WO2025121411A1 - Altered proximity-dependent modification enzyme - Google Patents

Abstract

[Problem] A proximity-dependent biotinylated enzyme for an analysis method for interaction between molecules has to be fused with an analysis target substance while maintaining a non-specific biotin labeling activity. Therefore, such an analysis method has been limited in terms of analysis target substances that can be used. [Solution] The present invention was accomplished as a result of confirming that it is possible to use low-molecular weight compounds and natural organic substances as analysis target substances, in an analysis method for interaction between molecules, using an altered proximity-dependent modification enzyme in which lysine on the surface is substituted and in which a tag including lysine is directly or indirectly fused to the N-terminus or the C-terminus if necessary.

Description

Engineered proximity-dependent modification enzymes

The present disclosure relates to engineered proximity-dependent modifying enzymes for use in analyzing interactions between biomolecules and between drugs (small molecules, peptides, nucleic acids, etc.) and proteins.
This application claims priority to Japanese Application No. 2023-205732, which is incorporated herein by reference.

In fields such as disease research and drug discovery, the analysis of interactions between biochemical substances and chemical substances is widely used as an extremely important approach in drug discovery research. In particular, protein-protein interactions are a general term for interactions that occur between proteins in vivo. These interactions are well known to be involved in the control of protein structural changes and mechanisms that are fundamental to life, such as signal transmission, transport, and metabolism. These interactions have extremely diverse patterns, and are characterized by an enormous variety in the softness and breadth of the surface of action, the length of contact life, and the presence or absence of structural changes, which vary depending on the protein type.

Many methods for measuring protein-protein interactions have been developed. One of these, immunoprecipitation, uses extracts from crushed cells, so there is a possibility of false positives and weak binding that dissociates during the washing process cannot be detected.
Therefore, BioID, a proximally dependent biotinylation enzyme that can randomly biotin-label lysine residues of nearby proteins by adding a mutation (R118G) to E. coli BirA, is developed. The intermediate biotinyl-5'-AMP is released from the active site, and BioID can randomly biotin-label lysine residues of nearby proteins. After that, the activity and non-specificity of the labeling were improved, and TurboID, AirID, etc. were developed. The present inventors have also applied this to non-denaturing protein array technology (see Patent Document 1 and Non-Patent Document 1). In particular, the greatest features of BioID are that it can analyze interactions of cells and individuals of various biological species in their living state, and that it can analyze transient, dynamic, and weak interactions.

WO/2022/009994

Proteome Letters 2021;6:9-16

A method for analyzing molecular interactions using a proximity-dependent biotinylation enzyme is a very useful tool. However, the enzyme needs to be fused with the target substance while maintaining its non-specific biotin labeling activity. For example, it is difficult for the enzyme to be fused with low molecular weight compounds or natural organic matter while maintaining its non-specific biotin labeling activity. Therefore, the target substances that can be used with this analysis method are limited.

The present disclosure has been completed by confirming that a method for analyzing interactions between molecules using a modified proximity-dependent modifying enzyme in which lysines on the surface have been replaced and in which a tag containing lysine is fused directly or indirectly, preferably to the N-terminus or C-terminus, can be used to analyze low molecular weight compounds and natural organic matter. That is, the present disclosure is as follows.

1. Engineered proximity-dependent modifying enzymes in which surface lysines have been substituted.
2. The modified proximity-dependent modification enzyme according to the preceding paragraph 1, wherein the proximity-dependent modification enzyme is a peptide having the following amino acid sequence (SEQ ID NO: 3: AirID):
MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGYCLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGRRGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAET LQKLGADGVRVKWPNDLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDRNQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQGALLLEQDGVIKPWIGGEISLRSA.
3. The modified proximity-dependent modification enzyme according to the preceding paragraph 2, wherein the lysine substitution position is any one or more of the following, and the substituted amino acid is arginine, histidine, glutamic acid, aspartic acid, or serine:
1) K2, 2) K38, 3) K41, 4) K56, 5) K71, 6) K122, 7) K163, 8) K194, 9) K244R, 10) K277, and 11) K307.
4. The modified proximity-dependent modification enzyme according to claim 2, wherein the lysine substitution is one or more of the following:
1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.
5. The modified proximity-dependent modification enzyme according to claim 2, wherein the lysine substitution is:
1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.
6. The modified proximity-dependent modification enzyme according to the preceding paragraph 1, wherein the proximity-dependent modification enzyme is a peptide having the following amino acid sequence (SEQ ID NO: 2: TurboID):
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEAL RKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK.
7. The modified proximity-dependent modification enzyme according to the preceding paragraph 6, wherein the lysine substitution positions are one or more lysines other than K172 and K183 as underlined:
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRV K WPNDLYLQDR K LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK.
8. The modified proximity-dependent modifying enzyme according to any one of the preceding aspects 1 to 7, wherein a tag comprising one or more lysines is fused directly or indirectly to the N-terminus or C-terminus.
9. The modified proximity-dependent modifying enzyme according to any one of the preceding aspects 1 to 7, wherein any one of the following tags is fused directly or indirectly to the N-terminus or C-terminus:
1) DYKDDDDK (N-terminus)
2) DYKDDDDK (C-terminus)
3) DYKDHDGDYKDHDIDYKDDDDK (N-terminus)
4) GGKKKGKK (C-terminus)
5) KKKDKKDD (N-terminus)
6) DDKKKDKK (C-terminus)
10. A modification proximity-dependent modifying enzyme-labeled analyte, which is labeled with the modification proximity-dependent modifying enzyme according to any one of the preceding items 1 to 9.
11. A modification proximity-dependent modifying enzyme-labeled analysis target substance, which is labeled with the modification proximity-dependent modifying enzyme according to the preceding item 5.
12. The modified proximity-dependent modifying enzyme-labeled analyte according to item 10 above, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus.
13. The modified proximity-dependent modifying enzyme-labeled analyte according to the preceding paragraph 11, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus.
14. A method for evaluating an interaction between an immobilization substance directly or indirectly immobilized on a substrate and a modified proximity-dependent modification enzyme-labeled analyte, comprising the steps of:
(1) adding the modified proximity-dependent modification enzyme-labeled analyte according to the preceding paragraph 12 or 13 to an immobilized substance immobilized directly or indirectly on a substrate in the presence of a label;
(2) detecting the labeling substance;
Evaluation method.
15. The evaluation method according to item 14, further comprising a step of cleaning the substrate between the step (1) and the step (2).
16. The evaluation method according to item 14 or 15, wherein the modification proximity-dependent modification enzyme-labeled analyte is the modification proximity-dependent modification enzyme-labeled analyte according to item 12.
17. A method for evaluating a protein, which is an immobilized substance indirectly immobilized on an array via magnetic beads, and a modified proximity-dependent modifying enzyme-labeled analyte, comprising the steps of:
(1) adding the modified proximity-dependent modifying enzyme-labeled analyte according to the preceding paragraph 12 or 13 in the presence of biotin to an immobilized substance indirectly immobilized on an array via magnetic beads;
(2) detecting the biotin;
Evaluation method.
18. The evaluation method according to claim 17, further comprising a step of washing the array between the step (1) and the step (2).
19. The evaluation method according to item 17 or 18, wherein the modification proximity-dependent modification enzyme-labeled analyte is the modification proximity-dependent modification enzyme-labeled analyte according to item 13.
20. A method for introducing a modified proximity-dependent modification enzyme-labeled analyte into a cell, comprising introducing the modified proximity-dependent modification enzyme-labeled analyte according to item 12 or 13 into the cell by electroporation.
21. The evaluation method according to item 14 above, wherein the substance to be analyzed maintains water solubility.
22. The evaluation method according to item 17 above, wherein the substance to be analyzed maintains its water solubility.
23. The evaluation method according to item 14 above, wherein the target substance to be analyzed has an alkynyl group and the modified proximity-dependent modifying enzyme has an azide group.
24. The evaluation method according to item 14 above, wherein the target substance to be analyzed has an azide group and the modified proximity-dependent modifying enzyme has an alkynyl group.

