Molecular circuits and their design methods, as well as plant diagnostic kits

Abstract

This invention provides a method for designing molecular circuits that can maintain a constant concentration of molecules that produce an output value. [Solution] A method for designing a molecular circuit that takes molecule D as input and molecule I as output, wherein a simplified model of the molecular circuit uses molecule Ba, molecule Bb, molecule I, molecule M, and molecule D to form the following three reaction systems: TIFF2026069922000011.tif3665 A method comprising a reaction system represented by , wherein the initial concentrations of molecules Ba, Bb, I, M, and D are [Ba]0, [Bb]0, [I]0, [M]0, and [D]0, respectively, and the parameters of Tb and Ti are set such that all of the following conditions are satisfied: (A1) Tb > 0, (A2) Ti > 0, (A3) [Ba]0 + [Bb]0 = Tb, (A4) [I]0 + [M]0 + [D]0 = Ti, and (A5) Ti / Tb << 1.

Description

[Technical Field]

[0001] This invention relates to a molecular circuit and a method for designing the same. It also relates to a plant diagnostic kit that uses the molecular circuit to diagnose the health status of plants. [Background technology]

[0002] Recent advances in nanotechnology have accelerated the development of various molecular systems driven by chemical operating principles (Non-Patent Literature 1). This has led to the development of intelligent molecular robots equipped with sensors and actuators designed at the molecular level, increasing the demand for molecular circuits that perform complex information processing, such as feedback controllers.

[0003] Currently, the most common method for designing molecular circuits is to utilize DNA computing. DNA computing is a theory and technology for artificially designing chemical reaction systems (DNA circuits) that perform desired information processing (such as logical operations) using nucleic acid molecules such as DNA and RNA. The greatest features of DNA circuits are their ability to directly receive the RNA signal to be detected as an input signal (seamlessness), their operation using only chemical energy (energy efficiency), and their ability to implement complex logical operations (high functionality).

[0004] Over the past 20 years, a variety of DNA circuits have been developed, from logic circuits to arithmetic circuits. The "signals" flowing through these circuits are defined by the concentration of DNA strands involved in the reaction system. For example, in logic circuits, whether the concentration of DNA strands is higher or lower than a threshold has qualitative significance, while in arithmetic circuits, the concentration of DNA strands has quantitative significance.

[0005] However, molecular circuits, including DNA circuits, are known to be highly retrospective, making it difficult to connect and operate multiple circuits in conjunction (Non-Patent Literature 2). For example, in the design of feedback controllers, it has been reported that when a controller that was confirmed to operate independently is connected to a controlled plant, it becomes unable to operate normally due to a rapid decrease in the concentration of DNA strands involved in the reaction system. When applying a logically high input (a concentration higher than the threshold) to a logic circuit, its polarity must remain constant throughout the reaction process. However, the concentration of the input DNA strands decreases rapidly over time once the reaction begins, so measures are needed to maintain that concentration. Therefore, in logic circuits that maintain the input concentration within a range that does not cross the threshold (e.g., entropy-driven circuits and seesaw gates), design methods that amplify the input concentration using autocatalytic reactions are widely employed. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] N. Srinivas, J. Parkin, G. Seelig, E. Winfree, Erik and D. Soloveichik, Enzyme-free nucleic acid dynamical systems, Science, Vol. 358, No. 6369, 2017. [Non-Patent Document 2] DD Vecchio, RM Murray, Biomolecular Feedback Systems, Princeton Univ. Pr., 2014. [Non-Patent Document 3] Y. Kawakatsu et. al., Microfluidic Device for Simple Diagnosis of Plant Growth Condition by Detecting miRNAs from Filtered Plant Extracts, Plant Phenomics, 6, 2024. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] However, in the case of arithmetic circuits, the input concentration has a value (such as a 2-input, 1-output adder), so a decrease or fluctuation in concentration is undesirable in arithmetic circuits. A decrease or fluctuation in concentration means that the countermeasures are insufficient for use as a logic circuit. Therefore, there was a need for circuit design that strictly maintains the input concentration at a specified level, that is, the design of a signal (input signal) generator that outputs a specified constant value.

[0008] On the other hand, disease control is essential when growing crops. In principle, pesticides are used because eliminating pests and diseases can prevent disease. In recent years, with the growing "food safety consciousness" of farmers and consumers, efforts are being made to reduce reliance on pesticides. The biggest benefit for farmers is the increased added value achieved through reduced-pesticide cultivation; for example, the retail price of komatsuna (Japanese mustard spinach) and cabbage can be set at 5 to 10 times higher or more.

[0009] However, there is a negative correlation between "amount of pesticide use" and "damage caused by disease," so in order to prevent disease and ensure a reliable yield, it is necessary to spray standard amounts of pesticides. This is where farmers face a dilemma. For example, powdery mildew, a typical disease affecting vegetables, is caused by infection with filamentous fungi, and when the infection spreads severely, white mold appears on the leaves. Currently, detection and countermeasures are carried out based on unwritten rules and know-how derived from each farmer's intuition and experience. In the early stages of infection, visual detection is impossible, so pesticides are currently sprayed for infection prevention or for treatment after infection, which is nothing more than a loss of added value and lost opportunities.

