Method for determining the activity of a methyltransferase enzyme

Abstract

The purpose of this invention is to provide a novel method for determining the activity of S-adenosylmethionine-dependent methyltransferase. In one embodiment, the invention provides the following technical solution: A method for determining the activity of methyltransferase, wherein the method comprises: (1) mixing the following: a protein having S-adenosylmethionine-dependent methyltransferase activity, S-adenosylmethionine, a methyl receptor, and S-adenosylhomocysteine ​​deaminase; and (2) determining the amount of the substrate and / or product of the S-adenosylmethionine-dependent methyltransferase activity.

Description

Technical Field

[0001] This invention relates to a method for determining the activity of methyltransferase. Background Technology

[0002] The quantification of S-adenosylmethionine (SAM)-dependent methyltransferase (MT) activity is widely used in drug development, medical diagnostics, and biotechnology fields, from basic research to applied research, making its technological improvement a crucial technical issue. For example, changes in MT activity are closely related to abnormal DNA methylation patterns in cancer or neurodegenerative diseases, and quantitative MT activity systems play an important role in basic research or the exploration of novel therapeutic targets. Accurate assessment of MT activity is also indispensable in the optimization or process control of genetic modification in the production of biological materials. While existing methods for MT activity determination, such as radioisotope assays (RI) or high-performance liquid chromatography (HPLC), demonstrate very high sensitivity and reliability, they are limited by factors such as radioactive material handling, equipment cost, and analysis time. In recent years, homogeneous assay techniques corresponding to high-throughput screening (HTS) have been developed, enabling efficient and rapid determination of MT activity. Representative technologies include BellBrook Labs' AptaFluor (Patent Document 1), Promega's MTase-Glo (Patent Document 2), and Revvity's HTRF EPIgeneous Methyltransferase Assay Kit (Homogeneous Time-Resolved Fluorescence (HTRF) Universal Methyltransferase Activity Assay Kit).

[0003] [Existing Technical Documents] [Patent Literature]

[0004] Patent Document 1: International Publication No. 2017 / 044494 Patent Document 2: U.S. Patent Application Publication No. 2013 / 0109037 Summary of the Invention

[0005] [The technical problem that the invention aims to solve]

[0006] As described above, various methods exist for determining the enzyme activity of S-adenosylmethionine-dependent methyltransferases. However, there is still room for improvement in existing methods. For example, the products of S-adenosylmethionine-dependent methyltransferase activity have properties that inhibit the enzyme activity itself. This property makes it difficult to accurately quantify S-adenosylmethionine-dependent methyltransferase activity using kinetic detection methods that show the linear enzyme reaction process. Therefore, the object of the present invention is to provide a new method for determining the enzyme activity of S-adenosylmethionine-dependent methyltransferases.

[0007] [Technical solutions used to solve technical problems]

[0008] To achieve the above objectives, the present invention includes the following invention in one embodiment.

[0009] (Invention 1) A method for determining the activity of methyltransferase, wherein, The method includes: (1) The following steps are to be combined. Proteins with S-adenosylmethionine-dependent methyltransferase activity S-adenosylmethionine, methyl receptors, S-adenosylhomocysteine ​​deaminase; and (2) The step of determining the amount of substrate and / or product of the S-adenosylmethionine-dependent methyltransferase enzyme activity.

[0010] (Invention 2) According to the method of invention 1, wherein, Step (1) also includes mixing nucleic acid aptamer beacons. In step (2), the luminescence of the nucleic acid aptamer beacon is measured.

[0011] (Invention 3) According to the method of Invention 2, the nucleic acid aptamer beacon is capable of detecting S-adenosylmethionine and S-adenosylhomocysteine.

[0012] [Invention Effects]

[0013] In one embodiment, the invention measures methyltransferase activity in an environment containing S-adenosylhomocysteine ​​deaminase. This reduces the accumulation of S-adenosylhomocysteine ​​caused by methyltransferase activity. S-adenosylhomocysteine ​​inhibits methyltransferase activity. Therefore, by reducing the accumulation of S-adenosylhomocysteine, enzyme activity can be appropriately measured. Attached Figure Description

