An evaluation method for evaluating enzymes translated from mRNA, an evaluation kit used in the evaluation method, and evaluation reagents comprising the evaluation kit and a first component for use in the evaluation reagents.

Abstract

This invention provides a method for efficiently evaluating (selecting) enzymes with desired characteristics from an mRNA display. [Solution] A method for evaluating an enzyme translated from mRNA 1 using an evaluation reagent 4 containing a first substrate 4b that reacts with enzyme 3 and a first component 4a that interacts with the obtained reactant. The method includes the steps of: forming a first complex by hybridizing mRNA with a second component 2 containing a hybridizing region 2a and a capture region 2b that captures the translated enzyme; forming a second complex by capturing the enzyme translated in a cell-free protein synthesis system into the first complex; forming a third complex 10c by binding the first component of the evaluation reagent to the second complex; and evaluating the enzyme by detecting an interacting product 6 formed in a reactant interaction step, in which the reactant obtained in the first substrate reaction step, in which the enzyme contained in the third complex reacts with the first substrate, and the reactant interacts with the first component.

Description

【Technical Field】 【0001】 The disclosure in the present application relates to an evaluation method for evaluating an enzyme translated from mRNA, an evaluation kit used in the evaluation method, evaluation reagents constituting the evaluation kit, and a first component for use in the evaluation reagents. 【Background Art】 【0002】 Evolutionary molecular engineering, a technique for creating molecules with desired properties and functions, is known. Evolutionary molecular engineering is a technique that experimentally reproduces, in a test tube, the cycle of evolution that has occurred on Earth, which is the repetition of mutation and selection in organisms, and improves the biological functions of molecules (such as nucleic acids and proteins). 【0003】 As an example of a technique for associating genotypes and phenotypes, the mRNA display method is known as a tool for advancing evolutionary molecular engineering (see Patent Document 1). The mRNA display method is a technique for integrating genotypes and phenotypes by covalently bonding mRNA as a genotype and peptide molecules as a phenotype using a cell-free translation system (in vitro protein synthesis system). As an example of the mRNA display method, a method of linking a peptide molecule translated from mRNA and the mRNA encoding the peptide via puromycin, which is an analog of the 3'-terminal portion of tyrosyl tRNA, is known. 【Prior Art Documents】 【Patent Documents】 【0004】 【Patent Document 1】 International Publication No. 98 / 016636 【Summary of the Invention】 【Problems to be Solved by the Invention】 【0005】 When improving enzymes using evolutionary molecular engineering techniques, for example, the mRNA encoding the enzyme is generally generated by in vitro transcription from a DNA library containing various mutations. Therefore, when evaluating (selecting) enzymes with desired characteristics using mRNA display methods, it is necessary to evaluate (select) mRNA displays with the desired characteristics from among mRNA displays where genotype and phenotype are integrated. However, currently, there is no known method for efficiently evaluating (selecting) enzymes with desired characteristics from among mRNA displays. 【0006】 The disclosures in this application are made to solve the above problems. The inventors have conducted diligent research and found that (1) Using an evaluation reagent containing a first substrate that reacts with an enzyme translated from mRNA, and a first component that interacts with the reactant obtained by the reaction of the enzyme and the first substrate, (2) The mRNA display (a complex of mRNA and enzyme) in which the genotype and phenotype are integrated forms a complex with the first component, (3) The enzyme translated from mRNA is reacted with the first substrate to form an interacting product with the first component, (4) Detect the interacting substances, This has led to a new discovery: it is possible to evaluate enzymes translated from mRNA. 【0007】 In other words, the purpose of the disclosure in this application is to provide an evaluation method for evaluating enzymes translated from mRNA, an evaluation kit used in the evaluation method, an evaluation reagent constituting the evaluation kit, and a first component for use in the evaluation reagent. [Means for solving the problem] 【0008】 The disclosures of this application relate to an evaluation method for evaluating enzymes translated from mRNA, an evaluation kit used in the evaluation method, and evaluation reagents and a first component for use in the evaluation reagents that constitute the evaluation kit. 【0009】 (1) An evaluation method for evaluating enzymes translated from mRNA using an evaluation reagent, The evaluation reagent comprises a first substrate that reacts with the enzyme, and a first component that interacts with the reactant obtained by the reaction of the enzyme and the first substrate. The aforementioned evaluation method is A first complex formation step involves hybridizing a second component, which includes a hybridizing region for hybridizing to the mRNA and a capture region for capturing the translated enzyme, to the mRNA, thereby forming a first complex in which the mRNA and the second component are complexed. A second complex formation step is performed in which the mRNA of the first complex is translated using a cell-free protein synthesis system, and the translated enzyme is captured in the capture region, thereby forming a second complex in which the first complex and the enzyme are complexed. A third complex formation step, in which the first component contained in the evaluation reagent is bound to the second complex to form a third complex, A first substrate reaction step in which the enzyme contained in the third complex reacts with the first substrate, A reactant interaction step in which at least the reactant obtained in the first substrate reaction step is made to interact with the first component to form an interacting product, An evaluation step in which the enzyme is evaluated by detecting the interacting product formed in the reactant interaction step, Evaluation methods, including those mentioned above. (2) The interacting material formed in the reactant interaction step binds to the first component of the third complex, The aforementioned evaluation process, The interacting material and an isolation carrier that reacts with the interacting material are reacted together. The evaluation is based on whether or not the third complex to which the interacting substances are bound can be isolated by the isolation carrier. The evaluation method described in (1) above. (3) The first component is an enzyme for evaluation, The reactant interaction step is The present invention further comprises a second substrate that interacts with the aforementioned enzyme for evaluation, By allowing the reactant and the second substrate to interact with the evaluation enzyme, the second substrate reaction product obtained from the second substrate binds to the first component of the third complex. The aforementioned evaluation process, The second substrate reactant is reacted with an isolation carrier that reacts with the second substrate reactant, The evaluation is based on whether or not the third complex to which the second substrate reactant is bound can be isolated by the isolation carrier. The evaluation method described in (1) above. (4) The first component is an enzyme for evaluation, The