[0010] Furthermore, in response to the global trend of expanding and increasing the organic market, the Ministry of Agriculture, Forestry and Fisheries in Japan is considering measures to increase the number of organic farmers in its "Green Food System Strategy." The biggest advantage of adopting organic farming is the increase in added value of agricultural products, with retail prices for komatsuna (Japanese mustard spinach) and cabbage sometimes being set at 8 to 13 times higher. In addition, against the backdrop of the recent global increase in concern for "reducing environmental impact," it is also expected to enhance the social credibility and brand power of organic farmers. However, generally speaking, organic farming prohibits the use of chemically synthesized pesticides and fertilizers, making soil management, disease prevention and countermeasures, and securing stable production volumes more difficult compared to chemical farming. Currently, there are no effective and efficient cultivation management methods, so cultivation management is carried out based on the intuition, experience, and labor of farmers. This is a major barrier to entry for farmers who want to start organic farming.

[0011] Methods for diagnosing the presence or absence of diseases that have a significant impact on crops before they manifest can be classified into two types: "contract services" carried out by "specialized institutions such as research facilities" and "test kits" carried out by "agricultural workers themselves."

[0012] In contract-based diagnostic services, farmers harvest a specified amount of plant fragments to be diagnosed and submit them to the service provider using a prescribed method (usually by delivery). The service provider then uses the received samples to extract, detect, and diagnose microRNAs. This entire process is carried out by specialized technical staff using expensive experimental equipment, and the diagnostic results are returned to the farmer as a report on a designated medium.

[0013] As for diagnostic kits, kits that utilize microRNA-based simple diagnostic techniques are well known. Generally, kits for simple microRNA-based diagnostic techniques almost always employ microfluidic devices (Non-Patent Literature 3). This is because it is necessary to efficiently trap minute amounts (on the order of pM to nM) of microRNA secreted from plant fragments.

[0014] While contract-based diagnostic services are the most common method for diagnosing the presence of diseases that significantly affect crops before they manifest, they require the submission of samples and are time-consuming. Furthermore, because microRNAs secreted from plant fragments are extremely minute (on the order of pM to nM), expensive measuring instruments (real-time PCR, plate readers, fluorescence spectrophotometers, etc.) are necessary for diagnosis. Diagnostic kits using microfluidic devices automate some of the manual work performed by technical staff, but the overall measurement protocol still requires manual work such as connecting the microfluidic device to the pump and capturing detection results with a CCD camera, making it complex for farmers to perform on-site and independently. In addition, the measurement time is highly dependent on the operator's skill level. Therefore, there are high hurdles to popularizing contract-based and diagnostic kits, and the presence of pathogens can only be determined after the disease has manifested.

[0015] Under these circumstances, the present invention aims to provide a molecular circuit and a method for designing the same that can maintain a constant concentration of molecules that produce an output value.

[0016] Moreover, the present invention aims to provide a plant diagnosis kit that can easily diagnose the health status of plants.

Means for Solving the Problems

[0017] As a result of intensive studies to solve the above problems, the present inventor has found that the following invention meets the above object, and has arrived at the present invention. That is, the present invention relates to the following invention.

[0018] <1> A method for designing a molecular circuit that takes molecule D as an input and outputs molecule I, where the simple model of the molecular circuit uses molecule Ba, molecule Bb, the molecule I, molecule M, and the molecule D in the following three reaction systems: [[ID=!18]]

Number

[0019] <6> This is a molecular circuit that takes molecule D as input and molecule I as output. The aforementioned molecular circuit, in its simplified model, uses molecules Ba, Bb, I, M, and D to form the following three reaction systems:

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[0020] <11> The aforementioned <6> from <10> An information processing system comprising one or more molecular circuits as described in any of the above. <12> For detecting molecule D, <11> The information processing system described above.

[0021] <13> This is a plant diagnostic kit for diagnosing the health status of a target plant, and the above <10> A plant diagnostic kit comprising the molecular circuit described above, wherein molecule D is a microRNA of the diagnostic item, and the health status is diagnosed based on the presence or absence of the production of molecule I. <14> The molecular circuit includes a determination means for determining whether or not molecule I has been generated, <13> The plant diagnostic kit described above. <15> The determination means includes a DNA diagnostic circuit and a DNA visualization circuit. <14> The plant diagnostic kit described above. [Effects of the Invention]

[0022] According to the present invention, a molecular circuit and a method for designing the same are provided, which can maintain a constant concentration of molecules that produce an output value.

[0023] Furthermore, the present invention provides a plant diagnostic kit that can easily diagnose the health status of plants. [Brief explanation of the drawing]

[0024] [Figure 1] This diagram illustrates the use of a conventional DNA circuit as a signal generator. [Figure 2] This figure illustrates the case in which the molecular circuit according to the present invention is used as a signal generator. [Figure 3] This is an example of connecting multiple molecular circuits of the present invention. [Figure 4] This is a specific scheme for the reaction systems of equations (1a), (2a), (3a), and (3b). [Figure 5] This is the simulation result for implementation example 1. [Figure 6] This is the simulation result for implementation example 2. [Figure 7] This is an example of an information processing system including the molecular circuit of the present invention. [Figure 8] This figure illustrates a diagnostic method using the plant diagnostic kit of the present invention. [Modes for carrying out the invention]

[0025] The embodiments of the present invention will be described in detail below, but the description of the constituent elements described below is just one example (representative example) of an embodiment of the present invention, and the present invention is not limited to the following unless its gist is changed. In this specification, when the expression "~" is used, it is used to mean an expression that includes the numerical value or physical property value before and after it.