[0014] Figure 1 This describes a methyltransferase (MT) activity assay system using a DNA aptamer beacon in one embodiment. The abbreviations are as follows: SAM (S-adenosylmethionine); MT (methyltransferase); SAH (S-adenosylhomocysteine); SAHDA (Adenosylhomocysteine ​​deaminase); SIH (S-inosine homocysteine). As the adenosine-responsive DNA aptamer interacts with the adenosine site within the SAM molecule, a quencher-labeled strand is released, exhibiting a fluorescence intensity corresponding to the SAM concentration. Under the action of methyltransferase (MT), SAM is converted to SAH. Although the interaction between the adenosine site within the SAH molecule and the DNA aptamer beacon enhances fluorescence, the addition of SAH deaminase (SAHDA) to the assay system immediately degrades SAH to S-inosine homocysteine ​​(SIH). The DNA aptamer beacon does not respond to SIH. SAH typically provides feedback inhibition to the MT reaction even at low concentrations, but the addition of SAHDA suppresses this reaction inhibition, thereby enabling more accurate measurement of MT activity. The dashed line represents the reaction that is avoided by adding SAHDA.

[0015] Figure 2 This describes the principle of the nucleic acid aptamer beacon in one implementation method.

[0016] Figure 3 This represents the response of DNA aptamer beacons to adenosine-related compounds. The responses of DNA aptamer beacons to adenosine (A), SAM (B), SAH (C), and inosine (D) are compared. Data are expressed as mean ± standard deviation (n=3).

[0017] Figure 4 Represent the standard curve and Hanes-Woolf scatter plot of SAM. (A) Standard curve of SAM. Data are expressed as mean ± standard deviation (n=3). (B) Coefficient of determination (R²) on the Hanes-Woolf scatter plot of the data used in the preparation of (A) above the lower limit of quantitation. 2 The regression line was constructed using data points in the concentration range (2-20 μM) above 0.99.

[0018] Figure 5 This section shows the SDS-PAGE analysis of SAHDA and MT proteins. Crude extracts of *E. coli* expressing each fusion protein and purified SAHDA and BamMHT (fused with mCherry) were separated by SDS-PAGE and analyzed by CBB staining. Arrows indicate bands of purified fusion proteins.

[0019] Figure 6The effects of SAHDA on SAH degradation and SAM quantification were demonstrated. (A) After mixing DNA aptamer beacons with 10 μM SAH, the fluorescence intensity was measured and the SAH concentration was quantified with and without the addition of SAHDA. (B) The effect of SAHDA on the SAM standard curve was evaluated by adding SAHDA to the SAM solution. All data are expressed as mean ± standard deviation (n=3).

[0020] Figure 7 This indicates the quantification of MT activity using a DNA aptamer beacon. To determine the methyl iodide transferase activity of BamMHT, the residual SAM concentration over time in a reaction solution containing 20 μM SAM and 0.5 mM KI as substrates was measured using an MT detection system with a DNA aptamer beacon. (A) Fluorescence intensity (F-F0) over time with and without BamMHT. (B) Rate of SAM consumption caused by BamMHT. All data represent averages (n=3).

[0021] [Explanation of reference numerals in the attached figures] 10: Region of the first substance; 20: Region of the second substance; 30: Region of the first nucleic acid; 40: Region of the second nucleic acid; 50: Binding region of the target substance; 60: Target substance. Detailed Implementation

[0022] The following describes specific embodiments for carrying out the invention. This description is intended to facilitate understanding of the invention and is not intended to limit the scope of the invention.

[0023] 1. Method Overview In one embodiment, the present invention relates to a method for determining the activity of a methyltransferase. The method includes the following steps.

[0024] (1) The following steps are to be mixed. Proteins with S-adenosylmethionine-dependent methyltransferase activity S-adenosylmethionine, methyl receptors, S-adenosylhomocysteine ​​deaminase; and (2) The step of determining the amount of substrate and / or product of S-adenosylmethionine-dependent methyltransferase activity.

[0025] The following details each step.

[0026] 2. The steps of mixing As described above, the various components are mixed. This allows specific enzymatic reactions to occur. Specifically, enzymatic reactions involving proteins exhibiting S-adenosylmethionine-dependent methyltransferase activity and S-adenosylhomocysteine ​​deaminase activity are performed.

[0027] During enzyme transfer of methyl groups, methyl acceptors have the function of receiving the methyl group (or the property of being methylated). While not limited, examples of methyl acceptors may include any and more of the following: nucleic acids (e.g., RNA, DNA, etc.), phospholipids (e.g., phosphatidylethanolamine), amino acids (e.g., amino acids having an amino group at the residue portion, such as lysine, arginine), catecholamines, and halides. Halides may include, for example, any and more of the following: iodides, bromides, and chlorides. Furthermore, methyl acceptors may be, for example, organic compounds or inorganic compounds. Examples of inorganic halides may include, for example, any and more of the following: potassium iodide, potassium bromide, potassium chloride, etc.