reactant interaction step is The present invention further comprises a second substrate that interacts with the aforementioned enzyme for evaluation, By allowing the reactant and the second substrate to interact with the evaluation enzyme, a second substrate reaction product, which is a fluorescent substance obtained by modifying the second substrate, is obtained. The evaluation step is evaluated by the fluorescence intensity of the second substrate reactant. The evaluation method described in (1) above. (5) The reactant interaction step is carried out with the third complex confined in a microspace, The isolation step further includes isolating the microspace in which the third complex is confined using a sorter, based on the evaluation results of the evaluation step described above. The evaluation method described in (4) above. (6) The first component is a peptide, The first substrate binds to the peptide by reacting with the enzyme. The evaluation method described in (2) above. (7) The first component binds to the mRNA or the hybridized region. The evaluation method described in any one of the above (1) to (6). (8) The first component includes a linker, and the first component indirectly binds to mRNA via the linker. The evaluation method described in (7) above. (9) The first component comprises DNA that hybridizes to the mRNA and a protein that binds to the DNA, The linker binds to the protein, By binding the first component to the linker, the first component indirectly binds to the mRNA via the linker The evaluation method according to (8) above. (10) Including a sequence analysis step of analyzing the sequence of the mRNA contained in the isolated third complex The evaluation method according to any one of (2), (3), (5), and (6) above. (11) An evaluation kit used in an evaluation method for evaluating an enzyme translated from mRNA, the evaluation kit comprising: An evaluation reagent comprising a first substrate that reacts with the enzyme and a first component that interacts with a reaction product obtained by the reaction of the enzyme and the first substrate; A second component comprising a hybridization region that hybridizes to the mRNA and a capture region for capturing the translated enzyme; Comprising Evaluation kit. (12) An evaluation reagent constituting an evaluation kit used in an evaluation method for evaluating an enzyme translated from mRNA, The evaluation reagent comprises a first substrate that reacts with the enzyme and a first component that interacts with a reaction product obtained by the reaction of the enzyme and the first substrate. Evaluation reagent. (13) The first component is a peptide or an evaluation enzyme, The first component comprises a linker that binds to the peptide or evaluation enzyme and a protein that binds to the linker. The evaluation reagent according to (12) above. (14) The first component further comprises DNA that hybridizes to the mRNA, By binding the protein to the DNA, the peptide or evaluation enzyme is indirectly connected to the mRNA. The evaluation reagent according to (13) above. (15) The first component is an evaluation enzyme, The evaluation reagent according to (13) or (14) above, further comprising a second substrate that interacts with the evaluation enzyme. The evaluation reagent according to (13) or (14) above. (16) The protein is scCRO, The enzyme used for evaluation is APEX2. The peptide contains glutamine An evaluation reagent as described in any one of the above (13) to (15). (17) A first component for use in the evaluation reagent described in (12) above, The first component is a peptide or an enzyme for evaluation. The first component comprises a linker that binds to the peptide or evaluation enzyme, and a protein that binds to the linker. Component 1. (18) The protein is scCRO, The enzyme used for evaluation is APEX2. The peptide contains glutamine The first component as described in (17) above. (19) Further comprising DNA that hybridizes to mRNA and binds to the protein The first component as described in (17) or (18) above. [Effects of the Invention] 【0010】 The evaluation kit or evaluation reagent disclosed in this application can be used to evaluate (select) the enzyme in the mRNA-enzyme complex. [Brief explanation of the drawing] 【0011】 [Figure 1A] Figure 1A is a schematic diagram illustrating each step of the evaluation method according to the embodiment. [Figure 1B] Figure 1B is a schematic diagram illustrating each step of the evaluation method according to the embodiment. [Figure 1C] Figure 1C is a schematic diagram illustrating each step of the evaluation method according to the embodiment. [Figure 2A] Figure 2A is a schematic diagram illustrating the reactant interaction step (ST5) and evaluation step (ST6) according to the embodiment. [Figure 2B]Figure 2B is a schematic diagram illustrating the reactant interaction step (ST5) and evaluation step (ST6) according to the embodiment. [Figure 2C] Figure 2C is a schematic diagram illustrating the reactant interaction step (ST5) and evaluation step (ST6) according to the embodiment. [Figure 3A] Figure 3A is a schematic diagram illustrating the general structure of the first component 4a. [Figure 3B] Figure 3B is a schematic diagram illustrating the general structure of the first component 4a. [Figure 3C] Figure 3C is a schematic diagram illustrating the general structure of the first component 4a. [Figure 4] Figure 4 is a graph showing the fluorescence intensity measured in Example 1 and Comparative Example 1. [Figure 5] Figure 5 is a schematic diagram showing an overview of the procedure in Example 2. [Figure 6] Figure 6 is a schematic diagram showing the general procedure of Example 2. [Figure 7] Figure 7 is a photograph used as a substitute for a drawing, and it shows the agarose electrophoresis performed in Example 2. [Figure 8] Figure 8 is a schematic diagram showing an overview of the procedure in Example 3. [Figure 9] Figure 9 is a photograph used as a substitute for a drawing, and it shows the agarose electrophoresis performed in Example 3. [Modes for carrying out the invention] 【0012】 The following describes in detail the evaluation method for evaluating enzymes translated from mRNA disclosed in this application (hereinafter sometimes simply referred to as the "evaluation method"), the evaluation kit used in the evaluation method, and the evaluation reagents and the first component for use in the evaluation reagents that constitute the evaluation kit. (1) A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits, respectively. (2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "identical" or "same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field. (3) When "approximately XX shape" is written, it includes not only the exact XX shape but also a shape that can be understood as approximately XX shape. This is how it is interpreted. 【0013】 (Embodiment of the evaluation method) The evaluation method according to the embodiment will be described with reference to Figures 1A to 1C. Figures 1A to 1C are schematic diagrams illustrating each step of the evaluation method according to the embodiment. 【0014】 The evaluation method according to the embodiment is carried out using an evaluation reagent. The evaluation reagent includes a first substrate that reacts with the enzyme, and a first component that interacts with the reactant obtained by the reaction of the enzyme and the first substrate. 【0015】 The evaluation method includes a first complex formation step, a second complex formation step, a third complex formation step, a first substrate reaction step, a reactant interaction step, and an evaluation step. 