[0026] <Molecular circuits and their design methods> The present invention relates to a molecular circuit that takes molecule D as input and molecule I as output. A simplified model of this molecular circuit uses molecules Ba, Bb, I, M, and D to form the following three reaction systems:

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[0027] Furthermore, the three reaction systems described above satisfy all of the following conditions. (A1)Tb>0 (A2)Ti>0 (A3)[Ba]0+[Bb]0=Tb (A4)[I]0+[M]0+[D]0=Ti (A5)Ti / Tb<<1 Here, [Ba]0, [Bb]0, [I]0, [M]0, and [D]0 are the initial concentrations of molecules Ba, Bb, I, M, and D, respectively, while Tb and Ti are concentration parameters with units.

[0028] The inventors investigated a reaction system in which molecule I is automatically supplied by molecule Ba and molecule M controls the concentration of molecule I and molecule D to maintain a predetermined relationship, in order to design a molecular circuit that maintains a constant output concentration. To realize such a reaction system, the reaction system shown in equations (1) to (3) above was considered as an abstracted simplified model. The reaction system shown in equations (1) to (3) can be described in ordinary differential equations as follows, where the rate constant of the reaction proceeding to the right in equation (1) is km, the rate constant of the reaction proceeding to the left in equation (1) is kl, and the rate constant of the reactions in equations (2) and (3) is kf.

[0029]

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[0030] Here, [Ba], [Bb], [I], [M], and [D] represent the concentrations of molecules Ba, Bb, I, M, and D, respectively, while km, kl, and kf represent the rate constants.

[0031] The inventors performed an analysis of the ordinary differential equation based on singular perturbation theory and derived a design guideline that, by setting various parameters in the three reaction systems as described above, it is possible to create a molecular circuit in which molecule I is produced when molecule D is added, and moreover, a molecular circuit in which a constant amount of molecule I is produced without fluctuations over time.

[0032] Furthermore, the theoretical analysis described above led to the conclusion that the velocity constant should be set as follows. (B1)km=[D]0·kl (B2)kf=(Tb / Ti)·kl The units for km, kl, and kf are 1 / (nM·s).

[0033] kl is the reference rate and is an arbitrary value. kl is usually greater than 0 and less than or equal to 10, and is set appropriately depending on the type of molecule, etc. For example, in a reaction system containing proteins, kl is 1.0 × 10⁻⁶. -7 Preferably, the value is 1.0 or less, and in the case of a DNA reaction system, kl is 1.0 × 10 -6 The above 1.0 × 10 -2 The following are preferable.

[0034] The simplified model described above is a reaction system in which molecule Ba automatically supplies molecule I, and molecule M works to reduce the difference between the concentration of molecule I and the concentration of molecule D or its amplified / attenuated concentration. In such a reaction system, by setting various parameters as described above, the molecular circuit of the present invention can control the concentration of molecule I (output value), which is the output concentration, according to the concentration of molecule D (input value), and can also maintain a constant output value.

[0035] As described above, conventional DNA circuits have the problem of being difficult to output a constant value even when used as signal generators. As shown in Figure 1, when a conventional DNA circuit is connected to a DNA circuit 12 as a signal generator 10, the concentration of molecules that serve as the input signal to the DNA circuit 12 decreases over time, affecting the output of the DNA circuit 12.

[0036] On the other hand, the molecular circuit of the present invention generates molecule I in such a way that the concentration of molecule I [I] is maintained at a constant level. Therefore, the molecular circuit of the present invention can be used as a signal generator that outputs a constant value. For example, as shown in Figure 2, the molecular circuit of the present invention can be connected to a DNA circuit 12 as a signal generator 11, and the concentration of molecule I [I] generated by the signal generator 11 can be used as the input signal to the DNA circuit 12. Since a constant value is input to the DNA circuit 12, the output from the DNA circuit 12 will be ideal without being affected by fluctuations in the input signal. In this case, by setting the parameters so that the concentration of molecule I [I] is greater than the initial concentration of molecule D [D] 0, the input value input to the molecular circuit of the present invention can be amplified, and it can be used as a signal enhancer that outputs a constant value.

[0037] For example, the molecular circuit of the present invention can be used as a signal amplifier by setting [Ba]0 to a value greater than [Bb]0, and as a signal attenuator by setting [Ba]0 to a value less than [Bb]0.

[0038] <Reaction System> The molecular circuit of the present invention takes molecule D as input and molecule I as output, and a simplified model thereof includes the following reaction system.

[0039]

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[0040] The molecular circuit of the present invention should be designed such that, when abstracted, its simplified model includes the aforementioned reaction system. The reaction system in equation (1) is a model of a reaction system in which molecule I is produced by molecule Ba. In implementation, the reaction is not limited to the separation of molecule Ba into molecule Bb and molecule I. Molecule Ba may also react with other molecules to produce molecule Bb and molecule I. The reaction system in equation (2) is a model of a reaction system in which molecule I, produced in equation (1), affects the concentration of molecule M. In implementation, equation (2) is not limited to a reaction in which molecule I and molecule M directly react to produce two molecules M. The reaction system in equation (3) is a model of a reaction system in which molecule D affects the concentration of molecule M. In implementation, equation (3) is not limited to reactions in which molecule M and molecule D directly react to produce molecule D. Furthermore, the reaction systems in equations (2) and (3) do not need to be completely irreversible reactions; they may be reversible reactions as long as the reaction appears to proceed in one direction. In other words, if the equilibrium is skewed and the reaction proceeds with a strong force in one direction, even if it is formally a reversible reaction, it can be approximated as an irreversible reaction, and thus can be represented as the reaction systems in equations (2) and (3).