The method of analyzing intermolecular interactions using an engineered proximity-dependent modifying enzyme in which lysines on the surface of the present disclosure have been substituted has one or more of the following advantages compared to conventional methods of analyzing intermolecular interactions using proximity-dependent modifying enzymes:
1) The water solubility of the target substance can be maintained. 2) There is no limitation on the type of target substance.

Analysis of the interaction between AirID-modified enzyme-IκBα and FG-RelA 1 Analysis of the interaction between AirID-modified enzyme-IκBα and FG-RelA 2 Diagram of the AirID surface lysine mutations fused to each tag Yields after synthesis and purification of surface lysine mutant AirIDs fused with each tag Analysis results using surface lysine mutation AirID fused with each tag 1 Preparation results of geldanamycin-AirID ² C Interaction of geldanamycin-AirID ² C with immobilized materials 1 Geldanamycin-AirID ² C interaction with immobilized materials 2 Checking the storage stability of geldanamycin-AirID ² C Comparison of solubility of AirID(native) and AirID modified forms after reaction with the target substance Analysis results for the low molecular weight substance thalidomide Electrophoresis results of proteins used 1 Binding reaction between an analyte containing an alkynyl group and a modified AirID containing an azide group (analytical results using JQ1 as the analyte) Electrophoresis results of proteins used 2 Protein array analysis results using AirID ² C Analysis results using surface lysine mutation AirID fused with each tag 2 Binding reaction between an azide-containing target substance and an alkynyl-containing modified AirID (analysis results using pomalidomide or thalidomide as the target substance) Electrophoresis results of proteins used

(Summary of the Disclosure)
Conventionally, in order to directly bind a target substance to AirID, which is a proximity-dependent modifying enzyme, chemical reactions were carried out at room temperature using the lysine or thiol group of the target substance, which is a protein, as a reaction scaffold. Reactions using cysteine as a scaffold can be performed using alkylation reactions with the α-iodo(bromo)acetamide terminal, perfluoroarylation reactions, maleimide Michael addition, etc., but cross-reactivity with lysine, which is the same nucleophilic residue, is a concern. In particular, of the two cysteines present in AirID, one is inside the protein folding structure, and only one cysteine is actually usable, making it insufficient as a scaffold. In addition, there is a method of fusing a peptide fragment containing cysteine as a tag to the N-terminus or C-terminus to increase the reaction scaffold. However, cysteine is not preferable because it often modifies the properties of proteins.
Therefore, in the present invention, it is considered preferable to select lysine, which has an ε-amino group and is highly reactive, as a scaffold. In this case, functional groups such as NHS (N-hydroxysuccinimide), isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imide ester, carbodiimide, anhydride, and fluoroester, which have high reactivity with lysine, can be used for binding with lysine.
In particular, the reaction between NHS and lysine proceeds under physiological conditions to slightly alkaline conditions (pH 7.2 to 9), and a wide variety of reaction reagents are commercially available, making this method preferable.
In the following example, the 13 lysines in AirID (SEQ ID NO: 3) were converted to arginines to investigate which arginines are involved in the proximal-dependent biotin enzyme activity. As a result, it was confirmed that the proximal-dependent biotin enzyme activity was lost when K172R and K183R were substituted. It was confirmed that the other lysines, even if substituted with arginine, did not affect the proximal-dependent biotin enzyme activity, although the activity was slightly stronger or weaker. Furthermore, it was confirmed that the conversion of the remaining 11 lysines could be set to any number, since there was no effect even if all of the remaining 11 lysines were replaced with arginines. This makes it possible to set the number of binding sites of the test compound to the proximal-dependent biotinylation enzyme according to the physical properties of the test compound, greatly expanding the range of conditions for intermolecular interaction analysis.
As with AirID, we confirmed that converting lysines other than K172 and K183 to arginines does not affect the proximally-dependent biotin enzyme activity of TourboID.
In both AirID and TourboID, computational predictions using AlphaFold2 (an AI that predicts the three-dimensional structure of protein molecules) also show that K172 and K183 are located inside the folded structure. K172 and K183 are likely lysines necessary for maintaining the three-dimensional structure.
In the following examples, lysine was replaced with arginine, but it is also possible to replace it with glutamic acid, histidine, aspartic acid, or serine, which are amino acids with properties similar to those of lysine. Note that arginine replacement is preferred in that it does not significantly change the physical properties of the proximity-dependent biotinylation enzyme, such as the surface charge.
Furthermore, since there was no substantial change in the proximity-dependent biotinylation enzyme when all lysines were changed to arginines, by converting all lysines to arginines and then binding a peptide fragment containing lysine (reactive scaffold tag) to the N-terminus or C-terminus, it is possible to ensure (maintain) the activity of the proximity-dependent biotinylation enzyme and then bind the substance to be analyzed to the scaffold tag.
The scaffold tag preferably contains one or more lysines, but in the following examples, it may contain about two to five lysines.
The present disclosure will be described in detail below, but the present disclosure is not limited to the following description.