【0016】 The first complex formation step (ST1) involves hybridizing component 2, which includes a hybridizing region 2a for hybridizing to mRNA1 and a capture region 2b for capturing the translated enzyme, to mRNA1, thereby forming a first complex 10a in which mRNA and component 2 are combined. 【0017】 mRNA1 can be generated by in vitro transcription from a DNA library in which various mutations have been introduced into the DNA encoding the target enzyme. In other words, the mRNA encoding the target enzyme may be an mRNA library in which various mutations have been introduced. There are no particular restrictions on the target enzyme, but examples include D-amino acid oxidase (DAAO), which can be used for D-amino acid measurement; transglutaminase (TG), which is attracting attention in the food and pharmaceutical industries and forms peptide bonds between glutamine residues and lysine residues, etc.; glucose oxidase; kinase; polymerase, ligase, choline oxidase, arditol oxidase, etc. Alternatively, mRNA1 used in the first complex formation step (ST1) may be of a single type. For example, mRNA1 may be evaluated type by type by performing the evaluation method disclosed in this application on each type of mRNA1 that has undergone point mutations. 【0018】 The second component 2 is not particularly limited as long as it includes a hybridization region 2a that hybridizes to mRNA1 and a capture region 2b for capturing the translated enzyme. The hybridization region 2a can be appropriately designed according to the sequence of the mRNA encoding the target enzyme. The capture region 2b only needs to be able to capture the enzyme translated from the mRNA, and examples include puromycin, which has already been used in mRNA display methods, and a structure in which an amino acid is ester-bonded to an oligoRNA consisting of the base sequence ACCA as described in Patent Document 1. The hybridization region 2a and the capture region 2b only need to be connected via a linker 2c. The second component 2 may be fabricated to have the above characteristics, or a commercially available product (e.g., puromycin linker) may be used. 【0019】 In the second complex formation step (ST2), mRNA1 of the first complex 10a is translated using a cell-free protein synthesis system, and the translated enzyme 3 is captured in the capture region 2b, thereby forming the second complex 10b, which is a complex of the first complex 10a and enzyme 3. A commercially available cell-free protein synthesis kit can be used as the cell-free protein synthesis system. 【0020】 The third complex formation step (ST3) involves forming the third complex 10c by binding the first component 4a contained in the evaluation reagent 4 to the second complex 10b. In the example shown in Figure 1B, the first component 4a is bound to mRNA1. Alternatively, the first component 4a may bind to any of the components constituting the second complex 10b: the hybridization region 2a, the capture region 2b, the linker 2c, or the enzyme 3. The binding method of the first component 4a will be described later, but the first component 4a may bind directly to the components constituting the second complex 10b, or it may bind indirectly via a linker or the like. 【0021】 The first substrate reaction step (ST4) involves reacting enzyme 3, contained in the third complex 10c, with the first substrate 4b, contained in the evaluation reagent 4. The reaction between enzyme 3 and the first substrate 4b yields the reactant 5. 【0022】 The reactant interaction step (ST5) involves interacting the reactant 5 obtained in at least the first substrate reaction step (ST4) with the first component 4a to form an interacting product 6. 【0023】 The evaluation step (ST6) evaluates the enzyme 3 by detecting the interacting product 6 formed in the reactant interaction step (ST5). 【0024】 <Embodiments of the reactant interaction step (ST5) and evaluation step (ST6)> Next, various embodiments of the reactant interaction step (ST5) and evaluation step (ST6) will be described with reference to Figures 2A to 2C. Figures 2A to 2C are schematic diagrams illustrating the reactant interaction step (ST5) and evaluation step (ST6) according to the embodiment. 【0025】 (First embodiment) Figure 2A shows an example in which reactant 5, formed in the reactant interaction step (ST5), binds to the first component 4a as an interacting substance 6. More specifically, in the example shown in Figure 2A, enzyme 3 is transglutaminase (TG), the first component 4a is a peptide containing glutamine (Q), and the first substrate 4b is biotinamine. In the example shown in Figure 2A, in the reactant interaction step (ST5), a peptide bonding reaction occurs between the biotinamine of the first substrate 4b and the glutamine of the first component 4a by transglutaminase 3, and reactant 5, from which the amino group of the biotinamine of the first substrate 4b has been removed, binds to the glutamine of the first component 4a as an interacting substance 6. Then, in the evaluation step (ST6), streptavidin 7a adsorbed on magnetic beads, which are isolation carriers 7, is reacted with biotin 6a of the interacting substance 6, and the magnetic beads are recovered with a magnet. If enzyme 3, transglutaminase, is active, peptide bonds are formed, and the interacting product 6 (third complex 10c) is isolated together with the magnetic beads. 【0026】 On the other hand, if the transglutaminase is not functioning, no peptide bond is formed. Therefore, since the interactor 6 does not bind to the peptide, which is the first component 4a, the third complex 10c is not isolated together with the magnetic beads. As described above, whether or not the interactor 6 (third complex 10c) is isolated by the isolation carrier 7 can be used to evaluate whether or not the enzyme has the desired phenotype. The evaluation method according to the first embodiment can also be described as a method for isolating (selecting) the third complex 10c having the desired phenotype. 【0027】 The mRNA contained in the isolated third complex 10c can be used to form cDNA using reverse transcriptase. This cDNA can then be amplified by PCR and used in further evolutionary molecular engineering techniques. 【0028】 Note that the enzyme 3, first component 4a, first substrate 4b, interacting substance 6, and isolation carrier 7 mentioned above are merely examples, and other components may be used. For example, instead of biotin-streptavidin binding, antigen-antibody reactions, protein complementation (Nanoluc complementation), spytag-spycatcher, etc., may be used. Alternatively, instead of magnetic beads, streptavidin may be immobilized on a column to recover the third complex 10c on the column. Other methods that can be used include fractionating this complex into emulsions and separating them by fluorescence properties, as well as centrifugation and chromatography methods involving various principles. 【0029】 (Second embodiment) Figure 2B shows an example where the first component 4a is an evaluation enzyme used to evaluate enzyme 3, and the second substrate reactant 8a obtained from the second substrate 8 binds to the first component 4a. More specifically, in the example shown in Figure 2B, enzyme 3 is D-amino acid oxidase (DAAO), the first component 4a is APEX2 (formally named "Ascorbate peroxidase," an enzyme involved in DNA repair), the first substrate 4b is D-Ala or D-Pro, and the second substrate 8 is biotinphenol. In the example shown in Figure 2B, in the reactant interaction step (ST5), D-amino acid oxidase 3 generates reactant 5, H2O2, from the first substrate 4b, D-Ala or D-Pro. Then, by allowing the H2O2 of reactant 5 and the biotinphenol of the second substrate 8 to interact with APEX2, which is the first component 4a, the second substrate reactant 8a, from which the hydrogen of biotinphenol has been removed, binds to APEX2 and the tyrosine of nearby proteins. In other words, in the example shown in Figure 2B, the second substrate reactant 8a corresponds to the interacting product 6 formed in the reactant interaction step (ST5). 