[0028] Therefore, additional components for carrying out the above-mentioned enzymatic reaction can be mixed in. For example, oxidizing agents, reducing agents (e.g., DTT), inorganic salts (other than the methyl acceptor mentioned above), and / or pH buffers can be added. Furthermore, the temperature conditions are not particularly limited, as long as they are adjusted to a suitable temperature for the enzymatic reaction. For example, the temperature for the enzymatic reaction can be in the range of 25°C to 40°C, preferably 27°C to 33°C.

[0029] exist Figure 1 The principle of the assay method of the present invention in one embodiment is shown. S-adenosylmethionine-dependent methyltransferases (MTs) (e.g., methyl halide transferases) have the function of synthesizing methyl halides from S-adenosylmethionine (SAM) and halides acting as methyl acceptors. In this case, the methyl group of S-adenosylmethionine is transferred. Furthermore, the result of the methyl transfer is the formation of S-adenosylhomocysteine ​​(SAH). Here, S-adenosylhomocysteine ​​has the function of inhibiting methyltransferases. Therefore, when the methyltransferase reaction is carried out, the methyltransferase reaction is stopped due to S-adenosylhomocysteine ​​as a reaction product. In fact, it is possible to stop the methyltransferase reaction in the initial stage. Then, when the reaction is stopped, it may hinder proper evaluation related to the performance of the methyltransferase.

[0030] Here, one component of the mixture contains S-adenosylhomocysteine ​​deaminase (SAHDA). This enzyme has the function of abstracting the amino group from S-adenosylhomocysteine, resulting in the formation of S-inosylhomocysteine ​​(SIH). That is, this enzyme changes a substance that inhibits methyltransferase activity into another substance. Then, S-inosylhomocysteine, as this other substance, does not inhibit the methyltransferase reaction. Thus, the possibility of stopping the methyltransferase reaction can be reduced.

[0031] Furthermore, S-adenosylhomocysteine ​​deaminase is known to be widely distributed in biological species. Therefore, there are no particular limitations on the biological species from which S-adenosylhomocysteine ​​deaminase can be sourced. Typically, S-adenosylhomocysteine ​​deaminase derived from species such as *Methanocaldococcus jannaschii* and *Thermotoga maritima* can be used.

[0032] 3. Procedures for the measurement To determine the enzymatic activity of the aforementioned methyltransferases, changes in the amount of the enzyme's substrate and / or the amount of the enzyme's product can be measured. There are no particular limitations on the measurement method. For example, the following methods can be used: radioisotope assay (RI), high-performance liquid chromatography (HPLC), antibody-based methods, mass spectrometry methods, and methods using nucleic acid aptamer beacons. The method using nucleic acid aptamer beacons will be described later.

[0033] In one embodiment, a method can be used to specifically detect S-adenosylmethionine, which is a substrate for methyltransferases. A decrease in the amount of substrate indicates that the enzyme reaction is in progress.

[0034] In another embodiment, a method can be used to specifically detect S-adenosylhomocysteine, which is a product of methyltransferase. An increase in the amount of product indicates that the enzyme reaction is in progress.

[0035] The performance of enzymes such as Km can be determined by specifically measuring changes in either the amount of substrate or the amount of product.

[0036] Furthermore, the aforementioned assay methods can be based on the identification and detection of S-adenosylmethionine and S-adenosylhomocysteine, or they can be based on not identifying and detecting S-adenosylmethionine and S-adenosylhomocysteine. This is because, even in the latter method, S-adenosylhomocysteine ​​will ultimately be transformed into other substances by S-adenosylhomocysteine ​​deaminase.

[0037] Therefore, even if the above-mentioned assay method is based on not recognizing and detecting S-adenosylmethionine and S-adenosylhomocysteine, it will not hinder the determination of the enzyme activity of methyltransferase.

[0038] However, when the above-described assay method is used to detect S-inosine homocysteine, it may interfere with the determination of methyltransferase activity. Therefore, in a preferred embodiment, the assay method used in the assay step satisfies the following conditions (1) and (2): (Condition 1) At least S-adenosylmethionine must be detected.