[0041] In implementation, the reaction system is usually designed to be approximately equivalent to the reaction system described above. Furthermore, each reaction system only needs to have a simplified model that can be represented by the above reaction system, and may involve multiple reaction pathways. Each reaction system may also involve molecules other than Ba, Bb, M, I, and D (auxiliary molecules). Specifically, a reaction system can be constructed in which molecule I is supplied by molecule Ba, and molecules I and D affect the concentration of molecule M. By appropriately adjusting the initial concentrations of each molecule as described above, the molecular circuit of the present invention can be designed.

[0042] Alternatively, the simplified model may be replaced with an approximately equivalent simplified model during the design process. For example, taking reaction system (2) as an example, it can be considered as M + I + Xi → M + M + Xi. In this case, since Xi appears before and after the reaction, Xi remains approximately unchanged and can be considered equivalent to M + I → M + M.

[0043] <Methods for designing molecular circuits> The molecular circuit of the present invention is designed by a method having the following step A.

[0044] (Step A) Step A is the step of setting the parameters Tb and Ti. The units of Tb and Ti are concentrations. Tb and Ti are set so that all of the following conditions (A1) to (A5) are satisfied, given that the initial concentrations of molecules Ba, Bb, I, M, and D are [Ba]0, [Bb]0, [I]0, [M]0, and [D]0, respectively. (A1)Tb>0 (A2)Ti>0 (A3)[Ba]0+[Bb]0=Tb (A4)[I]0+[M]0+[D]0=Ti (A5)Ti / Tb<<1

[0045] <Molecules Ba, Bb, M, I, D> Molecules Ba, Bb, M, I, and D can be nucleic acids, amino acids, proteins, or any other substance capable of achieving the above reaction system, but nucleic acids are preferred from the viewpoint of ease of design. For example, the above reaction system can be implemented using nucleic acid strand exchange reactions. Due to the chemical properties of DNA and RNA strands, which specifically bind to other DNA or RNA strands with complementary base sequences, a reaction system in which all reactions occur as planned can be rationally developed. Nucleic acids include single-stranded DNA, double-stranded DNA, RNA strands, microRNAs, etc. By using nucleic acids as molecular circuits (i.e., DNA circuits), it is possible to detect, remember, and output the presence of nucleic acids derived from biological cells, for example.

[0046] Molecule D is an input molecule added to operate the molecular circuit of the present invention, and the concentration of molecule D [D] becomes the input signal. When only the concentration of the output molecule I [I] is considered, the structure of molecule D is not particularly limited and can be designed and selected as appropriate. Molecule D may be artificially synthesized or naturally occurring. When used for the detection of molecule D, the molecule to be detected becomes molecule D. For example, when used in plant diagnosis as described later, the molecular circuit of the present invention is designed with the microRNA of the diagnostic item as molecule D.

[0047] Molecule I is the output molecule generated as the reaction progresses, and the concentration of molecule I [I] becomes the output signal. When the molecular circuit of the present invention is used as a signal generator or signal intensity converter, the molecular circuit of the present invention is connected to other molecular circuits (subsequent molecular circuits). In this case, since [I] becomes the input value of the subsequent molecular circuit, the structure of molecule I is appropriately designed and selected, taking into consideration the structure of the subsequent molecular circuit.

[0048] Molecules Ba and Bb are molecules that are present from the beginning in the reaction system of the molecular circuit of the present invention, and the reaction that results in the above-mentioned reaction system proceeds according to the structures of molecules D and I. Furthermore, each reaction system of the molecular circuit of the present invention may be configured to proceed through multiple reaction pathways, as long as it is designed to become the above-mentioned reaction system when abstracted. The molecular circuit of the present invention is usually constructed using molecules Ba, Bb, M, I, and D, and molecules other than these molecules (auxiliary molecules). Therefore, molecule M is not limited to a structure that directly reacts with molecules I and D, molecule I is not limited to a structure that directly reacts with molecule M, and molecule D is not limited to a structure that directly reacts with molecule M. For example, molecule M may not directly react with molecule I or D, and the reaction product of molecule M and auxiliary molecules may be designed to react with molecule I or D.

[0049] <Initial concentration of each molecule> The initial concentration of each molecule is set to satisfy all of the above conditions (A1) to (A5), depending on the required performance. The Ti / Tb ratio is usually greater than 0 and 1.0 × 10⁻⁶, depending on the required performance. -1 The following is true: 1.0 × 10-8 Above 2.0×10 -3 Below is preferred, 1.0×10 -8 Above 1.0×10 -3 Below is more preferred, 1.0×10 -6 Above 1.0×10 -3 Below is even more preferred. The concentration of each molecule, in the case of DNA or protein, is, for example, in the range of 1 fM to 100 mM, 1 pM to 100 μM, 1 nM to 10 μM, and can be adjusted to satisfy the above numerical range of Ti / Tb.

[0050] For example, when [D]0 is increased, Ti increases. At this time, since the condition of (A5) also needs to be satisfied, [Ba]0 and [Bb]0 also need to be increased. That is, when small values are set for [Ba]0 and [Bb]0, only a correspondingly small input value (i.e., [D]) can be accepted. For example, when [Ba]0 = [Bb]0 = 100 nM and [D]0 = 1 nM are set, the performance as a signal generator can be obtained as expected. However, when [Ba]0 = [Bb]0 = 10 nM and [D]0 = 1 nM, depending on the usage scenario, the performance as a signal generator may not be sufficient. [D]0 can be 0.1 or less of [Bb]0, 0.01 or less of [Bb]0, 0.001 or less of [Bb]0, etc. The higher the required performance, the more preferably [D]0 is set to a small value relative to [Bb]0.