(Proximity-dependent modifying enzymes)
In this disclosure, a proximity-dependent labeling enzyme refers to an enzyme that has the ability to bind a molecule that can be detected as a marker (referred to as a "labeling substance" in this disclosure) to an immobilized substance when an intermolecular interaction occurs between the analyte and an immobilized substance on an array, and the immobilized substance is present in the vicinity of the proximity-dependent labeling enzyme bound to the analyte.
Proximity-dependent labeling enzymes can be exemplified by enzymes that have been modified to weaken their substrate specificity. Examples of such enzymes include transferases, lyases, and ligases. Methods for weakening substrate specificity include amino acid conversion or chemical modification, such as introducing a mutation into the substrate binding site or introducing the sequence of a closely related enzyme.
The bond between the labeling substance and the protein that is the immobilized substance is preferably stronger than the interaction between the substance to be analyzed and the immobilized substance, and is preferably a covalent bond in that the interaction is not lost during B/F (B(Bound)/F(Free)) separation.
A fusion molecule (proximity-dependent labeling enzyme-labeled analyte) in which a proximity-dependent labeling enzyme is bound to an analyte is brought into contact with a non-denatured protein array, and the labeling substance is bound by covalent or strong binding force to a protein, which is an immobilized substance on the protein array with which the proximity-dependent labeling enzyme-labeled analyte interacts. The labeling substance does not substantially fall off or break away from the immobilized substance even after the B/F separation process.
Furthermore, after washing, the labeled substance bound to the immobilized substance can be detected and quantified by biochemical techniques (mass spectrometry, electrophoresis, etc.).
A preferred proximity-dependent labeling enzyme of the present disclosure is a proximity-dependent biotin ligase obtained by modifying a part of the amino acid sequence of the BirA protein, which is a biotin ligase of Escherichia coli. The BirA protein recognizes a specific amino acid sequence as a substrate and has the function of specifically binding biotin to the lysine residue in the amino acid sequence. On the other hand, the proximity-dependent biotin ligase loses substrate specificity and has the function of binding biotin to the lysine residue on the surface of all substances, including immobilized substances, within the proximity range.
For example, BioID (SEQ ID NO: 1), TurboID (SEQ ID NO: 2), AirID (SEQ ID NO: 3), etc. have been reported as proximity-dependent biotin ligases (Choi-Rhee., et al., Protein Sci, 2004 (Doi 10.1110/ps.04911804); Roux, K., et al., JCB, 2012 (Doi 10.1083/jcb.201112098); Branon, TC., et al., Nat Biotech, 2018 (Doi: 10.1038/nbt.4201); Kido, K., et al., eLife, 2020 (Doi 10.7554/eLife.54983)).
The proximity-dependent labeling enzyme used in the present disclosure may be a recombinant gene product or a chemically synthesized product, and may be a derivative or fragment, or may be modified, substituted, deleted, or added, as long as the function is not impaired.
An example of such a method is to prepare a fusion protein (AirID-labeled protein) by genetic engineering based on the base sequence of the protein to be analyzed and the base sequence information of the gene encoding AirID. That is, a gene in which a gene encoding the substance to be analyzed and a gene encoding AirID are linked is cloned, and the gene is expressed in a cell-free synthesis system, thereby preparing a fusion protein of the substance to be analyzed and AirID.
Alternatively, the protein to be analyzed may be indirectly bound to the AirID via a substance that binds to the substance to be analyzed (support substance for the substance to be analyzed), forming an AirID-support substance for the substance to be analyzed-protein. Also, a spacer may be inserted between the AirID and the substance to be analyzed.

(BioID: SEQ ID NO: 1)
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEV LRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK
(TurboID: SEQ ID NO:2)
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEA LRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK
(AirID: SEQ ID NO: 3)
MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGYCLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGRRGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAE TLQKLGADGVRVKWPNDLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDRNQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQGALLLEQDGVIKPWIGGEISLRSA

Engineered Proximity-Dependent Modification Enzymes
The "engineered proximity dependent modified enzymes" of the present disclosure are characterized in that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 30, 40, 50 or all of the lysines displayed on the surface of the enzyme have been substituted.
Modified AirID
In the amino acid sequence shown in SEQ ID NO: 3, the lysine substitution position is any one or more of the following, and the substituted amino acid is arginine, glutamic acid, aspartic acid, or serine.
1) K2, 2) K38, 3) K41, 4) K56, 5) K71, 6) K122, 7) K163, 8) K194, 9) K244R, 10) K277, and 11) K307.
The lysine substitution is one or more of the following:
1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.
The lysine substitutions are:
1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.
Modified TurboID
In the amino acid sequence shown in SEQ ID NO:2, the lysine substitution positions are any one or more of the lysines other than those underlined below, and the substituted amino acid is arginine, histidine, glutamic acid, aspartic acid, or serine.
The positions of lysine substitutions are any one or more of the underlined lysines other than K172 and K183, and the substituted amino acid is arginine.
The lysine substitution positions are all of the lysines other than those underlined below, and the substituted amino acid is preferably arginine.
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRV K WPNDLYLQDR K LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK (Sequence number 2)
The "modified proximity-dependent modifying enzyme" of the present disclosure also includes mutants that contain substitutions other than the lysine substitutions shown above, which do not have mutations at K172R and K183, but may include the following mutations:
In detail, in addition to the lysine substitutions shown above,
1) A polypeptide (mutant) consisting of an amino acid sequence having one or two amino acids substituted, deleted, inserted and/or added in the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3, and having substantially the same proximity-dependent modification enzyme activity as the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3.
2) A polypeptide (mutant) consisting of an amino acid sequence having 95% or more identity (particularly, 96% or more, 97% or more, 98% or more, or 99% or more is preferred) with the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3, and having substantially the same proximity-dependent modification enzyme activity as the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3.
From the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, enzymatic activity, etc.) of the peptide when introducing mutations into the peptide, for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) can be easily envisioned.
The degree of the "proximity-dependent modification enzymatic activity substantially equivalent to that of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3" may be stronger or weaker than the proximity-dependent modification enzymatic activity of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 2. For example, the degree of the proximity-dependent modification enzymatic activity may be about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, or about 150% compared to the proximity-dependent modification enzymatic activity of the amino acid sequence set forth in SEQ ID NO: 2 or 3.
Alternatively, identity can be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default or initial setting parameters).
As used herein, polypeptide includes proteins, polypeptides and oligopeptides, the minimum size of which is two amino acids.
In this specification, the term "protein" includes its degradation products, fragmented peptides, and the like.

The engineered proximity-dependent modifying enzymes of the present disclosure are preferably fused directly or indirectly to a tag comprising one or more lysines at the N-terminus and/or C-terminus. Preferred tags are exemplified below.
1) DYKDDDDK (N-terminus - SEQ ID NO: 4)
2) DYKDDDDK (C-terminus: SEQ ID NO: 4)
3) DYKDHDGDYKDHDIDYKDDDDK (N-terminus - SEQ ID NO: 5)
4) GGKKKGKK (C-terminus: SEQ ID NO: 6)
5) KKKDKKDD (N-terminus - SEQ ID NO: 7)
6) DDKKKDKK (C-terminus: SEQ ID NO: 8)

The modified proximity-dependent modifying enzyme of the present invention may be directly or indirectly bound to the target substance by using a click chemistry technique. For example, if an azide (alkyne) is directly or indirectly bound to the modified proximity-dependent modifying enzyme, then an alkyne (azide) may be directly or indirectly bound to the target substance.

(Method for evaluating the interaction between an immobilized substance directly or indirectly immobilized on a substrate and a modified proximity-dependent modifying enzyme-labeled analyte)
The present disclosure relates to a method for evaluating the interaction between an immobilized substance immobilized directly or indirectly to a substrate and an analyte labeled with a modified proximity-dependent modification enzyme (hereinafter sometimes referred to as the "evaluation method of the present disclosure"), which comprises the following steps:
(1) adding a target substance labeled with a modified proximity-dependent modification enzyme to an immobilized substance that has been directly or indirectly immobilized on a substrate in the presence of a labeling substance; and (2) detecting the labeling substance. Note that evaluation of the interaction between the immobilized substance and the target substance includes detecting or quantifying the transient or persistent binding between the immobilized substance and the target substance.
Preferably, the method includes a step of cleaning the substrate between the above steps (1) and (2).