[0039] (Condition 2) S-inosine homocysteine ​​is not detected.

[0040] 4. Nucleic acid aptamer beacons In one embodiment, the above-described assay method can be based on nucleic acid aptamer beacons. For example, the method for measuring methyltransferase activity may further include mixing nucleic acid aptamer beacons in the mixing step (1), and the assay step (2) may include measuring the luminescence of the nucleic acid aptamer beacons.

[0041] The following is an overview of nucleic acid aptamer beacons.

[0042] In terms of function, nucleic acid aptamer beacons have the following functions: when they bind to a target substance, they cause structural changes in the nucleic acid and can provide a prompt in a way that is easy to identify.

[0043] In one embodiment, the nucleic acid aptamer beacon has the following components (see reference). Figure 2 ).

[0044] • Substance 1 (10) • Substance 2 (20) • Region of the first nucleic acid (30) • Region of the second nucleic acid (40) • Target material binding region (50).

[0045] Substance 1 (10) has the function of fluorescence. Here, fluorescence means absorbing light of a certain wavelength and emitting light of another wavelength. In addition, from the viewpoint of the phenomenon known as FRET (Fluorescence Resonance Energy Transfer), substance 1 (10) can act as a donor molecule.

[0046] The second substance (20) has the function of regulating the luminescence of the first substance (10). For example, the second substance (20) may function as a quenching agent (also referred to as an extinction agent in this specification). Specifically, the second substance (20) is able to absorb light of a specific wavelength emitted by the first substance (10), and may not emit light of that wavelength or other wavelengths. Thus, the luminescence of the first substance (10) can be extinguished by the second substance (20).

[0047] Furthermore, from the perspective of a phenomenon known as FRET (Fluorescence Resonance Energy Transfer), the second substance (20) can function as an acceptor molecule. Specifically, the second substance (20) can absorb light of a specific wavelength emitted by the first substance (10), and can also emit light of other wavelengths.

[0048] The first nucleic acid region (30) is a region of nucleic acid that is directly or indirectly linked to the first substance (10). Moreover, the second nucleic acid region (40) is a region of nucleic acid that is directly or indirectly linked to the second substance (20).

[0049] In one example, "directly linked" means, for example, that the first substance (10), which is a substance other than nucleic acid, is not linked to the region (30) of the first nucleic acid, which is nucleic acid, without any other atoms or molecules. The same applies to the second substance (20).

[0050] In other examples, “indirect linking” means, for example, that a first substance (10), which is a substance other than a nucleic acid, is linked to a region (30) of a first nucleic acid via other substances (e.g., other atoms, other molecules, such as other nucleic acids or other linking molecules). The same applies to the second substance (20).

[0051] The target substance binding region (50) is a nucleic acid region that binds to the target substance (60). Preferably, it is a nucleic acid region that specifically binds to the target substance (60). Although not limited, specific binding means, for example, a degree that indicates a statistically significant difference in the properties of binding to the target substance (60) (e.g., binding constant, etc.) compared to a control.

[0052] Combination Figure 2 To illustrate how the above-mentioned elements contribute to the detection of the target substance (60).

[0053] In one example, the region (30) of the first nucleic acid binds complementary to the region (40) of the second nucleic acid. This shortens the distance between the first substance (10) and the second substance (20) which are respectively linked to these regions. Therefore, the luminescence from the first substance (10) is modulated by the second substance (20). For example, when the second substance (20) functions as a quencher, the luminescence from the first substance (10) is extinguished.

[0054] However, in the presence of the target substance (60), the target substance (60) binds to the target substance binding region (50). This causes at least a localized structural change in the nucleic acid aptamer beacon. Furthermore, this structural change hinders the complementary binding of the region (30) of the first nucleic acid and the region (40) of the second nucleic acid. This increases the distance between the first substance (10) and the second substance (20). Therefore, the luminescence from the first substance (10) is not modulated by the second substance (20).

[0055] The above is just one example, and there are other possibilities based on... Figure 2 The various modification patterns of the structure are shown.

[0056] 4-1. Types of nucleic acids (Change of pattern 1) The nucleic acid used can be DNA, RNA, or a combination of both (e.g., the region (30) of the first nucleic acid is DNA and the region (40) of the second nucleic acid is RNA or the region (30) of the first nucleic acid is RNA and the region (40) of the second nucleic acid is DNA).