[0051] Also, the molecular circuit of the present invention can control the amount of molecule I generated by [Ba]0, [Bb]0, and [D]0.

[0052] For example, if [Ba]0=[Bb]0 and [D]0=d are set, molecular I will be generated such that its concentration is equal to the initial concentration [D]0 of molecular D. By setting the initial concentration in this way, molecular I can be generated such that [I]=d (i.e., the output value is d), so that the molecular circuit of the present invention can be used as a signal generator that produces an output of the same intensity as the input value. In such a case, d is set to, for example, the desired intensity (concentration of molecular I [I]) that you want to output from the molecular circuit of the present invention. Alternatively, as one method of setting the initial concentration, [D]0=d and Ti / Tb is set to 0.001 to find the approximation rate of [I] with respect to d, and if this approximation rate is insufficient, the value of Ti / Tb can be gradually decreased (the values ​​of [Ba]0 and [Bb]0 can be gradually increased) to increase the approximation rate of [I] with respect to d and adjust it to achieve the desired performance (for example, an approximation rate of 99% or more).

[0053] If we set [Ba]0 to X times [Bb]0 (where X is a number greater than 0) and [D]0=d, then molecule I can be generated at a concentration X times the initial concentration [D]0 of molecule D. In other words, molecule I is generated such that [I] is X times d (i.e., the output value is X times d).

[0054] For example, if X>1 is set, [I] will be a value greater than [D]0 and can serve as a signal amplifier. When X>1 is set and the molecular circuit of the present invention is connected to other molecular circuits, even when the initial concentration [D]0 of molecule D is small, the concentration [I] of molecule I, which serves as the input signal for other molecular circuits, can be increased. For example, if [Ba]0 is set to a value 1000 times that of [Bb]0, the output value (the input value for other molecular circuits) will be 1000 times the initial concentration [D]0 of molecule D. Therefore, even when it is difficult to increase the initial concentration [D]0 of molecule D, sufficient output intensity ([I]) can be ensured. By making such a setting, the reaction system becomes simpler compared to the case where the molecular circuit of the present invention is connected to other molecular circuits having an amplification function. That is, since the molecular circuit of the present invention functions as a signal generator and a signal amplifier (generates a signal of a certain value with sufficient intensity), the reaction system becomes simple.

[0055] Also, as shown in FIG. 3, when two molecular circuits of the present invention are connected in series, in the front-stage molecular circuit, [Ba]0 is set to Xa times ([Xa] is a number greater than 1) of [Bb]0, and in the rear-stage molecular circuit, [Ba]0 is set to Xb times ([Xb] is a number greater than 1) of [Bb]0. Then, the output value from the rear-stage molecular circuit is amplified to Xa×Xb times the input value of the front-stage molecular circuit. For example, if [Ba]0 is set to 100 times [Bb]0 in the front-stage molecular circuit and [Ba]0 is set to 1000 times [Bb]0 in the rear-stage molecular circuit, the concentration [I] (output value) of molecule I in the rear-stage molecular circuit of the present invention can be 100,000 times the initial concentration [D]0 of molecule D in the front-stage molecular circuit of the present invention. Therefore, when it is desired to further enhance the signal, a structure in which two or more molecular circuits of the present invention are connected may be used.

[0056] On the other hand, when X = 1, [I]=[D]0, and it can serve as a signal generator where the input intensity = output intensity. When 0<X<1 is set, [I] will be a value smaller than [D]0 and can serve as a signal attenuator.

[0057] In one embodiment, the initial concentration of molecule I can be set to [I]0=0, but [I]0 is not limited to this as long as all of the above conditions (A1) to (A5) are met. Also, in one embodiment, the initial concentration of molecule M can be set to [M]0≒0 or [M]0=0, but [M]0 is not limited to this as long as all of the above conditions (A1) to (A5) are met. Furthermore, the initial concentrations of auxiliary molecules that appear in implementation (realization) are also adjusted as appropriate.

[0058] (Step B) The molecular circuit of the present invention may have step B. Step B is a step in which the rate constants are set, and in this step, if the rate constant for the reaction proceeding to the right in equation (1) is km, the rate constant for the reaction proceeding to the left in equation (1) is kl, and the rate constants for the reactions in equations (2) and (3) are kf, then km, kl, and kf are determined as follows. (B1)km=[D]0·kl (B2)kf=(Tb / Ti)·kl The units for km, kl, and kf are 1 / (nM·s). kl is the reference rate and can be any value. kl is usually greater than 0 and less than or equal to 10. In reaction systems involving proteins, kl is 1.0 × 10⁻⁶. -7 It is preferable to set the value within the range of 1.0 or less, and in the case of a DNA reaction system, kl is 1.0 × 10 -6 The above 1.0 × 10 -2 It is preferable to set it within the following range.

[0059] <Implementation Example 1> The following shows an example of the implementation of the molecular circuit of the present invention.

[0060]

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[0061] Figure 4 shows a more specific scheme of the reaction system for equations (1a), (2a), (3a), and (3b). In equations (1a), (2a), (3a), and (3b), Ba, Bb, I, M, D, xm2, xm3, sD, xd1, xd2, and xd3 are molecules. In Implementation Example 1, each molecule was designed using DNA, as shown in Figure 4.

