chain reaction (PCR) annealing procedure. The annealing procedure included heating the sample to 90°C and holding for 5 minutes, then cooling to 85°C at a rate of -1°C per minute, and finally cooling to 25°C at a rate of -0.5°C per minute and holding for gel electrophoresis or fluorescence experiments. For gel electrophoresis, a 20 μL sample was prepared by inputting DNA, substrate, and λExo, incubated at 30°C for 1 hour, and then run on an electrophoresis apparatus at 80V for 2.5 hours. For fluorescence experiments, a 50 μL sample was prepared by inputting DNA, substrate, and λExo, and all fluorescence measurements were performed in real-time at 30°C using a real-time fluorescence PCR instrument. PCR annealing, gel electrophoresis, and fluorescence experiments were all performed using a Bio-Rad instrument.

[0035] Example 1

[0036] like Figure 1The working principle of the signal selection module is demonstrated: when Input 1 or Input 2 is added to the substrate, a strand displacement reaction occurs between the input and the core unit, forming Waste 1 or Waste 2, and Intermediate 1 or Intermediate 2. Then, λExo recognizes the 5' phosphorylated concave end structure in the intermediate and hydrolyzes the DNA strand with the ec or e*b* domain along the 5'-3' direction until output strands A, B* or C, D* are completely released. This core unit-based operation was verified using natural polyacrylamide gel electrophoresis (PAGE) experiments (e.g., ...). Figure 2 Outputs B* or D* are single chains modified with phosphate, which will be hydrolyzed by λExo after a certain time. Outputs A or C will undergo a chain substitution reaction with reporter A or reporter C, generating signal 1 or signal 2. Signal selection was validated through fluorescence experiments. Different inputs 1 and 2 were added to the substrate, and fluorescence signal 1 (e.g., ...) was monitored over 60 minutes. Figure 3 (A) or signal 2 (e.g.) Figure 3 (B)

[0037] Example 2

[0038] like Figure 4 The diagram illustrates the working principle of the signal self-reset module: When input 1 is added to the substrate, a chain substitution reaction with the core unit forms waste 1 and intermediate product 1. These are then digested by an enzyme to obtain output A and output B*. Output B* undergoes chain substitution with the self-reset unit, inducing the release of the signal chain and enhancing the fluorescence signal. The self-reset unit at this point forms a hydrolyzable substrate that λExo can interact with. After hydrolysis, the signal chain rehybridizes with the resulting receiver, achieving fluorescence re-quenching and completing the signal self-reset process. In this module, λExo is not only used to generate output B* but also serves as fuel in the signal reset process. Figure 5 The fluorescence verification of the self-reset module is shown: A quantitative input 1 was added to the substrate three times at 60-minute intervals. Experimental results show that the signal follows a single oscillation mode and completes the signal self-reset task in all three cycles. Figure 6 The image shows the fluorescence curves of the signal self-reset module at different enzyme concentrations. As the enzyme concentration increases, the required signal self-reset time gradually decreases.

[0039] Example 3

[0040] like Figure 7The diagram illustrates the working principle of the signal cross-catalysis module: When input 1 is added to the substrate, it undergoes a substitution reaction with the core unit 1 chain, forming waste 1 and intermediate product 1. These are then digested by an enzyme to yield input 2 and output B chain. Input 2 chain can then trigger a reaction with core unit 2, generating input 1 and output E chain. At this point, input 1 and input 2 chains can cross-catalyze the generation of output B, which in turn substitutes with the reporter B chain to produce fluorescence signal 1, and output E substitutes with the reporter E chain to produce fluorescence signal 2. This cross-catalysis module not only achieves catalytic amplification of signals but also enables multiple inputs and outputs of signals.

[0041] like Figure 8 The fluorescence verification of the signal-crossing catalytic module is shown: Input 1 and Output 2, with concentrations ranging from 0 to 400 nM, were added to the substrate, and the fluorescence curves of Signal 1 and Signal 2 were monitored over 180 minutes. When the concentrations of Input 1 and Output 2 were 400 nM, 320 nM, and 240 nM, the fluorescence intensity curves all reached essentially the same fluorescence value and tended to equilibrium, demonstrating the realization of the cross-catalytic module with multiple input-output characteristics.

[0042] Example 4

[0043] like Figure 9 The diagram illustrates the working principle of the signal delay module: Output B is generated using a cross-catalysis module. Output B only induces fluorescence signal enhancement after completely consuming threshold unit 1 and undergoing a chain displacement reaction with reporter B, thus achieving delayed signal triggering. In this module, the signal delay time can be continuously adjusted by changing the concentration of threshold unit 1, providing greater flexibility and accuracy for the constructed signal delay network.