[0057] 4-2. Functions of Substance 1 and Substance 2 (Change Mode 2) In one example, the first substance (10) and the second substance (20) may be a combination that causes the donor fluorescence to be extinguished via FRET. Here, extinction also includes the case of localized fluorescence attenuation. For example, the first substance (10) has the function of emitting light of a certain wavelength. Moreover, the second substance (20) has the function of absorbing light of that wavelength or near that wavelength (and the second substance (20) does not have the function of emitting light).

[0058] In other examples, the first substance (10) and the second substance (20) may be a combination of receptor-induced fluorescence expression via FRET (Fluorescence Resonance Energy Transfer). Here, the first substance (10) functions as a donor molecule, and the second substance (20) functions as an acceptor molecule. For example, the first substance (10) has the function of emitting light of a certain wavelength. Moreover, the second substance (20) has the function of absorbing light of that wavelength or near that wavelength. In addition, the second substance (20) can utilize light of that wavelength or near that wavelength as excitation light and has the function of emitting light of other wavelengths.

[0059] As another example, the first substance (10) and the second substance (20) can also act as donors and acceptors in PET (Photoinduced Electron Transfer).

[0060] 4-3. Regions of the first and second nucleic acids (modification of pattern 3) The regions of the first nucleic acid (30) and the second nucleic acid (40) can be integrated or exist separately. Figure 2 This is an example of the latter. In the case of integration, for example, the region of the first nucleic acid (30) and the region of the second nucleic acid (40) can exist separated by the target substance binding region (50) and / or other nucleic acid regions. When the two are at least locally complementary, the nucleic acid aptamer beacon can have a ring structure.

[0061] 4-4. Distance between Substance 1 and Substance 2 (Change Mode 4) exist Figure 2 In one example, when the target substance (60) is absent, the distance between the first substance (10) and the second substance (20) is brought closer, and when the target substance (60) is present, the distance between the first substance (10) and the second substance (20) is increased. In other examples, when the target substance (60) is absent, the distance between the first substance (10) and the second substance (20) can be increased, and when the target substance (60) is present, the distance between the first substance (10) and the second substance (20) can be brought closer.

[0062] As mentioned above, nucleic acid aptamer beacons can be modified in various alteration patterns or combinations thereof.

[0063] 4-5. Combination of Substance 1 and Substance 2 The combination of the first and second substances is not particularly limited; for example, it can be a combination capable of inducing FRET. When considering homogeneous FRET, the combination of the first and second substances can include not only combinations of different kinds of substances but also combinations of the same kind of substances. However, typically, the combination of the first and second substances is a combination of different kinds of substances. Additionally, the second substance may contain a matting agent.

[0064] Although there is no limitation, records may include, for example, combinations of the following.

[0065] CFP and YFP GFP (including EGFP) and mCherry mTurquoise2 and mVenus mNeonGreen and mRuby3 Fluorescein and Tetramethylrhodamine Fluorescein and Black Hole Quencher (registered trademark) 1 (BHQ-1) IAEDANS (5-[2-iodoacetyl]aminonaphthalene-1-sulfonic acid) and fluorescein EDANS (5-(2-aminoethylamino)naphthalene-1-sulfonic acid) and Dabcyl Cy3 and Cy5 Alexa Fluor 488 and Alexa Fluor 594.

[0066] In other examples, it could be a combination that can induce PET. Although there is no limitation, records may include, for example, combinations of the following.

[0067] Porphyrins and quinones Organic pigments and amines.

[0068] The term "nucleic acid" as used in this specification may include naturally occurring nucleic acids, or it may not include naturally occurring nucleic acids. Alternatively, the term "nucleic acid" as used in this specification may include chemically modified nucleic acids, or it may not include chemically modified nucleic acids. The terms "DNA" and "RNA" are used interchangeably in this specification. In addition, there is no particular limitation on the types of chemical modifications, but for example, it may include any one or more of the following: fluorescent labeling (e.g., FAM, TAMRA, ROX, etc.), methylation (e.g., methylation of 5-methylcytosine, methylation of the 2'-OH group of RNA, etc.), introduction of base analogs (e.g., 5-bromouracil (BrU), 6-thioguanine (6-TG), pseudouridine, etc.), fluorine modification (e.g., 2'-fluoroRNA (2'-F RNA)), introduction of linkers (e.g., PEGylation, biotin binding, etc.), poly(A) tail, morpholinonucleotide (PMO), peptide nucleotide (PNA), locked nucleotide (LNA), bridged nucleotide (BNA), unlocked nucleotide (UNA), glycerol nucleotide (GNA), thiophosphate ester bond, alkylphosphonate diester modification, etc.