【0030】 Then, in the evaluation step (ST6), streptavidin 7a adsorbed on magnetic beads, which are the isolation carrier 7, is reacted with biotin 8b contained in the second substrate reaction product 8a, and the magnetic beads are recovered with a magnet. If the enzyme 3, D-amino acid oxidase, is active, H2O2, which is the substrate of APEX2, is provided, and biotinphenol binds to APEX2, so the second substrate reaction product 8a (third complex 10c) is isolated together with the magnetic beads. On the other hand, if D-amino acid oxidase 3 is not functional, H2O2 is not provided, and the third complex 10c is not isolated together with the magnetic beads. The isolated third complex 10c can be used in further evolutionary molecular engineering techniques, similar to the first embodiment. 【0031】 Note that the enzyme 3, first component 4a, first substrate 4b, isolation carrier 7, and second substrate 8 described above are merely examples, and other components may be used. In addition to the examples of the second embodiment, for example, a peroxidase such as horseradish peroxidase may be used as the first component 4a. Also, a peptide or a sugar such as digoxin may be used instead of biotin in the second substrate 8, in which case an antibody or lectin may be used as the isolation carrier 7. 【0032】 (Third embodiment) Figure 2C shows an example in which the first component 4a is an evaluation enzyme for evaluating enzyme 3, and the fluorescence intensity of the reactant of the second substrate 8 is evaluated. More specifically, in the example shown in Figure 2C, enzyme 3 is D-amino acid oxidase (DAAO), the first component 4a is APEX2, the first substrate 4b is a D-amino acid, and the second substrate 8 is 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red). In the example shown in Figure 2C, in the reactant interaction step (ST5), D-amino acid oxidase 3 generates reactant 5, H2O2, from the first substrate 4b, a D-amino acid. Then, by interacting the H2O2 of reactant 5 and the second substrate 8, 10-acetyl-3,7-dihydroxyphenoxazine, with APEX2, the first component 4a, the second substrate 8 is transformed into a fluorescent substance (Resorufin). In the example shown in Figure 2C, the second substrate reactant 8a, which is a fluorescent substance obtained by changing the second substrate 8, corresponds to the interacting product 6 formed in the reactant interaction step (ST5). 【0033】 In the evaluation step (ST6), the fluorescence intensity of the second substrate reaction product 8a should be measured. If enzyme 3, D-amino acid oxidase, is active, H2O2, the substrate of APEX2, is provided. Combining H2O2 with 10-acetyl-3,7-dihydroxyphenoxazine causes 10-acetyl-3,7-dihydroxyphenoxazine to transform into a fluorescent substance and emit fluorescence. On the other hand, if D-amino acid oxidase 3 is not functional, H2O2 is not provided, and therefore 10-acetyl-3,7-dihydroxyphenoxazine does not emit fluorescence. Thus, enzyme 3 can be evaluated by measuring the fluorescence intensity. 【0034】 Note that the enzyme 3, first component 4a, first substrate 4b, and second substrate 8 mentioned above are merely examples, and other components may be used. For example, enzyme 3 may be an enzyme that generates hydrogen peroxide, such as glucose oxidase. The first substrate 4b may be one suitable for each enzyme. The second substrate 8 may be a substrate that emits light (such as luminol and its derivatives) or produces color (such as o-phenylenediamine, 3,3',5,5'-tetramethylbenzidine, etc.). 【0035】 The reactant interaction step (ST5) may be performed with the third complex 10c confined in a microspace within the emulsion. In this case, an isolation step is performed in which the microspace containing the third complex 10c is isolated using a sorter based on the evaluation results of the evaluation step (ST6), thereby enabling the isolation of the third complex 10c using the fluorescence intensity in the evaluation step as an indicator. 【0036】 The evaluation method according to this embodiment has the following effects. (1) In the evaluation method according to the embodiment, the enzyme can be evaluated at the single-molecule level by adding the first component 4a contained in the evaluation reagent to the second complex (mRNA display) in which the genotype and phenotype are integrated, and then adding the substrate of at least the enzyme (phenotype) translated from mRNA. Therefore, the enzyme can be evaluated at the single-molecule level by obtaining cDNA from the complex evaluated at the single-molecule level by reverse transcription and analyzing the DNA sequence. Furthermore, mutations and the like can be added, so evolutionary molecular engineering techniques can be efficiently implemented. 【0037】 (Regarding optional additional configurations for which evaluation methods can be applied) Next, with reference to Figures 3A to 3C, optional additional configuration examples in which the evaluation method according to the embodiment can be employed will be described. Figures 3A to 3C are schematic diagrams illustrating the outline of the first component 4a. The first component 4a contained in the evaluation reagent may indirectly bind to the components constituting the second complex 10b via the linker 4a1. 【0038】 Figure 3A shows an example in which the first component 4a binds to mRNA1 via linker 4a1. In the example shown in Figure 3A, the end of linker 4a1 opposite the first component 4a may be formed by a component that binds to mRNA, such as DNA or RNA that hybridizes with mRNA, an aptamer, etc. 【0039】 The first component 4a may further include DNA 4a2 that hybridizes to mRNA1 and protein 4a3 that binds to DNA 4a2, as shown in Figure 3B. One end of linker 4a1 binds to protein 4a3 and the other end binds to the first component 4a, and protein 4a3 binds to DNA 4a2, so that the first component 4a indirectly binds to mRNA via linker 4a1. 【0040】 In the example shown in Figure 3A, linker 4a1 can be directly bound to mRNA, but when aptamers are used, the sequences that hybridize to mRNA are limited. On the other hand, in the example shown in Figure 3B, DNA 4a2 that hybridizes to mRNA is designed, and protein 4a3 is bound to DNA 4a2 that has hybridized to mRNA1. In the example shown in Figure 3B, DNA 4a2 can be hybridized to any location on the mRNA, and the length of linker 4a1 can also be designed arbitrarily, which has the effect of positioning the first component 4a close to enzyme 3. The location for hybridizing DNA 4a2 can be determined by analyzing the three-dimensional structure from the mRNA sequence information, the information of the first component 2, and the sequence information of enzyme 3. Alternatively, as shown in Figure 3B, oligo DNA may be bound to the first component 4a, and the oligo DNA may be hybridized to mRNA1. By designing the oligo DNA sequence to bind to a desired position on the mRNA based on the mRNA sequence information, the first component 4a can be positioned close to enzyme 3. Furthermore, as shown in Figure 3C, even when a peptide is used as the first component 4a, the peptide may be indirectly bound to mRNA1 using linker 4a1, DNA 4a2, and protein 4a3. 【0041】 DNA4a2 should be designed according to the characteristics of protein 4a3. For example, if scCro (single-chain Cro protein) is used as the protein, it should be designed to have a double-stranded region recognized by scCro and a hybridization region that hybridizes to mRNA1. Alternatively, the DNA-binding protein Tus and its recognition sequence may be used. 