[0044] like Figure 10 The following is the fluorescence verification and analysis of the signal delay module: the time corresponding to half of the maximum increment of each fluorescence curve is T. 1 / 2 This is used as a comparable metric for signal delay time. The figure shows an example of a fluorescence curve with a threshold concentration of 600 nM, illustrating T. 1 / 2 The method of finding (e.g.) Figure 10 (A). Experimental results show that the signal delay time increases with the increase of the concentration of threshold unit 1, and the concentration of threshold unit 1 is related to the corresponding T. 1 / 2 The values ​​are basically linearly related (e.g.) Figure 10 (B). Theoretically, as long as sufficient output B is generated, the signal delay module can achieve timed triggering of the signal.

[0045] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A signal processing method based on a modular DNA reaction network driven by an exonuclease, characterized in that; The method described is not for diagnostic purposes and specifically includes the following steps: Step 1: Construction and operation of the signal selection module The substrate of the signal selection module consists of a core unit and two reporters. The core unit is a symmetrical structure formed by annealing two 5' phosphate-modified DNA strands and two unphosphate-modified DNA strands. The two 5' phosphate-modified DNA strands hybridize to form two 3' overhangs, which then hybridize with the two unphosphate-modified DNA strands to form a core unit with two 5' overhangs. When an input is added to the core unit, the two input strands select the two 5' overhangs of the core unit respectively, and through sequential DNA strand substitution reactions and enzymatic digestion by lambda exonuclease, two different sets of output strands are obtained, each set containing... One non-phosphate modified chain and one phosphate modified chain ensure the scalability of subsequent modular network design. Two reporters are used to receive the two non-phosphate modified output chains produced by the core unit. One reporter consists of two complementary chains modified with ROX fluorophores and BHQ2 quenchers, and the other reporter consists of two complementary chains modified with FAM fluorophores and BHQ1 quenchers. The two reporters split the fluorescence and quenching groups of the reporter according to the two different output chains received, thus producing two different fluorescence signals. The signal selection module can generate two different fluorescence signals according to the two different input chains to realize the signal selection function. Step 2: Construction and Operation of the Signal Self-Reset Module The substrate of the signal self-reset module consists of a core unit and a self-reset unit. The core unit is the same as that of the signal selection module. The self-reset unit consists of three DNA strands, one of which is modified with a ROX fluorescent group at its 3' end and the other with a BHQ2 quenching group at its 5' end. Both of these DNA strands hybridize with the unmodified third DNA strand. During the operation of the signal self-reset module, the core unit generates a phosphate-modified output strand. Then, the output strand and the self-reset unit undergo a DNA strand substitution reaction, inducing the release of the signal strand and generating an enhanced fluorescence signal. The induced signal strand is the single-stranded DNA of the self-reset unit with the ROX fluorescent group modified at its 3' end. At this time, the self-reset unit forms a hydrolyzable substrate that can be acted upon by λ Exo. After hydrolysis is completed, the signal strand rehybridizes with the hydrolyzed substrate to complete the re-quenching of fluorescence and realize the signal self-reset function. Step 3: Construction and operation of the signal cross-catalysis module The signal cross-catalysis module consists of two identical core units, two reporters for real-time monitoring of the cross-catalysis signal, and two threshold units. The structural design of the two core units is the same as that of the signal selection module, but the sequence design ensures that the input of any one core unit is the output of the other. Furthermore, compared to the core unit designs of the signal selection and signal self-resetting modules, one of the phosphate-modified output strands of the core unit in the signal cross-catalysis module is replaced with an unmodified strand to prevent the phosphate strand from being hydrolyzed by λ Exo over a long period. The two reporters are designed using the same method as the self-resetting unit in the signal self-resetting module, with fluorescent quenching groups modified by ROX and BHQ2, and FAM and BHQ1, respectively. The two threshold units lack the 3' end-modified fluorescent DNA strand compared to the reporter structure. During operation, regardless of which core unit is added to the substrate, a cross-catalytic closed loop is formed, continuously catalyzing the generation of two input strands and two output strands until all core units in the substrate are consumed. The signal cross-catalysis module not only amplifies the signal but also enables multiple input and output signals. Step 4: Construction and operation of the signal delay module The signal delay module was further implemented by utilizing the substrate of the signal cross-catalysis module. After the output is generated using the cross-catalysis module, the output will preferentially hybridize with the threshold unit because the output and the threshold unit have a faster reaction rate, and no fluorescence signal enhancement will be generated at this time. If a small amount of output undergoes a chain displacement reaction with the reporter, which is slower than hybridization, the signal displaced by the chain will still hybridize with the threshold unit to form the reporter, and this process will not generate an increase in fluorescence intensity. Only when the threshold unit is completely consumed can the output undergo a chain displacement reaction with the reporter to induce fluorescence signal enhancement, thereby realizing signal delay triggering. In the signal delay module, the signal delay time can be continuously adjusted by changing the concentration of the threshold unit.

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