[0069] 4-6. Procedures for the measurement As described above, step (2) of the measurement can include measuring the luminescence of the nucleic acid aptamer beacon. Step (2) of the measurement can, for example, include irradiating the mixture with light at or near the excitation wavelength of the first substance. Furthermore, step (2) can measure the luminescence from the first substance or the second substance using an enzyme-linked immunosorbent assay (ELISA) reader, or it can measure the effect of the extinction of the second substance.

[0070] 4-7. Nucleic acid aptamer beacons in methods for determining the enzyme activity of methyltransferases Return to reference Figure 1 To illustrate how nucleic acid aptamer beacons can aid in assays, as mentioned above, there exist S-adenosylmethionine and S-adenosylhomocysteine ​​corresponding to the substrates and products of methyltransferases. The fluorescently labeled aptamer chains constituting the nucleic acid aptamer beacons can bind to these substances. On the other hand, the fluorescently labeled aptamer chains cannot bind to S-inosylhomocysteine. In other words, the fluorescently labeled aptamer chains have the function of specifically binding to the adenosine site.

[0071] When S-adenosylmethionine and S-adenosylhomocysteine ​​are absent, the fluorescently labeled aptamer chain binds complementary to the quencher chain. As a result, the fluorescent portion of the first substance (in...) Figure 2 (shown as "F") and the quenching agent portion as the second substance (in) Figure 2 The light emitted from the fluorescent portion is extinguished. (The text then shows a "Q" symbol, which appears to be a typo and should be removed.)

[0072] When more than one of S-adenosylmethionine and S-adenosylhomocysteine ​​is present, the fluorescently labeled aptamer chain undergoes a structural change, resulting in the release of the quencher chain. Furthermore, the fact that the quencher moiety exists away from the fluorescently labeled aptamer allows for the detection of luminescence from the fluorescent moiety.

[0073] Normally, because fluorescently labeled aptamer chains specifically bind to both the substrate and product of methyltransferases, enzyme activity cannot be determined solely based on nucleic acid aptamer beacons. However, S-adenosylhomocysteine ​​is converted to S-inosylhomocysteine ​​in the presence of S-adenosylhomocysteine ​​deaminase. Therefore, as a result, the fluorescently labeled aptamer chain detects only the substrate of the methyltransferase. Thus, enzyme activity can be measured.

[0074] While not limited, nucleic acid aptamer beacons can contain the following nucleic acid sequences as target binding regions. The following sequences correspond to regions that specifically recognize adenosine sites.

[0075] 5'-ACGACGTTTGCGATGAGAAACGTATGGTTTCGAAGGTCGT-3' (Serial Number 1) [Example]

[0076] 1. Preparation of DNA aptamer beacons In this embodiment, a DNA aptamer beacon (Ding and Liu, 2023) specifically for adenosine detection was used as a model. The DNA aptamer beacon consisted of a fluorescein (FAM)-labeled aptamer strand and a quencher (Black HoleQuencher1, BHQ1)-labeled strand (Table 1), both synthesized by Eurofins Genomics Co., Ltd. A solution of the DNA aptamer beacon was prepared by mixing 1 μM of the FAM-labeled aptamer strand, 2 μM of the quencher-labeled strand, 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 20 mM MgCl2, heating at 95°C for 2 minutes, and then cooling to 25°C for 30 minutes to allow the two DNA strands to bind complementaryly.

[0077] Table 1. Base sequences and modification sites of the oligoDNA used.

[0078] [Table 1]

[0079] 2. Analysis of the response of DNA aptamer beacons to adenosine-related compounds The detection of adenosine-related compounds using DNA aptamer beacons was performed in a polystyrene 96-well plate format, and fluorescence measurements were performed at 30°C using a microplate reader (Synergy H1, BioTek) (Ex 480 nm, Em 530 nm). Each well contained 100 μL of adenosine-related compounds (at various concentrations), a DNA aptamer beacon (20 nM based on FAM-labeled aptamer strands), and 100 mM Tris-acetate (pH 7.5). Adenosine, S-adenosylmethionine (SAM), S-adenosylhomocysteine ​​(SAH), and inosine were used as adenosine-related compounds.