【0042】 The evaluation method may include a sequence analysis step in which the sequence of mRNA1 contained in the isolated third complex 10c is analyzed. The sequence analysis step is not particularly limited as long as it can analyze the mRNA sequence, and any known method may be used. Although not limited, sequence analysis may be performed directly from the mRNA contained in the isolated third complex 10c, or cDNA may be reverse transcribed using reverse transcriptase, amplified as necessary, and then sequence analysis may be performed. 【0043】 (Evaluation kit embodiment) As described in the evaluation method according to the embodiment, the evaluation method disclosed in this application allows for the evaluation of enzymes translated from mRNA containing mutations by using an evaluation reagent in a known mRNA display method. Since mRNA libraries are generally prepared by the user, an evaluation kit containing the evaluation reagent may be provided as a kit for use in an evaluation method for evaluating enzymes translated from mRNA. 【0044】 The evaluation kit may include an evaluation reagent comprising a first substrate 4b that reacts with enzyme 3, and a first component 4a that interacts with the reactant 5 obtained by the reaction of enzyme 3 and the first substrate 4b, and a second component 2 comprising a hybridizing region 2a that hybridizes to mRNA 1 and a capture region 2b for capturing the translated enzyme 3. The evaluation reagent may optionally include a second substrate 8. The second component 2, first component 4a, first substrate 4b, and the optional additional elements, a linker 4a1 bound to the first component 4a, protein 4a3 and DNA 4a2, isolation carrier 7, and second substrate 8, which are at least included in the evaluation kit, have been described in the evaluation method according to the embodiment. Therefore, a detailed description will be omitted to avoid redundancy. 【0045】 (Embodiment of evaluation reagent) The second component 2 included in the evaluation kit can be a commercially available product such as puromycin linker. Similarly, a commercially available magnetic bead, which is an example of an isolation carrier 7, can also be used. Therefore, the evaluation reagents included in the evaluation kit, more specifically the first component 4a and the first substrate 4b, or the first component 4a, the first substrate 4b, and the second substrate 8, may be provided as evaluation reagents. 【0046】 (Embodiment of the first component) The first substrate 4b included in the evaluation reagent can also be a commercially available one. Therefore, only the first component 4a included in the evaluation reagent may be provided as a component for use in the evaluation reagent. 【0047】 Examples are provided below to specifically illustrate the embodiments disclosed in this application. These examples are solely for illustrative purposes and are not intended to limit or restrict the scope of the invention disclosed in this application. [Examples] 【0048】 <Example 1> We conducted an experiment where the enzyme translated from mRNA was D-amino acid oxidase (DAAO). The experimental procedure is shown below. <Fabrication of the third complex> (1) DNA amplification DNA was amplified by PCR using the composition shown in Table 1 below. The template sequence and primer sequence are shown in Table 1. SEQ.ID No. 3 in Table 1 shows the plasmid sequence containing the D-amino acid oxidase (DAAO) encoding sequence. SEQ.ID No. 4 shows the template sequence with the D-amino acid oxidase (DAAO) encoding sequence, T7 promoter, T7 terminator, and RBS sequences added. The KOD One master mix for DNA amplification was obtained from TOYOBO, Osaka, Japan. PCR was performed 20 times with the cycle of "1 minute at 95°C, 10 seconds at 98°C, 5 seconds at 60°C, 5 seconds at 68°C, and infinity at 20°C". [Table 1] 【0049】 [Table 2] 【0050】 (2) In vitro transfer The DNA amplified in (1) above, along with the Promega RiboMAX kit, was incubated at 37°C for 2 hours using the composition shown in Table 3 below to transfer mRNA from the DNA in vitro. 【0051】 [Table 3] 【0052】 (3) Binding of the second component to mRNA (formation of the first complex) For the second component, puromycin linker (Epsilon Molecular Engineering) was used. Using the purchased puromycin linker (referred to as "cnvK linker" in the table below, or simply as "linker") and the mRNA obtained in (2) above, a first complex was formed in the composition shown in Table 4, in which mRNA and linker were combined. In the composition shown in Table 4, the linker:mRNA was mixed in a 1:1 ratio. [Table 4] 【0053】 More specifically, the first complex was prepared using the following procedure. a: The compositions shown in Table 4 above were annealed at 90°C for 1 minute, 70°C for 1 minute, and 25°C for infinity. b: Photocrosslinking was performed at 365nm for 4 minutes. 【0054】 (4) Creation of mRNA display (formation of complex 2) Using the cell-free protein synthesis kit PUREfrex® 2.1 (GeneFrontier), DAAO was translated from mRNA in the composition shown in Table 5 below. The translated DAAO was then bound to puromycin to form the second complex. More specifically, the solution shown in Table 5 (where "mRNA-Linker" represents the "first complex") was incubated in a 37°C water bath for 30 minutes, EDTA was added to a total volume of 0.5 M, and the mixture was incubated in a 37°C water bath for 5 minutes. [Table 5] 【0055】 (5) Fabrication of the third complex (5-1) Preparation of the first component (APEX2-scCro) The first component (4a-4a1-4a3) shown in Figure 3B was prepared using the following procedure. a: DNA amplification DNA was amplified using the forward primer (SEQ.ID No. 5), reverse primer (SEQ.ID No. 6), and template plasmid (pRSET-APEX2-scCro; SEQ.ID No. 7) shown in Table 6, following the same procedure as in "(1) DNA amplification" above. The sequence of APEX2-scCro is shown in SEQ.ID No. 8. 【0056】 [Table 6] 【0057】 b: Translation of the first component (APEX2-scCro) APEX2-scCro was translated using the template (APEX2-scCro) obtained in (a) above and the cell-free protein synthesis kit PUREfrex® 2.1 (GeneFrontier) in the compositions shown in Table 7 below. More specifically, the solutions shown in Table 7 were incubated in a 37°C water bath for 2 hours. [Table 7] 【0058】 (5-2) Preparation of DNA4a2, the first component that hybridizes to mRNA1 For the DNA4a2 hybridized with mRNA, we used the DNA shown in SEQ.ID No. 9 below (ORC hairpin linker2). SEQ.ID No.9: GATCCtatcaccgcgggtgataGTACGTTTTTTCGTACtatcacccgcggtgataGGATCAAATGATGATGATGGCTGCCT 【0059】 The DNA was converted into a double-stranded DNA with a hairpin structure by performing the following cycle: "3 minutes at 95°C → cooling at 0.4°C / second → 1 minute at 70°C → infinity at 20°C" with the composition shown in Table 8 below. The composition of the binding buffer is as follows. <2×Binding buffer> 20mM Tris-HCl, pH 8.0 2mM EDTA 2M NaCl 0.2% Tween 20 [Table 8] 【0060】 (5-3) Binding of the second complex to DNA4a2 15 μL of the second complex prepared in (4) above and 11.5 μL of DNA4a2 prepared in (5-2) above were mixed and annealed in the following cycle: "25°C for 1 minute → increase temperature at 0.4°C / sec → 40°C for 1 minute → decrease temperature at 0.1°C / sec → 25°C indefinitely". 【0061】 (5-4) Immobilization of the second complex onto purification beads 20 μL of purification beads (Dynabeads Streptavidin MyOne C1) were placed in a tube and washed three times with 200 μL of 1x Binding buffer. Next, 26.5 μL of 2x Binding buffer and 26.5 μL of the second complex were added and incubated at 25°C for 30 minutes. Then, the second complex, to which hairpin DNA4a2 was bound, was immobilized onto the purification beads by washing once with 200 μL of 1x Binding buffer and once with 200 μL of PBS. Note that the ends of the puromycin linker contain biotin, and this biotin binds to the streptavidin in Dynabeads Streptavidin MyOne C1, causing the second complex to bind to the purification beads. 