(substrate)
The substrate may be a substrate known per se for detecting the binding between the immobilized substance and the substance to be analyzed. The substrate may be flat or may be in the form of a so-called ELISA plate. The substrate may also be a substrate having a micro-dimple-shaped depression formed on the flat surface of the substrate, or a substrate having a porous membrane or a nitrocellulose membrane formed on the surface of the substrate. It is also possible to form a pad for mounting a protein. As a method for carrying out such processing, molding, lithography, etc. can be appropriately selected according to the substrate material. The substrate is preferably made of a material with a low background so as not to affect the luminescence or fluorescence detection used for the subsequent detection of interactions. For example, suitable substrate materials include non-fluorescent glass, amorphous carbon, quartz, polystyrene, polycarbonate, polymethyl methacrylate, polyolefin, polyethylene terephthalate, cycloolefin copolymer, etc.

(array)
The array of the present disclosure is an array of immobilized substances directly or indirectly immobilized on a substrate (preferably on a substrate on which positioning information is specified). The array allows simultaneous evaluation of interactions of the analysis target substance with all of the immobilized substances arranged.

(immobilized substance)
The immobilization substance is not particularly limited as long as it can be directly or indirectly immobilized on the substrate, and examples thereof include proteins, antibodies, nucleic acids (including DNA, RNA, etc.), peptides, low molecular weight compounds, medium molecular weight compounds, cell extracts, tissue extracts, sugars, lipids, physiologically active substances, and complexes thereof. The immobilization substance may be a single molecule or a mixture, a natural product, a genetically modified product, or a chemically synthesized product, or a derivative or fragment. Modification, substitution, deletion, and addition may be performed.

(Substances to be analyzed)
The substance to be analyzed is not particularly limited as long as it can be directly or indirectly labeled with a proximity-dependent modifying enzyme, and examples thereof include proteins, antibodies, nucleic acids (including DNA, RNA, etc.), peptides, low molecular weight compounds, medium molecular weight compounds, cell extracts, tissue extracts, sugars, lipids, physiologically active substances, complexes thereof, etc. According to the examples below, low molecular weight compounds that are usually difficult to analyze are preferred.
A specific example of a complex of an analyte is a complex formed between a protein A and a compound B, which enables interaction with an immobilized substance C or enhances the strength of the interaction. The analyte may be a single molecule or a mixture, a natural product, a recombinant product, or a chemically synthesized product, or may be a derivative or a fragment. Modification, substitution, deletion, or addition may be performed.

(Direct or indirect immobilization of immobilized substances onto a substrate)
The immobilization of the immobilization substance to the substrate directly or indirectly can be performed by a known immobilization method as long as the immobilization substance does not substantially flow out in the substrate washing step (B/F separation washing step) of the evaluation method of the present disclosure. For example, it is necessary to bind the immobilization substance by a suitable physical or chemical method depending on the material of the substrate. Note that indirect immobilization to the substrate means that the immobilization substance is fixed to the substrate via some substance (e.g., beads). Note that immobilization means that the substance is physically or chemically bound to the substrate.
When the substance to be immobilized is a tag fusion protein fused with a tag, a ligand that specifically binds to the tag, an antibody that recognizes the tag, a metal chelate that binds to the tag, or the like may be formed on the surface of the substrate. By using the substrate with the surface and the tag fusion protein, the substance to be immobilized can be directly or indirectly immobilized on the substrate by tag-ligand binding, tag-antibody binding, or tag-chelate binding. More specifically, examples of such binding include His tag and Ni-NTA, GST tag and glutathione, MBP tag and dextrin, biotin and avidin, biotin and streptavidin, biotin and neutravidin, FLAGTM tag and anti-FLAGTM antibody, GST tag and anti-GST antibody, HA tag and anti-HA antibody, etc.
When an inorganic substrate such as glass is used and a protein not fused with a tag is used as the immobilization substance, it is preferable to treat the substrate surface with a silane coupling agent having a functional group (e.g., an epoxy group, an active ester, an amino group, an acid anhydride group, an isocyanate group, etc.) that can bind to an amino group or a carboxyl group. A solution containing an immobilization substance (particularly a protein) can be spotted onto the treated substrate, and the immobilization substance can be immobilized on the substrate surface by covalent bonding at the N-terminus or C-terminus of the protein. Silane coupling agents with various chain lengths are commercially available, and any of them can be used as long as they do not affect the structure of the protein. It is also possible to adjust the bond distance between the immobilization substance (particularly a protein) and the substrate using a linker.
Other examples of suitable linkers for immobilizing proteins on metal surfaces include an aminooxy linker having a hydrophobic alkyl and a thiol group, and a hydrazide linker.

(Proteins as immobilized substances)
It is known that proteins as immobilized substances immobilized on a substrate or immobilized or mounted on an array are greatly affected by the physical and chemical macro- and micro-environments and are denatured. In particular, at liquid/solid interfaces and liquid/gas interfaces, such denaturation easily and irreversibly progresses, and as a result, the protein is likely to become inactive in that it loses its original function. In conventional methods, this inactive state makes it difficult to evaluate the interaction between the protein as the immobilized substance and the protein as the target substance to be analyzed. In other words, it is preferable that the protein as the immobilized substance is kept in a non-denatured state.
It is also known that the proteins immobilized at each designated position on the array as the immobilized substance only need to retain at least a portion of their ability to interact with the target substance. This depends on the type of protein mounted, but as described in the literature on protein-protein (Song, G. et al., Mol cell Proteomics.2019, Al-Mulla, F., et al., Cancer Res., 2011, etc.) and nucleic acid-protein (Hu S et al., Cell, 2009, Liu, L., et al., Nucleic Res., 2019, etc.) interaction analysis, if some function remains, it can be a substantially non-denatured protein array. In other words, for molecular species such as kinases, as long as the substrate protein of the kinase maintains a certain degree of structure, the intermolecular interactions can be evaluated even if the protein does not have the structure that it should have as a whole. Thus, the non-denatured state of the protein as the immobilized substance means that the site that interacts with the target substance maintains at least its shape or function.

(Method of synthesizing proteins as substances to be analyzed and substances to be immobilized)
The protein synthesis method as the analysis target substance and the immobilization substance can be a method known per se, but it is convenient to use a commonly used recombinant protein. For example, Escherichia coli, Bacillus subtilis, Sf9 insect cells, CHO cells, human cells, yeast, Brevibacillus, filamentous fungi (Azotobacter), tobacco BY-2 cells, or a plant transient expression system such as Nicotiana besamiana, lettuce, tomato (fruit and leaves), rice, barley, Phalaenopsis orchid, or red pepper, or a cell-free protein synthesis system can be used. For example, suitable examples of cell-free protein synthesis systems include Escherichia coli, Escherichia coli reconstituted system, wheat, insects, yeast, tobacco, rabbit reticulocytes, and human cells. For the purpose of comprehensively obtaining a wide variety of proteins, the wheat cell-free system is particularly excellent, has an extremely high probability of synthesizing proteins in a soluble state, and is very advantageous in terms of cost. In particular, cell-free protein synthesis using the WEPRO7240 series (Cell-Free Sciences) uses reagents from which GST-like proteins have been removed in advance, allowing for easy purification with glutathione beads to obtain highly pure proteins. This is one of the most preferred methods for preparing a wide variety of purified GST-tagged fusion proteins.