[0080] 3. Preparation of DNA constructs and transformation in E. coli A plasmid containing a DNA fragment encoding the SAH deaminase (SAHDA, TM0936) derived from *Thermotoga maritima* (Hermann et al., 2007) was obtained from the DNASU plasmid repository (Table 2). To express SAHDA in *E. coli* by adding a 7-amino acid tag (Met-His-His-His-His-His) to the N-terminus, the DNA fragment encoding SAHDA was amplified by PCR using the plasmid as a template. The amplified fragment was cloned downstream of the T7 promoter in the pDEST17 vector and introduced into *E. coli* strain BL21(DE3).

[0081] Methyl halide transferase (BamMHT) from Batis maritima (Ni and Hager, 1998) was used as a model for quantifying MT activity (Table 2). The DNA fragment encoding BamMHT was synthesized by Integrated DNA Technologies. To express the protein in *E. coli* in the form of a 7-amino acid fusion protein (mCherry) with a 6xHis tag added to the N-terminus and a 10-amino acid fusion protein (Gly-Ser-Ala-Gly-Ser-Ala-Ala-Gly-Ser-Gly) as a linker, the protein was cloned downstream of the T7 promoter of the pDEST17 vector and introduced into *E. coli* strain BL21(DE3).

[0082] Table 2. Amino acid sequences of the proteins used.

[0083] [Table 2]

[0084] 4. Expression and purification of fusion proteins in E. coli Each fusion protein-expressing *E. coli* strain was cultured overnight at 30°C in ZYM5052 medium (Studier, 2005) containing 100 μg / ml ampicillin, and the *E. coli* were recovered. The recovered cells were resuspended in 15 ml of binding buffer according to the His-Bind Kit (Novagen) program and disrupted using a pressure cell disruptor (EmulsiFlex-B15, AVESTIN). After centrifugation (16000 g, 4°C, 10 min), the supernatant was used as the crude protein extract. The target protein was purified from the crude protein extract using a batch method with the resin of the His-Bind Kit. After desalting and concentration by ammonium sulfate precipitation and ultrafiltration (10 KD, GVS), the SAHDA and BamMHT solution was prepared as 0.1 M Tris-acetate pH 7.5, 1 mM MTT, 50% glycerol. The purified proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Coomassie Brilliant Blue (CBB) staining. The concentration of the purified proteins was determined using bovine serum albumin (BSA) as a standard protein according to the Bradford method.

[0085] 5. MT activity assay system using DNA aptamer beacons Quantification of MT activity using DNA aptamer beacons was performed in 96-well polystyrene plates. Fluorescence was measured at 30°C using a Synergy H1, BioTek microplate reader (Ex 480 nm, Em 530 nm). Each well contained 100 μL of a mixture of 20 μM SAM, a DNA aptamer beacon (20 nM based on FAM-labeled aptamer strand), 100 mM Tris-acetate (pH 7.5), 0.5 mM KI, 1 mM dithiothreitol (DTT), 4 μg purified SHAHDA, and 1 μg purified BamMHT. The enzyme reaction was initiated by the addition of KI, and MT activity was calculated using kinetic assays based on data from the range within which the rate of decrease in SAM concentration remained constant.

[0086] 6. Results Previously, a DNA aptamer beacon for quantifying adenosine (Ade1301b) was reported (Ding and Liu, 2023). This study aims to investigate whether this DNA aptamer beacon can be applied to… Figure 1 The detection system shown analyzed whether it responded to related compounds other than adenosine. Figure 3 The results indicate that DNA aptamer beacons respond to S-adenosylmethionine and S-adenosylhomocysteine, which have an intramolecular adenosine structure, at the same level as adenosine. Additionally, they indicate no response to inosine, which has a structure similar to adenosine but lacks an amino group.

[0087] Construct a standard curve of this DNA aptamer beacon against SAM and a Hanes-Woolf scatter plot based on it. Figure 4 The detection limit (3.3σ / S) and the lower limit of quantitation (10σ / S) were calculated based on the standard deviation (n=3) of the fluorescence intensity (F0) of the blank value (0 μM SAM) and the slope (S) of the linearly related concentration range (0-2 μM) in the standard curve (Table 3). Based on data from the SAM standard curve (…),… Figure 4 A) Hanes-Woolf scatter plot ( Figure 4 The regression line of B) was used to calculate the apparent dissociation constant (Table 3).