[0062] The initial concentrations of each molecule in equations (1a), (2a), (3a), and (3b) were set as follows: β is a concentration parameter with units. • [Ba]0 = [Bb]0 = β / 2 • [I] 0 = 0 • [xm2]0 = β / 2 • [M] 0 = 0 ·[xm3]0=0 ·[sD]0=0 ·[D]0=d • [xd10] = β / 2 ·[xd2]0=d • [xd3]0=0

[0063] Equation (1a) represents a reaction system involving molecules Ba, Bb, and I, and corresponds to the simplified model equation (1). With the above setup, molecule I is automatically generated. Equation (2a) represents a reaction system involving molecule M and molecule I, which is produced in equation (1a), and corresponds to the simplified model equation (2). With the above settings, the concentration of molecule I is adjusted by molecule M so that its concentration is the same as that of molecule D. In addition, the reaction rate kf = β × kl is reproduced by setting the initial concentration of xm2 to β / 2. For the reaction system represented by equation (3) in the simplified model, the reaction is reproduced in several stages (equations (3a) and (3b)) so that it can be reproduced at the domain level. Equations (3a) and (3b) represent reaction systems involving molecules M and D. To maintain the constant concentration of D, the initial concentrations of both molecules xd1 and xd2 are varied. With the above settings, molecule M adjusts molecule D to maintain its initial concentration.

[0064] Furthermore, in implementation example 1, equations (2) and (3) are designed as reversible reactions, but because the reaction rate constant is set to the initial concentration of the molecule, it is an approximation near the initial concentration, and there is no problem because the force flowing in one direction becomes strong near the initial concentration. One of the advantages of designing the initial concentration and reaction rate constant in this way is that there are no constraints on the reaction rate constant, and it is easier to design as only the initial concentration needs to be increased.

[0065] (Simulation results for implementation example 1) For implementation example 1, the experimental time was 3600 s, the target concentration was 10 nM (= d), and β = 1.0 × 10⁻¹⁰. 4 ε = Ti / Tb = 1.0 × 10⁻⁶ -3 kl = 1.0 × 10 -5 The output value (concentration of molecule [I]) was simulated under the experimental conditions. Figure 5 shows the experimental results. In Figure 5, the solid line represents the concentration of molecule I, and the dotted line represents the concentration of molecule D. The approximation rate of [I] to 100% of [D] was 99.6%.

[0066] <Implementation Example 2> In the reaction system of Implementation Example 1, the initial concentrations were set as follows. X is a factor. In order to maintain the constant concentration of D, the initial concentrations of molecules Ba, xd1, and xd2 were set to be X higher. [Ba]0 = X × β / 2 • [Bb] 0 = β / 2 • [I] 0 = 0 • [xm2]0 = β / 2 • [M] 0 = 0 ·[xm3]0=0 ·[sD]0=0 ·[D]0=d • [xd1]0 = X × β / 2 • [xd2]0 = X × d • [xd3]0=0

[0067] (Simulation results for implementation example 2) Regarding the molecular circuit of Implementation Example 2, with X = 3 (3-fold amplification), the experimental time, target concentration, β, ε, and kl were set to the same experimental conditions as in Implementation Example 1, and the output value (concentration of molecule [I]) was simulated. The results of the experiment are shown in Fig. 6. In Fig. 6, the solid line represents the concentration of molecule I, and the dotted line represents the concentration of molecule D. [I] was amplified to 2.98 times the target concentration, and the approximation rate of [I] to 300% of [D] was 297.6%.

[0068] In Implementation Example 2, although X = 3 and amplification is simulated, Implementation Example 2 is not limited to those that amplify signals. X in Implementation Example 2 is the magnification ([I] / [D]) of the concentration of molecule I with respect to the concentration of molecule D, and is a number greater than 0. If set to 0 < 1 < X, it can be a signal attenuator, and if set to X > 1, it can be a signal amplifier. When X = 1, it is Implementation Example 1.

[0069] <Information processing system> The molecular circuit of the present invention can be combined with various logic circuits and arithmetic circuits to construct an information processing system. For example, as shown in Fig. 7, two molecular circuits of the present invention can be used as signal generators 11A and 11B capable of implementing a constant value output, and each can be connected to an addition circuit 13 to form a system that performs addition processing.

[0070] In addition, an information processing system including one or more molecular circuits of the present invention can be a system for detecting molecule D, and for example, as will be described later, it can be used for diagnosing the health status of plants.

[0071] <Plant diagnosis kit> The plant diagnosis kit of the present invention is a plant diagnosis kit for diagnosing the health status of a target plant and includes the molecular circuit of the present invention.

[0072] The plant diagnostic kit of the present invention applies DNA computing technology to fundamentally resolve the problems in conventional plant diagnostics, and forms the basis of an inexpensive diagnostic kit that allows customer farmers to diagnose plant diseases in a simple manner. As mentioned above, most currently known diagnostic kits utilize microfluidic devices. The key to microfluidic devices is the efficient trapping of microRNAs, and various channel and trap structures are designed using MEMS microfabrication technology. On the other hand, the plant diagnostic kit of the present invention is innovative in that it is a "seamless information processing technology using DNA circuits," and therefore does not require a trap structure in the first place. Furthermore, by detecting, calculating, and visualizing molecular signals using molecular computation, highly efficient and speedy diagnosis becomes possible. Moreover, the plant diagnostic kit of the present invention enables speedy diagnosis regardless of the operator's skill level. In addition, by forming and utilizing a DNA circuit that can amplify microRNAs even with a small amount of sample, the plant diagnostic kit of the present invention can detect early signs of important diseases that are difficult to find in the field. Therefore, diagnosis is possible in a short time, simply, and accurately.