(Evaluation Method of the Present Disclosure)
An example of the evaluation method of the present disclosure is not particularly limited as long as it includes the steps of (1) adding an analyte labeled with a modified proximity-dependent modification enzyme to an immobilized substance immobilized directly or indirectly on a substrate in the presence of a labeling substance, and (2) detecting the labeling substance. A method using a non-denaturing protein array is exemplified below.
The immobilization substance is arranged and immobilized on a substrate. In this description, a non-denaturing protein array using a non-denaturing protein as the immobilization substance is used as a representative example.
In order to keep the immobilized proteins in a non-denatured state, the surface of the array or the inside of the wells of the array is constantly filled with a buffer.
During buffer exchange, it is preferable to slowly pour buffer into and out of the protein array to prevent the proteins immobilized on the magnetic beads from moving to adjacent wells together with the magnetic beads. In particular, when pouring buffer, it is preferable to use a syringe or the like to inject it toward the wall. During reaction and washing of the protein array, it is preferable to shake the array back and forth once per second to prevent the magnetic beads from moving.
The storage buffer in the protein array is removed, and the fusion protein of the modified AirID and the substance to be analyzed diluted with the reaction buffer is added to the protein array in the presence of biotin as a labeling substance. The addition may be any method as long as the immobilized substance and the substance to be analyzed can come into contact with each other. Furthermore, "in the presence of a labeling substance (biotin)" may be any method as long as the immobilized substance and the labeling substance (biotin) can come into contact with each other. For example, the labeling substance may be added to the array at any stage before, simultaneously with, or after the addition of the substance to be analyzed to the array. The storage buffer means a near-neutral buffer suitable for biological reactions that contains glycerol or the like to prevent protein aggregation or stabilize the structure, but is not particularly limited thereto. The reaction buffer means a near-neutral buffer suitable for biological reactions that contains a blocking agent to prevent the substance to be analyzed from being nonspecifically adsorbed to the substrate or immobilized substance, and a labeling substance (biotin) and an activation energy source (ATP) necessary for the reaction, but is not particularly limited thereto. The washing buffer means, but is not limited to, a near-neutral buffer that contains salts and surfactants and is suitable for biological reactions in order to remove the target substance that is free in the solution or bound to the substrate or immobilized substance.
In the presence of biotin (and ATP, if necessary), all of the proteins immobilized on the array interact with the modified AirID fusion protein, which is the substance to be analyzed, and when the immobilized substances and the substance to be analyzed bind, the modified AirID labels the lysine residues of the immobilized substances within close range with biotin. If the fusion protein does not contain a lysine residue, the protein may be modified to contain a lysine residue, if necessary.
In the interaction evaluation method of the present disclosure, biotin bound to a protein, which is an immobilized substance immobilized on an array, is detected, and therefore, an interaction can be detected even if a specific but weakly interacting analyte is removed by a washing operation.
After washing, biotin labeled proteins, which are immobilized substances on the array, are detected using a substance that specifically recognizes and binds to biotin, and the results of the interaction analysis are obtained as a measurement image.
Substances that specifically recognize and bind to biotin used to detect interactions include anti-biotin antibodies and streptavidin. Both substances are preferably HRP-labeled, AP-labeled, or fluorescently labeled. Anti-biotin antibodies or streptavidin are preferably diluted in a reaction buffer and placed in the protein array to perform a binding reaction with biotin. After the reaction, it is necessary to wash and remove free anti-biotin antibodies or streptavidin. After washing, if an HRP/AP-labeled substance is used, a chemiluminescent reagent is added, and the luminescence obtained by the reaction of the chemiluminescent reagent with HRP/AP is measured with a luminescence detection device. An example of a luminescence detection device is LAS (manufactured by GE). If a fluorescent label is used, the measurement is made with a fluorescence detection device. An example of a fluorescence detection device is Typhoon (manufactured by GE).
From the measured images, the presence or absence of interaction between the protein immobilized on each array and the protein to be analyzed is determined.
To determine the interaction, it is desirable to digitize the signal of each spot on the measurement image and determine that an interaction exists when the signal is equal to or greater than a certain value. It is desirable to use analysis software such as Array Pro Analyzer to digitize the measurement image. This makes it possible to measure the strength of interaction (binding strength) between multiple immobilized substances and the substance to be analyzed at once.

Preferred examples of the modified proximity-dependent modifying enzyme, the N-terminus of the modified proximity-dependent modifying enzyme, the C-terminus of the modified proximity-dependent modifying enzyme, the target substance, and the terminus of the target substance of the present disclosure are as follows, but are not particularly limited.
(1) Modified proximity-dependent modification enzymes: an enzyme in which all of K2, K38, K41, K56, K71, K122, K163, K194, K244, K277 and K307 are substituted (AirID_KR11), an enzyme in which only K2 is substituted, an enzyme in which only K38 is substituted, an enzyme in which only K41 is substituted, an enzyme in which only K56 is substituted, an enzyme in which only K71 is substituted, an enzyme in which only K122 is substituted, an enzyme in which only K163 is substituted, an enzyme in which only K194 is substituted, an enzyme in which only K244 is substituted, an enzyme in which only K277 is substituted, an enzyme in which only K307 is substituted, an enzyme having an azide group in these enzymes, and an enzyme having an alkynyl group in these enzymes (2) N-terminus of modified proximity-dependent modification enzymes: DYKDDDDK, DYKDHDGDYKDHDIDYKDDDDK or KKKDKKDD
(3) C-terminus of modified proximity-dependent modification enzyme: DYKDDDDK, GGKKKGKK, or DDKKKDKK (C-terminus)
(4) Target substance to be analyzed: a low molecular weight compound, a target substance to be analyzed having an azide group, a target substance to be analyzed having an alkynyl group (5) Terminus of target substance to be analyzed: an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus In the present disclosure, the examples described above in (1) to (5) can be combined.
The present disclosure will be described in more detail below with reference to examples. However, the following examples should be regarded as an aid in gaining a concrete understanding of the present disclosure, and the scope of the present disclosure is not limited in any way by the following examples.

(Confirmation of enzyme activity by lysine modification)
In this example, it was confirmed whether the lysine residue of AirID was modified to maintain the enzyme activity.

(AirID)
The interaction between AirID-modified enzyme-IκBα and FG-RelA was analyzed by in-tube GSH magnetic bead assay, and the results are shown in Figures 1 and 2.
Analysis of AF confirmed that mutations at either of the internally located lysines (Lys172 and Lys183) abolished biotinylation activity, while mutations at the other lysines (surface lysines) to arginine did not affect activity.
For the above reasons, in the following examples, a modified AirID was used in which lysines other than those believed to be located inside (Lys172, Lys183) were mutated to arginine.