[0088] Table 3. Characteristics of the SAM quantification system using DNA aptamer beacons

[0089] [Table 3] Detection limit (μM) 0.61 Limit of quantification (μM) 1.85 Apparent dissociation constant (μM) 7.09

[0090] The gene for the SAH-degrading enzyme (SAH deaminase) used to eliminate SAH generated by MT activity from the reaction system was expressed in E. coli, and the fusion protein was purified. Figure 5Additionally, the fusion BamMHT protein, used as a model for the MT detection system, was similarly expressed and purified in *E. coli*. Figure 5 Both proteins showed the expected molecular weight and were prepared with adequate purification.

[0091] The study investigated whether the prepared SAHDA fusion protein adequately decomposed SAH in the reaction system. The results showed that in the negative control (which did not contain SAHDA), the quantitative value of SAH in the system remained unchanged; in contrast, in the sample containing SAHDA, SAH was decomposed and disappeared. Figure 6 A). Furthermore, the addition of SAHDA did not affect SAM quantification using DNA aptamer beacons. Figure 6 B).

[0092] To confirm whether it can be built Figure 1 The MT activity quantification system shown was used to determine the methyl transfer activity of BamMHT protein, a model MT protein, relative to iodide ions. No decrease in fluorescence intensity over time was observed in the negative control (which did not contain BamMHT protein). However, a decrease in fluorescence value (F-F0) due to SAM consumption was observed in the sample containing BamMHT protein. Figure 7 A). When calculating SAM consumption using the SAM standard curve based on this fluorescence value, a linear increase in SAM consumption was observed ( Figure 7 B). Even in reaction systems showing a decrease in SAM concentration of at least 10 μM, the enzyme reaction was observed to proceed linearly, but this was the concentration range in which most of the MT activity was significantly inhibited by SAH accumulated in the absence of SAHDA. The reason why a decrease in fluorescence was not immediately observed after the start of the enzyme reaction is thought to be that it takes time for the interaction between SAM and the DNA aptamer to reach equilibrium.

[0093] This invention provides a novel solution to overcome various technical problems in the prior art, such as the difficulty in accurately calculating enzyme activity points due to reliance on endpoint determination, reproducibility issues caused by the stability of RNA or antibodies, and operational complexity or reproducibility problems due to multi-step enzymatic reactions. In particular, this invention is the first to discover that the DNA aptamer beacon (Ade1301b) (Ding and Liu, 2023) also responds to SAM and SAH. Furthermore, SAH is rapidly degraded by a single enzyme (SAHDA) within the reaction system, and the SIH product does not affect the DNA aptamer beacon. By removing SAH, the barrier to MT activity can be mitigated, and MT activity can be accurately quantified using a kinetic detection method exhibiting linear enzymatic reactions.

[0094] The following documents were referenced in the above embodiments.

[0095] References (Non-patent literature) Ding Y, Liu J (2023) Pushing adenosine and ATP SELEX for DNA Aptamers with nanomolar affinity. J Am Chem Soc 145: 7540-7547 Hermann JC, Marti-Arbona R, Fedorov AA, Fedorov E, Almo SC, Shoichet BK, Raushel FM (2007) Structure-based activity prediction for an enzyme of unknown function. Nature 448: 775-779 Ni X, Hager LP (1998) cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme. Proc Natl Acad Sci 95: 12866-12871 Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. ProteinExpr Purif 41: 207-234

[0096] The above describes specific embodiments of the invention. These embodiments are merely specific examples, and the invention is not limited to them. For example, the technical features disclosed in one embodiment can be applied to other embodiments. Furthermore, unless otherwise specified, the order of some steps in a particular method can be interchanged with other steps, and additional steps can be added between two specific steps. The scope of the invention is defined by the scope of the technical solution.

Claims

1. A method for determining the activity of methyltransferase, characterized in that, The method includes: (1) The following steps are to be combined. Proteins with S-adenosylmethionine-dependent methyltransferase activity S-adenosylmethionine, methyl receptors, S-adenosylhomocysteine ​​deaminase; and (2) The step of determining the amount of substrate and / or product of the S-adenosylmethionine-dependent methyltransferase enzyme activity.

2. The method according to claim 1, characterized in that, Step (1) also includes mixing nucleic acid aptamer beacons. In step (2), the luminescence of the nucleic acid aptamer beacon is measured.

3. The method according to claim 2, characterized in that, The nucleic acid aptamer beacon can detect S-adenosylmethionine and S-adenosylhomocysteine.

Images