【0062】 (5-5) Preparation of the third complex The entire amount of the second complex immobilized on the purification beads prepared in (5-4) above was mixed with 2.2 μL of the first component (APEX2-scCro) prepared in (5-1) above, 1 μL of 100 μM hemin, and 41.8 μL of PBS. The mixture was incubated at 4°C for 1 hour using a rotator, and unreacted material was removed by washing with 200 μL of PBS. 【0063】 (5-6) Purification of the third complex The entire amount of the third complex (immobilized on the purification beads) prepared in (5-5) above was mixed with 1 μL of 100 μM Hemin, 5 μL of RNase T1, and 44 μL of PBS. The mixture was incubated at 30°C for 1 hour using a rotator to separate the purification beads from the third complex. The supernatant was collected to prepare the third complex. The prepared third complex has the structure shown in Figure 3B. 【0064】 <First substrate reaction step, reactant interaction step, and evaluation step> The supernatant (supernatant) obtained in (5-6) above was used to evaluate the activity of DAAO in the third complex. The mechanism of the reactant interaction step in Example 1 is the same as in Figure 2C. The first substrate reaction step, reactant interaction step, and evaluation step were performed using the Amplex Red assay. The specific procedure is as follows. 【0065】 145 μL of 100 mM phosphate buffer (pH 7.5) and 50 μM Amplex red were added to a black 96-well plate. Next, 5 μL of 0.1 M D-Ala (in 100 mM phosphate buffer (pH 7.5)) was added to each well. The plate was immediately excited at a wavelength of 540 nm using a fluorescence plate reader, and fluorescence intensity was measured over time at a wavelength of 590 nm (every minute for 30 minutes). All measurements were repeated. 【0066】 <Comparative Example 1> In the <first substrate reaction step, reactant interaction step, and evaluation step> of Example 1 described above, the experiment was carried out in the same procedure as in Example 1, except that D-Ala, the first substrate, was not administered (NC). 【0067】 Figure 4 shows the fluorescence intensities measured in Example 1 and Comparative Example 1. The fluorescence intensities shown in Figure 4 are the values ​​obtained 30 minutes after the start of measurement. As is clear from Figure 4, fluorescence was observed in Example 1 by the Amplex red assay. In Example 1, since reaction product 5, H2O2, was generated from D-Ala in the first substrate reaction step, it is thought that H2O2 and the second substrate 8, Amplex red (10-acetyl-3,7-dihydroxyphenoxazine), interacted with the first component 4a, APEX2, causing Amplex red to be converted into the fluorescent substance Resorufin. On the other hand, in Comparative Example 1, since D-Ala was not administered, reaction product 5, H2O2, was not generated, and only background-level fluorescence was observed. Although not shown in the figure, in an experiment where 5 μL of 30 mM H2O2 (positive control; PC) was added instead of the first substrate D-Ala in Example 1, fluorescence was observed similarly to Example 1. From these results, we confirmed that by performing the first substrate reaction step and the reactant interaction step on the third complex disclosed in this application, it is possible to evaluate whether or not enzyme 3 translated from mRNA is functioning. 【0068】 Note that the mDAAO shown in Figure 4 is a modified version of DAAO in which the 255th amino acid R (arginine) is replaced with L (leucine). The results for mDAAO shown in Figure 4 were obtained by preparing and experimenting with the same procedure as in Example 1, except that the template DNA sequence was modified. mDAAO differs from wild-type DAAO in that it becomes a monomer by modifying the 255th amino acid, but as shown in Figure 4, it was confirmed to have almost the same activity as wild-type DAAO. Since the DAAO described in Example 2, which will be discussed later, refers to mDAAO, the results are shown in Figure 4 to demonstrate that mDAAO has enzymatic activity. 【0069】 <Example 2> Next, we conducted an experiment to confirm whether the evaluation method disclosed in this application could perform evaluation (separation of the third complex containing DAAO with enzymatic activity) in a situation where third complexes with different functions were mixed together. Figures 5 and 6 show an outline of the procedure in Example 2. The detailed procedure is described below with reference to Figures 5 and 6. 【0070】 (1) Amplification of the inactive DAAO gene A template was prepared in which the 232nd amino acid Y (tyrosine) of the D-amino acid oxidase (DAAO) from Example 1 was modified to be A (alanine) (hereinafter referred to as "Inactive DAAO gene"). (2) Fabrication of the third complex DNA of a DAAO gene (meaning the mDAAO mentioned above) with no modification at amino acid 232 was mixed with the inactive DAAO gene amplified in (1) above, in a ratio of DAAO gene:inactive DAAO gene = 1:10 and 1:100, i.e., the proportion of inactive DAAO gene was increased. By preparing the third complex using the mixed DNA in the same procedure as in Example 1, a mixture of the third complex containing active DAAO and the third complex containing inactive DAAO was obtained. 【0071】 Next, the supernatant (supernatant) of the prepared third complex mixture was incubated at 30°C for 30 minutes with the composition shown in Table 9 below to carry out the first substrate reaction step and reactant interaction step. The mechanism of the first substrate reaction step and reactant interaction step in Example 2 is the same as in Figure 2B, and the structure of the third complex is the same as in Figure 3B. [Table 9] 【0072】 Unreacted biotin styramide and D-Ala were removed from the reaction mixture after the reactant interaction step by sieving using a 10 kDA MWCO membrane. 【0073】 Next, 20 μL of Dynabeads SA MyOne C1 (isolation support 7 bound to streptavidin 7b, shown in Figure 2B) was added to the solution from which unreacted biotin styramide and D-Ala had been removed, and the mixture was incubated at 250°C for 30 minutes using a rotator. Then, the isolation support 7 was magnetically attracted using a magnet to separate it into a fraction in which DAAO was functional (enriched library, abbreviated as "EL") and a fraction that did not bind to the isolation support (unselected library, abbreviated as "UL"). 【0074】 Next, cDNA was prepared and amplified by reverse transcription of the mRNA of the 3rd complex from the EL and UL fractions using a standard method. Figure 5 shows a schematic of the reverse transcription. Reverse transcription was performed in two steps: in the first step, the sequence containing the DAAO gene and Inactive DAAO gene was amplified (PCR1: Nested_Fw1, 2ndR DAAO Rv1), and in the second step, a narrower sequence containing the DAAO gene and InactiveDAAO gene than in the first step was amplified (PCR2: InFusion DAO Fw, InFusion DAO Rv). Note that the “enzyme gene” shown in Figure 5 contains other sequences derived from the template before and after the DAAO gene and InactiveDAAO gene. Furthermore, "Nested_Fw2" shown in Figure 5 is the Fw primer used in the first step of the reverse transcription of "transglutaminase (TG)" in Example 3, which will be described later (the Rv primer is the same as in Example 2), while "gBlocks TG Fw" and "gBlocks TG Rv" are the primers used in the second step of the reverse transcription of "transglutaminase (TG)" in Example 3, which will be described later. Table 10 shows the primer sequences described in Figure 5. 