(TurboID)
The enzymatic activity of the lysine-modified TurboID was confirmed in the same manner as for the AirID described above.
It was confirmed that the activity was not affected even when the lysines other than the two underlined ones below (K172 and K183) were modified to arginines.
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRV K WPNDLYLQDR K LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

(Preparation and functional confirmation of surface lysine-mutated AirIDs fused with each tag)
In this example, surface lysine mutated AirIDs were prepared by fusing each tag shown in Figure 3. Next, the yields after synthesis and purification were confirmed depending on the difference in each tag. Furthermore, the detection sensitivity depending on the difference in each tag was confirmed.

The yields after synthesis and purification of surface lysine mutant AirIDs fused with each tag are shown in Figure 4. The yields after synthesis and purification were confirmed to be equivalent to those of the wild type.

The analysis results using the surface lysine-mutated AirID fused with each tag are shown in Figures 5 and 16. In detail, 1 mM Thalidomide-O-PEG4-NHS ester was bound to the surface lysine-mutated AirID with each lysine tag, and biotinylation during interaction with CRBN was analyzed.
We confirmed that the surface lysine mutant AirID (1) showed less CRBN biotinylation than the WT. We confirmed that the tagged surface lysine mutant AirIDs (4-7) containing 4-5 lysines showed more CRBN biotinylation than the WT. This is thought to depend on the amount of thalidomide bound to the lysines. We confirmed that the C-terminal tag (3) showed more CRBN biotinylation than the N-terminal tag (2).
In addition, surface lysine-mutated AirID (5, 7) fused with a tag containing five lysines showed strong activity.

(Analysis using geldanamycin-surface lysine mutation AirID)
In this example, geldanamycin-surface lysine mutant AirID_KR11 (geldanamycin-AirID ² C) was prepared. Furthermore, the surface lysine mutant AirID was evaluated using HSP90AB1, which specifically binds to geldanamycin.

(Preparation of Geldanamycin-AirID ² C)
The results of preparation of geldanamycin-AirID ² C are shown in Figure 6. It was confirmed that the prepared geldanamycin-AirID ² C was soluble.

(Confirmation of interaction between geldanamycin-AirID ² C and immobilized material)
The results of the interaction between geldanamycin-AirID2C and the immobilized material are shown in FIGS.
From the results in FIG. 7, it was confirmed that GM-AirID ² C prepared under the condition of 20 μM AirID_KR11 + 100 μM GM-NHS specifically interacted with HSP90AB1 and biotinylated it.
From the results in FIG. 8, it was confirmed that the reaction between GM-AirID ² C and HSP90AB1 was inhibited by adding unmodified geldanamycin as a competitor, which indicates that this is a specific interaction.
The interaction between Biotin-modified GM and HSP90AB1 without using AirID could not be detected by this assay. That is, it was confirmed that the evaluation method of the present disclosure is excellent because it is specific and highly sensitive.

(Shelf life of Geldanamycin-AirID ² C)
The results of using geldanamycin-AirID ² C stored at −80° C. are shown in Figure 9. It was confirmed that geldanamycin-AirID ² C stored at −80° C. retains its functionality.

(Comparison of solubility of AirID(native) and AirID modified bodies after reaction with the target substance)
In this example, the solubility of AirID (native) and modified AirID after reaction with a target substance was compared.
When (S,R,S)-AHPC-PEG4-NHS ester (VHL ligand) was reacted at a high concentration (5000 μM), a difference in solubility was confirmed between AirID and the AirID modified form (AirID_KR11) (see Figure 10).
They also succeeded in obtaining a "drug-binding specialized modified AirID = AirID ² C," which maintains stability even when bound to a drug.

(Analysis 1 using low molecular weight target substances)
In this example, thalidomide, a low molecular weight compound, was used as the substance to be analyzed.

(Analysis of molecular interaction between thalidomide-bound AirID modified by NHS ester reaction and CRBN)
In this example, we analyzed the interaction between thalidomide and CRBN, which are known to bind to each other. The details are as follows.
(Protein synthesis of immobilized material)
Template DNAs were synthesized in which BRD2, BRD3, BRD4, CRBN, and CRBN YW/AA were fused to FLAG ^TM tag protein and GST protein as immobilization substances (target proteins).
Using each of the synthesized template DNAs, FLAG ^™ -GST fusion proteins were synthesized in a wheat cell-free expression system.
(Binding of immobilized substances to magnetic beads and purification)
The FLAG ^- GST fusion immobilized material was bound to glutathione magnetic beads used in non-denaturing protein arrays and purified. The purified magnetic beads were dispensed and placed in tubes and immobilized by magnetic force, allowing for solution exchange during washing.
(Protein synthesis and purification of AirID variants)
A template DNA was synthesized by fusing the AirID variant (AirID_KR11) with a lysine-containing tag (containing multiple lysine residues) and a His tag protein. More specifically, a tag containing one or more lysines at the N-terminus and/or C-terminus (see paragraph "0015") was used. The present inventors have confirmed that the effect of this embodiment can be obtained if the tag contains one or more lysines.
Using the synthesized template DNA, lysine-containing tag and His-tag fused AirID variants were synthesized in a wheat cell-free expression system.
The synthesized AirID variants were bound to Ni resin and purified, and the AirID variants were eluted from the Ni resin using imidazole.
(Binding reaction between the target substance and modified AirID)
After purification, the eluted AirID variant and the target substance having NHS Ester were mixed in a solution and left to stand at 4-37°C for 1-6 hours to carry out the binding reaction. It was confirmed that the reaction temperature and time could be changed within a range that does not affect the properties of the protein. The concentration of the AirID variant was about 5-100 μM, but it was confirmed that tests at lower and higher concentrations are also possible. The concentration of the target substance having NHS Ester was about 100-1000 μM, which was higher than the concentration of the AirID variant, but it was confirmed that tests at lower and higher concentrations are also possible. The solvent used during the reaction was phosphate buffer adjusted to pH 7.4, but it was confirmed that the pH could be set between about 7.2 and 8.5, and other buffers could also be used. An AirID variant bound to the target substance was prepared.
The target substance containing unreacted NHS ester was removed by solution exchange through dialysis.
(Biotin labeling of immobilized substances in the presence of target substances)
The modified AirID bound to the substance to be analyzed (thalidomide) was diluted with a reaction buffer containing biotin and ATP and reacted with the FLAG ^™ -GST fusion immobilized substance on the magnetic beads to label the FLAG ^™ -GST fusion immobilized substance with biotin.
(Removal of substances to be analyzed)
In order to remove the analyte-binding AirID variants that were free in the reaction buffer or adsorbed onto the magnetic beads, the reaction buffer was removed and then washed multiple times with a washing buffer.
(Biotin detection using anti-biotin antibody)
The washing buffer was removed, and anti-biotin antibody diluted with reaction buffer was added and reacted with the FLAG ^TM -GST fusion immobilized substance on the magnetic beads. To remove free anti-biotin antibody, the reaction buffer was removed, and the beads were washed multiple times with washing buffer. After removing the washing buffer, a chemiluminescent reagent was added and reacted. The resulting luminescence was detected with LAS4000 (GE), and a measurement image was obtained.
(Biotin detection results)
The results are shown in Figure 11. Luminescence was observed between the thalidomide-bound AirID variant and CRBN. No luminescence was observed between the variants and other proteins.
Specifically, thalidomide-conjugated AirID ² C modified only CRBN with biotin. The biotin modification reaction of CRBN was inhibited by the addition of pomalidomide as a competitor, confirming that thalidomide-conjugated AirID ² C specifically bound to CRBN.
FIG. 12 shows the results of electrophoresis of the proteins used.
From the above, in this Example, it was confirmed that the analytical method of the present disclosure is capable of analyzing specific interactions between an immobilized substance and an analyte substance that is a low compound.