[0073] The plant diagnostic kit of the present invention diagnoses the health status of a plant based on the presence or absence of the production of molecule I, with molecule D being the microRNA of the diagnostic item. MicroRNAs are a type of non-coding RNA that does not contain genomic information, and are single-stranded RNAs with a length of approximately 19 to 23 nucleotides. They are small molecules present in all cells of all eukaryotes, regardless of whether they are plants or animals, and more than 4400 types of microRNAs have been identified. Their main role is the regulation of gene expression and post-transcriptional regulation within cells. In plants, microRNAs are known to function as regulatory factors for physiological functions closely related to agriculture, such as the control of development and differentiation, environmental stress response, disease response, and nutrient response, and databases of microRNAs have been established.

[0074] The microRNAs used as diagnostic items are involved in the control of development or differentiation, environmental stress response, disease response, and nutrient response. For example, when a plant is infected with a disease, an inflammatory response and an immune response occur, and specific microRNAs are secreted as a result of these responses. By determining the presence or absence of these specific microRNAs, the possibility of disease can be diagnosed. In the early stages of infection, the amount of microRNA secreted due to the inflammatory response and immune response is extremely small, but the amplification function of the molecular circuit of the present invention allows it to be converted into molecule I with a sufficient concentration, thus enabling the detection of abnormalities in the plant growth process (disease, stress, etc.) at a very early stage.

[0075] In one embodiment, the plant diagnostic kit of the present invention may include a container containing a solution comprising molecules constituting the molecular circuit of the present invention. The initial concentrations of each molecule in the solution are designed as described above. By adding a sample of a target plant (for example, a sample of the leaves of the target plant, prepared as powder or juice) to the container and bringing the sample into contact with the solution, if the target plant contains molecule D, molecule D in the sample is input into the molecular circuit, and molecule I is generated (output). If the target plant does not contain molecule D, nothing is input into the molecular circuit, and therefore molecule I is not generated (output). The presence or absence of generation of molecule I allows for the diagnosis of the plant's health.

[0076] In the plant diagnostic kit of the present invention, the molecular circuit of the present invention is set to [Ba]0 be X times (for example, 100 times or more, or 1000 times or more) [Bb]0, and functions to amplify the signal and output it at a constant value. Molecule D is a microRNA, but it is preferable that molecules Ba, Bb, M, I and auxiliary molecules are designed with DNA according to the structure of the microRNA of the diagnostic item.

[0077] The plant diagnostic kit of the present invention can, for example, modify molecule I with a fluorescent molecule, measure the fluorescence intensity of the solution after the reaction using a device such as a fluorescence spectrophotometer, and diagnose the health of the plant based on the presence or absence of fluorescence. Alternatively, the plant diagnostic kit of the present invention can include the molecular circuit of the present invention and a determination means for determining whether or not molecule I has been generated in the molecular circuit. The determination means can be implemented by combining existing DNA circuits such as logic circuits or visualization methods. By including the determination means, the health of the plant can be easily diagnosed visually without using special equipment to measure fluorescence intensity after the reaction.

[0078] For example, the plant diagnostic kit of the present invention may include a DNA amplification circuit, a DNA diagnostic circuit, and a DNA visualization circuit. The DNA amplification circuit is the molecular circuit of the present invention, and by setting [Ba]0 to X times (for example, 100 to 1000 times) [Bb]0, the microRNA of the diagnostic item is amplified to a sufficiently concentrated molecule I and output. As a result, the DNA amplification circuit can detect and store extremely small microRNA signals and amplify them as molecule I. The DNA diagnostic circuit and the DNA visualization circuit are means of judgment. The DNA diagnostic circuit performs a diagnosis based on the microRNA signal (molecule I generated due to microRNA). The DNA visualization circuit visualizes the diagnostic results.

[0079] Furthermore, the plant diagnostic kit of the present invention may include multiple molecular circuits of the present invention, and each of the multiple molecular circuits of the present invention may be connected to a determination means, and the health status of the plant may be diagnosed according to the pattern of signals output from the multiple molecular circuits of the present invention.

[0080] Figure 8 shows an example of a plant diagnostic method using the plant diagnostic kit of the present invention. The plant diagnostic kit 20 shown in Figure 8 contains a solution 24 in a tube 22 that includes each molecule (DNA) that constitutes the DNA amplification circuit 14 (molecular circuit of the present invention), the DNA diagnostic circuit 15, and the visualization circuit 16.

[0081] When diagnosing the health of a plant, a plant fragment 30 is cut into powder using a special jig and sealed in a tube 22 (Figure 8(A)). If the plant fragment powder contains microRNA of the diagnostic item, the DNA amplification circuit 14, DNA diagnostic circuit 15, and visualization circuit 16 autonomously perform logical calculations upon receiving the microRNA input, causing the solution 25 in tube 22 to change color and the diagnostic result to be visualized (Figure 8(B)). On the other hand, if the plant fragment powder does not contain microRNA of the diagnostic item, the DNA amplification circuit 14, DNA diagnostic circuit 15, and visualization circuit 16 are not executed, and the solution 26 in tube 22 does not change color.