【0075】 [Table 10] 【0076】 Next, as shown in Figure 6, the sequence containing the amplified DAAO gene (hereinafter sometimes simply referred to as "DAAO gene") and the sequence containing the inactive DAAO gene (hereinafter sometimes simply referred to as "Inactive DAAO gene") (both 1045 bp) were cut into 764 bp and 281 bp segments using the restriction enzyme Xhol, and the uncut segments were subjected to agarose electrophoresis. The results are shown in Figure 7. In Figure 7, "+" indicates that the sequence was treated with the restriction enzyme Xhol, and "-" indicates that it was not treated with the restriction enzyme Xhol. 【0077】 The left side of Figure 7 shows the electrophoresis results when DAAO gene and inactive DAAO gene were mixed in a ratio of 1:10. The right side of Figure 7 shows the electrophoresis results when DAAO gene and inactive DAAO gene were mixed in a ratio of 1:100, i.e., when the proportion of normal genes in the third complex was 1%. In the "EL-fraction" on the right side of Figure 7, a band was observed near the amplified DAAO gene (1045 bp), and in the "E.L+ fraction," bands were observed near 281 bp and 764 bp. On the other hand, in the "UL-fraction," a band was observed near the amplified DAAO gene (1045 bp), and in the "UL+ fraction," bands were observed near 281 bp and 764 bp. From these results, it was confirmed that the evaluation method disclosed in this application can selectively evaluate (enrich) mRNA encoding active DAAO even when different types of mRNA (active DAAO and inactive DAAO) are mixed. 【0078】 <Example 3> In this experiment, transglutaminase (TG) was used as the enzyme translated from mRNA, instead of DAAO as in Example 1. The procedure is as follows. (1) DNA amplification The experiment was carried out in the same manner as in Example 1, except that the sequences shown in Table 11 below were used. SEQ.ID No. 17 in Table 11 shows the sequence of the plasmid containing the sequence encoding transglutaminase (TG). SEQ.ID No. 18 shows the template sequence obtained by adding the T7 promoter, T7 terminator, and RBS sequences to the sequence encoding transglutaminase (TG). The primers used for DNA amplification were the same as in Example 1 (SEQ.ID No. 1 and SEQ.ID No. 2). [Table 11] 【0079】 (2) In vitro transcription and (3) Binding of the second component to mRNA The procedure was carried out in the same manner as in Example 1. 【0080】 (4) Creation of mRNA display (formation of complex 2) The mRNA display was prepared using the same procedure as in Example 1, except that the solution shown in Table 12 was used instead of the solution shown in Table 5 of Example 1. [Table 12] 【0081】 (5) Fabrication of the third complex (5-1) Preparation of the first component (peptide-scCro) Instead of the first component (APEX2-scCro) in Example 1, the first component (4a-4a1-4a3) shown in Figure 3C was prepared using the following procedure. a: DNA amplification The template plasmid shown in Table 13 (pRSET-peptide-scCro: SEQ.ID No. 19) was used. The forward and reverse primers were the same as in Example 1 (SEQ.ID No. 5 and SEQ.ID No. 6). The sequence of peptide-scCro is shown as SEQ.ID No. 20. Except for the difference in the template plasmid, DNA amplification was performed using the same procedure as in Example 1. [Table 13] 【0082】 b: Translation of the first component (peptide-scCro) Peptide-scCro was translated using the same procedure as in Example 1, except that it was translated using the Template (peptide-scCro) obtained in (a) above and the cell-free protein synthesis kit PUREfrex® 2.1 (GeneFrontier) with the composition shown in Table 14 below. [Table 14] 【0083】 (5-2) DNA4a2 hybridizes with mRNA1 It was prepared using the same procedure as in Example 1. 【0084】 (5-3) Binding of the second complex to DNA4a2 It was prepared using the same procedure as in Example 1. 【0085】 (5-4) Immobilization of the second complex onto purification beads It was prepared using the same procedure as in Example 1. 【0086】 (5-5) Preparation of the third complex The preparation was carried out using the same procedure as in Example 1, except that 100 μM hemin was not added. 【0087】 (5-6) Purification of the third complex Purification was carried out using the same procedure as in Example 1. The prepared third complex has the structure shown in Figure 3C. 【0088】 Figure 8 shows an outline of the procedure for Example 3. The detailed procedure will be described below with reference to Figure 8. <Production of the third complex (Inactive TG)> (1) Amplification of sequences containing the inactive TG gene A template containing the Inactive TG gene (hereinafter referred to as "Inactive TG gene") was prepared in which the 64th amino acid of TG in Example 3, C (cysteine), was modified to A (alanine). (2) Fabrication of the third complex A sequence containing a TG gene with no modification at the 64th amino acid and a sequence containing the inactive TG gene amplified in (1) above were mixed in a ratio of TG gene:inactive TG gene = 1:10 and 1:100, i.e., with a higher proportion of inactive TG gene. By preparing the third complex using the mixed DNA in the same procedure as in Example 1, a mixture of the third complex containing active TG and the third complex containing inactive TG was obtained. 【0089】 Next, the supernatant (supernatant) of the prepared third complex mixture was incubated at 37°C for 60 minutes with the composition shown in Table 15 below to carry out the first substrate reaction step and reactant interaction step. The mechanism of the first substrate reaction step and reactant interaction step in Example 3 is the same as in Figure 2A. [Table 15] 【0090】 After the reactant interaction step, the reaction solution was dereacted using the same procedure as in Example 2 to remove unreacted material. Dynabeads SA MyOne C1 (isolation carrier 7 bound to streptavidin 7b, shown in Figure 2A) was added, and the solution was fractionated by magnetic aspiration into an enriched library (EL) in which TG was functional and an unselected library (UL) in which the isolation carrier did not bind. 【0091】 Next, cDNA was prepared and amplified by reverse transcription of the mRNA of the third complex of the EL and UL fractions using a standard method. Then, as shown in Figure 8, the sequence containing the amplified TG gene (hereinafter sometimes simply referred to as "TG gene") and the sequence containing the inactive TG gene (hereinafter sometimes simply referred to as "Inactive TG gene") (both 1182 bp) were cleaved using the restriction enzyme Haell. The TG gene was cleaved into two segments of 853 bp and 329 bp, while the inactive TG gene was cleaved into three segments of 365 bp, 488 bp, and 329 bp. Agarose electrophoresis was performed on the DNA treated with restriction enzyme and the DNA that was not treated with restriction enzyme. The results are shown in Figure 9. In Figure 9, "+" means that the DNA was treated with the restriction enzyme Haell, and "-" means that the DNA was not treated with the restriction enzyme Haell. 【0092】 The Control reactions on the left side of Figure 9 show the electrophoresis results of Haell-treated samples for the Inactive TG gene and the TG gene, respectively. As shown in Figure 9, an 853 bp fragment was obtained from the TG gene, but no 853 bp fragment was observed from the Inactive TG gene. In the Inactive TG gene, two bands were observed, which is thought to be because 329 bp and 365 bp overlapped at almost the same position. 【0093】 Next, when TG gene and inactive TG gene were mixed in a ratio of 1:10 and 1:100, the electrophoresis results showed that the bands in the lanes for restriction enzyme addition ("+") and restriction enzyme removal ("-") were the same as in the control reactions for both the "EL fraction" and the "UL fraction." From these results, it was confirmed that the evaluation method disclosed in this application allows for the selective evaluation (enrichment) of functional mRNA even when different types of mRNA (active TG and inactive TG) are mixed. [Industrial applicability] 【0094】 The evaluation method disclosed in this application is useful as a tool for advancing evolutionary molecular engineering. Therefore, it is useful for the food industry and the medical industry. [Explanation of Symbols] 【0095】 1…mRNA, 2...Second component, 2a...Hybridization region, 2b...Capture region, 2c...Linker 3…Enzymes, 4…Evaluation reagent, 4a…First component, 4a1…Linker, 4a2…DNA, 4a3…Protein, 4b…First substrate 5…First reactant, 6... Interacting substances, 6a... Biotin, 7…Isolation carrier, 7b…Streptavidin 8...Second substrate, 8a...Second substrate reactant, 8b...Biotin 10a...first complex, 10b...second complex, 10c...third complex