(Analysis 2 using low molecular weight target substances)
In this example, JQ1, a low molecular weight compound, was used as the substance to be analyzed.
In this example, we analyzed the interaction between JQ1 and the BRD family or between Thalidomide and CRBN, which are known to bind to each other. Details are as follows.

(Protein synthesis of immobilized material)
Template DNAs were synthesized in which BRD2, BRD3, BRD4, CRBN, and CRBN YW/AA were fused to FLAG ^TM tag protein and GST protein as immobilization substances (target proteins).
Using each of the synthesized template DNAs, FLAG ^™ -GST fusion proteins were synthesized in a wheat cell-free expression system.
(Binding of immobilized substances to magnetic beads and purification)
The FLAG ^- GST fusion immobilized material was bound to glutathione magnetic beads used in non-denaturing protein arrays and purified. The purified magnetic beads were dispensed and placed in tubes and immobilized by magnetic force, allowing for solution exchange during washing.
(Protein synthesis and purification of AirID variants)
A template DNA was synthesized by fusing the AirID variant (AirID_KR11) with a lysine-containing tag (containing multiple lysine residues) and a His tag protein. More specifically, a tag containing one or more lysines at the N-terminus and/or C-terminus (see paragraph "0015") was used. The present inventors have confirmed that the effect of this embodiment can be obtained if the tag contains one or more lysines.
Using the synthesized template DNA, lysine-containing tag and His-tag fused AirID variants were synthesized in a wheat cell-free expression system.
The synthesized AirID variants were bound to Ni resin and purified, and the AirID variants were eluted from the Ni resin using imidazole.
(Binding reaction of Azide and modified AirID)
After purification, the eluted AirID variant and Azido-PEG4-NHSEster were mixed in a solution and left to stand for 1 to 6 hours at 4 to 37°C to carry out the binding reaction. It was confirmed that the reaction temperature and time could be changed within a range that does not affect the properties of the protein.
The AirID variant was tested at a concentration of 5-100 μM, but it was confirmed that lower and higher concentrations were also possible. The Azido-PEG4-NHSEster concentration was 100-1000 μM, which was equal to or higher than the concentration of the AirID variant, but it was confirmed that lower and higher concentrations were also possible. The solvent used during the reaction was phosphate buffer adjusted to pH 7.4, but it was confirmed that the pH could be set between 7.2 and 8.5, and other buffers could also be used. Azide-linked AirID variants were prepared.
Unreacted Azido-PEG4-NHS Ester was removed by solution exchange through dialysis.
(Binding reaction between the target substance and azide-bound AirID modified substance)
The prepared azide-bound (azide group-bearing) AirID variant was mixed with the target substance having an alkyne (alkynyl group), and the binding reaction was carried out by click chemistry reaction. During the click chemistry reaction, the target substance having an alkyne (alkynyl group) was added to the azide-bound AirID variant in a mixture of 250 μM THPTA, 50 μM CuSO4, 2.5 mM sodium ascorbate, and 1 mM aminoguanidine. The concentration of the target substance having an alkyne (alkynyl group) was 100 μM, but it was confirmed that there was no problem as long as the concentration exceeded the number of azides bound to the AirID variant. An AirID variant bound to the target substance was prepared. The target substance having an unreacted alkyne (alkynyl group) was removed by solution exchange via dialysis.
(Biotin labeling of immobilized substances in the presence of the substance to be analyzed)
The AirID variants bound to the substance to be analyzed (JQ1 or Thalidomide) diluted with a reaction buffer containing biotin and ATP were reacted with the FLAG ^™ -GST fusion immobilized substance on the magnetic beads to label the FLAG ^™ -GST fusion immobilized substance with biotin.
(Removal of substances to be analyzed)
In order to remove the analyte-binding AirID variants that were free in the reaction buffer or adsorbed onto the magnetic beads, the reaction buffer was removed and then washed multiple times with a washing buffer.
(Biotin detection using anti-biotin antibody)
The washing buffer was removed, and anti-biotin antibody diluted with reaction buffer was added and reacted with the FLAG ^TM -GST fusion immobilized substance on the magnetic beads. To remove free anti-biotin antibody, the reaction buffer was removed, and the beads were washed multiple times with washing buffer. After removing the washing buffer, a chemiluminescent reagent was added and reacted. The resulting luminescence was detected with LAS4000 (GE), and a measurement image was obtained.
(Biotin detection results)
The results are shown in Figure 13. Luminescence was observed between the JQ1-bound AirID variant and the BRD family, and between the Thalidomide-bound AirID variant and CRBN. No luminescence was observed between other compounds and proteins.
Specifically, JQ1-bound AirID ² C modified only the BRD family with biotin. JQ1, which was added as a competitor, inhibited the biotin modification reaction of the BRD family, confirming that JQ1-bound AirID ² C specifically bound to the BRD family.
FIG. 14 shows the results of electrophoresis of the proteins used.
In this example, it was confirmed that the analytical method of the present disclosure is capable of analyzing specific interactions between an immobilized substance and a substance to be analyzed.

(Protein array analysis using AirID ² C)
Using an E3 ligase array (loaded with approximately 570 proteins), we searched for proteins that interact with drug X. Drug X, added as a competitor, inhibited the biotin modification reaction of the positive protein (1), so AirID ² C_drug X can search for E3 ligases that specifically bind (see Figure 15).
As described above, the modified proximity-dependent modification enzymes of the present disclosure are useful in protein array analysis.