[0082] There are no restrictions on the part of the plant fragment used; leaves, seeds, seedlings, etc., can be selected as appropriate depending on the diagnostic requirements. Additionally, the sap extracted from the plant fragment may be added to tube 22 for diagnosis. Furthermore, the visualization circuit is not limited to the presence or absence of coloration; other methods such as turbidity may be used.

[0083] In the plant diagnostic kit shown in Figure 8, the only manual work required of farmers is "finely cutting plant fragments and sealing them in a special tube," which significantly reduces the technical and mental burden on farmers. Therefore, it can be easily used by vegetable farmers who want to overcome crop diseases while reducing pesticide use, or by those who want to monitor growth conditions from multiple angles and detect various abnormalities early. [Explanation of Symbols]

[0084] 10, 11, 11A, 11B signal generators 12 DNA Circuits 13 Adding Circuits 14 DNA amplification circuits 15 DNA diagnostic circuits 16 DNA visualization circuit 20 Plant Diagnostic Kits 22 tubes 24, 25, 26 solution 30 plant fragments

Claims

1. This is a method for designing a molecular circuit that takes molecule D as input and molecule I as output. The aforementioned molecular circuit, in its simplified model, uses molecules Ba, Bb, I, M, and D to form the following three reaction systems: [Math 1] Includes a reaction system represented by, The initial concentrations of the molecules Ba, Bb, I, M, and D are respectively [Ba] 0 [Bb] 0 [I] 0 [M] 0 [D] 0 In that case, the following conditions (A1) to (A5): (A1) Tb > 0 (A2) Ti > 0 (A3) (B) 0 +[Bb] 0 =Tb (A4) [I] 0 +[M] 0 +[D] 0 =Ti (A5) Ti / Tb<<1 A method comprising step A, setting the parameters of Tb and Ti such that all conditions are met.

2. The method according to claim 1, wherein the molecule Ba, the molecule Bb, the molecule I, the molecule M, and the molecule D are nucleic acids.

3. In step A, [Ba] 0 = [Bb] 0 [D] 0 The method according to claim 1 or 2, wherein the value is set to =d, and [I] becomes d.

4. In step A, [Ba] 0 [Bb] 0 Let X times (where X is a number greater than 0), and [D] 0 The method according to claim 1 or 2, wherein the value is set to =d and [I] is X times d.

5. If we let km be the rate constant for the reaction proceeding to the right in equation (1), kl be the rate constant for the reaction proceeding to the left in equation (1), and kf be the rate constant for the reactions in equations (2) and (3), The method according to claim 1 or 2, comprising step B of determining km, kl, and kf such that the following (B1) and (B2) are satisfied. (BB1)k=[D] 0 ・kl (B2) kf=(Tb / Ti)・kl

6. This is a molecular circuit that takes molecule D as input and molecule I as output. The aforementioned molecular circuit, in its simplified model, uses molecules Ba, Bb, I, M, and D to form the following three reaction systems: 【Number 2】 Includes a reaction system represented by, A molecular circuit that satisfies all of the following conditions. (A1) Tb > 0 (A2) Ti > 0 (A3) (B) 0 +[Bb] 0 =Tb (A4) [I] 0 +[M] 0 +[D] 0 =Ti (A5) Ti / Tb<<1 Here [Ba] 0 [Bb] 0 [I] 0 [M] 0 [D] 0 These are the initial concentrations of molecules Ba, Bb, I, M, and D, respectively, and Tb and Ti are concentration parameters with units.

7. The aforementioned molecular circuit is the following reaction system: [Math 3] The reaction system is represented by (where xm2, xm3, sD, xd1, xd2, xd3 are molecules), [Ba] 0 =X×β / 2, [Bb] 0 = β / 2, [I] 0 = 0, [x m²] 0 = β / 2, [M] 0 = 0, [x m³] 0 = 0, [sD] 0 = 0, [D] 0 = d, [xd1] 0 =X×β / 2, [xd2] 0 = X × d, [x d 3] 0 = 0, β is a parameter whose unit is concentration. The molecular circuit according to claim 6, wherein X is the ratio of the concentration of molecule I to the concentration of molecule D ([I] / [D]), and is a number greater than 0.

8. The molecular circuit is a signal generator connected to other molecular circuits, The signal generator is configured such that [I] = d, [Ba] 0 = [Bb] 0 [D] 0 = d, The molecular circuit according to claim 6, wherein [I] is used as an input signal for the other molecular circuit.

9. The molecular circuit is a signal intensity transducer connected to other molecular circuits. The signal intensity converter is configured such that [I] is X times d, [Ba] 0 [Bb] 0 X times (where X is a number greater than 0), and [D] 0 = d, The molecular circuit according to claim 6, wherein [I] is used as an input signal for the other molecular circuit.

10. The molecular circuit according to claim 6, wherein the molecule Ba, the molecule Bb, the molecule I, the molecule M, and the molecule D are nucleic acids.

11. An information processing system comprising one or more molecular circuits according to any one of claims 6 to 10.

12. The information processing system according to claim 11 for detecting molecule D.

13. This is a plant diagnostic kit that diagnoses the health status of the target plant. The molecular circuit described in claim 10 is included, The aforementioned molecule D is a microRNA of the diagnostic item, A plant diagnostic kit that diagnoses the health status of plants based on the presence or absence of molecular I production.

14. The plant diagnostic kit according to claim 13, further comprising a determination means for determining whether or not molecule I has been generated in the molecular circuit.

15. The plant diagnostic kit according to claim 14, wherein the determination means includes a DNA diagnostic circuit and a DNA visualization circuit.

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