Claims

[Claim 1] An evaluation method for evaluating enzymes translated from mRNA using evaluation reagents, The evaluation reagent comprises a first substrate that reacts with the enzyme, and a first component that interacts with the reactant obtained by the reaction of the enzyme and the first substrate. The aforementioned evaluation method is A first complex formation step involves hybridizing a second component, which includes a hybridizing region for hybridizing to the mRNA and a capture region for capturing the translated enzyme, to the mRNA, thereby forming a first complex in which the mRNA and the second component are complexed. A second complex formation step is performed in which the mRNA of the first complex is translated using a cell-free protein synthesis system, and the translated enzyme is captured in the capture region, thereby forming a second complex in which the first complex and the enzyme are complexed. A third complex formation step, in which the first component contained in the evaluation reagent is bound to the second complex to form a third complex, A first substrate reaction step in which the enzyme contained in the third complex reacts with the first substrate, A reactant interaction step in which at least the reactant obtained in the first substrate reaction step interacts with the first component to form an interacting product, An evaluation step in which the enzyme is evaluated by detecting the interacting product formed in the reactant interaction step, Evaluation methods, including those mentioned above. [Claim 2] The interacting material formed in the reactant interaction step binds to the first component of the third complex, The aforementioned evaluation process, The interacting material and an isolation carrier that reacts with the interacting material are reacted together. The evaluation is based on whether or not the third complex to which the interacting substances are bound can be isolated by the isolation carrier. The evaluation method according to claim 1. [Claim 3] The first component is an enzyme for evaluation, The reactant interaction step is The present invention further comprises a second substrate that interacts with the aforementioned enzyme for evaluation, By allowing the reactant and the second substrate to interact with the evaluation enzyme, the second substrate reaction product obtained from the second substrate binds to the first component of the third complex. The aforementioned evaluation process, The second substrate reactant is reacted with an isolation carrier that reacts with the second substrate reactant, The evaluation is based on whether or not the third complex to which the second substrate reactant is bound can be isolated by the isolation carrier. The evaluation method according to claim 1. [Claim 4] The first component is an enzyme for evaluation, The reactant interaction step is The present invention further comprises a second substrate that interacts with the aforementioned enzyme for evaluation, By allowing the reactant and the second substrate to interact with the evaluation enzyme, a second substrate reaction product, which is a fluorescent substance obtained by modifying the second substrate, is obtained. The evaluation step is evaluated by the fluorescence intensity of the second substrate reactant. The evaluation method according to claim 1. [Claim 5] The reactant interaction step is carried out with the third complex confined in a microspace. The process further includes an isolation step in which the microspace in which the third complex is confined is isolated using a sorter based on the evaluation results of the evaluation step described above. The evaluation method according to claim 4. [Claim 6] The first component is a peptide, The first substrate reacts with the enzyme to bind to the peptide. The evaluation method according to claim 2. [Claim 7] The first component binds to the mRNA or the hybridized region. The evaluation method according to any one of claims 1 to 6. [Claim 8] The first component includes a linker, and the first component indirectly binds to mRNA via the linker. The evaluation method according to claim 7. [Claim 9] The first component comprises DNA that hybridizes to the mRNA and a protein that binds to the DNA. The linker binds to the protein, The first component bonds to the linker, The first component indirectly binds to mRNA via the linker. The evaluation method according to claim 8. [Claim 10] The process includes a sequence analysis step of analyzing the sequence of mRNA contained in the isolated third complex. The evaluation method according to any one of claims 2, 3, 5, or 6. [Claim 11] An evaluation kit used in an evaluation method for evaluating enzymes translated from mRNA, the evaluation kit is, An evaluation reagent comprising a first substrate that reacts with the enzyme, and a first component that interacts with the reactant obtained by the reaction of the enzyme and the first substrate, A second component comprising a hybridizing region for hybridizing to the mRNA and a capture region for capturing the translated enzyme, including Evaluation kit. [Claim 12] An evaluation reagent that constitutes an evaluation kit used in an evaluation method for evaluating enzymes translated from mRNA, The evaluation reagent comprises a first substrate that reacts with the enzyme, and a first component that interacts with the reactant obtained by the reaction of the enzyme and the first substrate. Evaluation reagent. [Claim 13] The first component is a peptide or an enzyme for evaluation. The first component comprises a linker that binds to the peptide or the enzyme used for evaluation, and a protein that binds to the linker. The evaluation reagent according to claim 12. [Claim 14] The first component further comprises DNA that hybridizes to the mRNA, The protein binds to the DNA, thereby indirectly connecting the peptide or evaluation enzyme to the mRNA. The evaluation reagent according to claim 13. [Claim 15] The first component is an enzyme for evaluation, The enzyme further comprises a second substrate that interacts with the aforementioned enzyme for evaluation. The evaluation reagent according to claim 13 or 14. [Claim 16] The aforementioned protein is scCRO, The enzyme used for evaluation is APEX2. The peptide contains glutamine The evaluation reagent according to claim 13 or 14. [Claim 17] A first component for use in the evaluation reagent described in claim 12, The first component is a peptide or an enzyme for evaluation. The first component comprises a linker that binds to the peptide or the enzyme used for evaluation, and a protein that binds to the linker. Component 1. [Claim 18] The aforementioned protein is scCRO, The enzyme used for evaluation is APEX2. The peptide contains glutamine The first component according to claim 17. [Claim 19] It further comprises DNA that hybridizes to mRNA and binds to the protein. The first component according to claim 17 or 18.

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