(Analysis of intermolecular interactions between pomalidomide- or thalidomide-binding AirID variants and CRBN)
In this example, we analyzed the interaction between CRBN and pomalidomide or thalidomide, which are known to bind to CRBN. We used modified AirIDs in which Azide and Alkyne were bound to pomalidomide, modified AirIDs in which Alkyne and Azide were bound to thalidomide, and modified AirIDs in which the target substance was bound by click chemistry reaction in different combinations. The details are as follows.
(Protein synthesis of immobilized material)
Template DNAs were synthesized in which CRBN, CRBN YW/AA, and BRD4 were fused to a FLAG ^TM tag protein and a GST protein as immobilization substances (target proteins).
Using each of the synthesized template DNAs, FLAG ^™ -GST fusion proteins were synthesized in a wheat cell-free expression system.
(Binding of immobilized substances to magnetic beads and purification)
The FLAG ^- GST fusion immobilized material was bound to glutathione magnetic beads used in non-denaturing protein arrays and purified. The purified magnetic beads were dispensed and placed in tubes and immobilized by magnetic force, allowing for solution exchange during washing.
(Protein synthesis and purification of AirID variants)
A template DNA was synthesized by fusing the AirID variant with a lysine-containing tag (containing multiple lysine residues) and a His tag protein. More specifically, a tag containing one or more lysines at the N-terminus and/or C-terminus (see paragraph "0015") was used. The present inventors have confirmed that the effect of this embodiment can be obtained if the tag contains one or more lysines.
Using the synthesized template DNA, lysine-containing tag and His-tag fused AirID variants were synthesized in a wheat cell-free expression system.
The synthesized AirID variants were bound to Ni resin and purified, and the AirID variants were eluted from the Ni resin using imidazole.
(Binding reaction of alkyne or azide with modified AirID)
After purification, the eluted AirID variant was mixed with Propargyl-PEG4-NHS ester or Azido-PEG4-NHS ester in a solution and left to stand at 4-37°C for 1-6 hours to carry out the binding reaction. It was confirmed that the reaction temperature and time could be changed within a range that does not affect the properties of the protein. The concentration of the AirID variant was about 5-100 μM, but it was confirmed that it can be carried out at lower or higher concentrations. The concentration of Propargyl-PEG4-NHS ester or Azido-PEG4-NHS ester was about 100-1000 μM, which was higher than the concentration of the AirID variant, but it was confirmed that it can be carried out at lower or higher concentrations. The solvent used during the reaction was phosphate buffer adjusted to pH 7.4, but it was confirmed that the pH could be set between about 7.2 and 8.5, and other buffers could also be used. AirID variants bound to alkynes or azides were prepared. Unreacted Propargyl-PEG4-NHS ester or Azido-PEG4-NHS ester was removed by solution exchange through dialysis.
(Binding reaction between the target substance and alkyne or azide-bound AirID modified substance)
The prepared alkyne or azide-bound AirID modified substance was mixed with the target substance having an alkyne group, and the binding reaction was carried out by click chemistry reaction. During the click chemistry reaction, the target substance having an azide group or alkynyl group was added to the alkyne or azide-bound AirID modified substance in a mixture of 250 μM THPTA, 50 μM CuSO4, 2.5 mM sodium ascorbate, and 1 mM aminoguanidine. The concentration of the target substance having an azide group or alkynyl group was 100 μM, but it was confirmed that there was no problem as long as the concentration exceeded the number of alkynes or azides bound to the AirID modified substance. The target substance was bound to the AirID modified substance. The unreacted target substance having an azide group or alkyne group was removed by solution exchange by dialysis.
(Biotin labeling of immobilized substances in the presence of target substances)
The AirID variants bound to the substance to be analyzed (pomalidomide or thalidomide) diluted with a reaction buffer containing biotin and ATP were reacted with the FLAG ^™ -GST fusion immobilized substance on the magnetic beads to label the FLAG ^™ -GST fusion immobilized substance with biotin.
(Removal of substances to be analyzed)
In order to remove the analyte-binding AirID variants that were free in the reaction buffer or adsorbed onto the magnetic beads, the reaction buffer was removed and then washed multiple times with a washing buffer.
(Biotin detection using anti-biotin antibody)
The washing buffer was removed, and anti-biotin antibody diluted with reaction buffer was added and reacted with the FLAG ^TM -GST fusion immobilized substance on the magnetic beads. To remove free anti-biotin antibody, the reaction buffer was removed, and the beads were washed multiple times with washing buffer. After removing the washing buffer, a chemiluminescent reagent was added and reacted. The resulting luminescence was detected with LAS4000 (GE), and a measurement image was obtained.
(Biotin detection results)
The results are shown in Figure 17. Luminescence was observed between the pomalidomide- or thalidomide-bound AirID variant and CRBN. No luminescence was observed between the variants and other proteins.
Specifically, AirID2C bound to pomalidomide or thalidomide modified only CRBN with biotin. The biotin modification reaction of CRBN was inhibited by pomalidomide added as a competitor, confirming that AirID2C bound to pomalidomide or thalidomide specifically bound to CRBN.
The results of electrophoresis of the proteins used are shown in Figure 18. It was confirmed that the AirID mutant was modified with an alkynyl group.
This Example and Example 6 confirmed that the analytical method of the present disclosure is capable of analyzing specific interactions between an immobilized substance and a target substance (especially a low molecular weight compound) using a target substance having an alkynyl group (azide group) and a modified proximity-dependent modifying enzyme having an azide group (alkynyl group).

The evaluation method of the present invention can analyze low molecular weight compounds, which was difficult to do with conventional evaluation methods.

Claims (24)

An engineered proximity-dependent modifying enzyme in which surface lysines have been substituted.
2. The modified proximity-dependent modifying enzyme of claim 1, wherein the proximity-dependent modifying enzyme is a peptide having the following amino acid sequence (SEQ ID NO:3: AirID):
MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGYCLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGRRGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAET LQKLGADGVRVKWPNDLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDRNQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQGALLLEQDGVIKPWIGGEISLRSA.
3. The engineered proximity dependent modification enzyme of claim 2, wherein the lysine substitution position is any one or more of the following and the substituted amino acid is arginine, histidine, glutamic acid, or aspartic acid:
1) K2, 2) K38, 3) K41, 4) K56, 5) K71, 6) K122, 7) K163, 8) K194, 9) K244R, 10) K277, and 11) K307.
3. The engineered proximity-dependent modification enzyme of claim 2, wherein the lysine substitution is any one or more of the following:
1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.
3. The engineered proximity dependent modification enzyme of claim 2, wherein the lysine substitution is:
1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.
2. The modified proximity-dependent modifying enzyme of claim 1, wherein the proximity-dependent modifying enzyme is a peptide having the following amino acid sequence (SEQ ID NO:2: TurboID):
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEAL RKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK.
The modified proximity-dependent modification enzyme of claim 6, wherein the lysine substitution positions are any one or more of lysines other than K172 and K183 as underlined.
MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRV K WPNDLYLQDR K LAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK.
8. The modified proximity-dependent modifying enzyme according to any one of claims 1 to 7, wherein one or more lysine-containing tags are fused directly or indirectly to the N-terminus or C-terminus.
The modified proximity-dependent modification enzyme according to any one of claims 1 to 7, wherein any one of the following tags is fused directly or indirectly to the N-terminus or C-terminus:
1) DYKDDDDK (N-terminus - SEQ ID NO: 4)
2) DYKDDDDK (C-terminus: SEQ ID NO: 4)
3) DYKDHDGDYKDHDIDYKDDDDK (N-terminus - SEQ ID NO: 5)
4) GGKKKGKK (C-terminus: SEQ ID NO: 6)
5) KKKDKKDD (N-terminus - SEQ ID NO: 7)
6) DDKKKDKK (C-terminus: SEQ ID NO: 8)
A modification proximity-dependent modifying enzyme-labeled analyte, which is labeled with the modification proximity-dependent modifying enzyme according to any one of claims 1 to 9.
A modification proximity-dependent modifying enzyme-labeled analyte, which is labeled with the modification proximity-dependent modifying enzyme according to claim 5 .
11. The modified proximity-dependent modifying enzyme labeled analyte of claim 10, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus.
12. The modified proximity-dependent modifying enzyme labeled analyte of claim 11, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus.
A method for evaluating an interaction between an immobilized substance directly or indirectly immobilized on a substrate and a modified proximity-dependent modifying enzyme-labeled analyte, comprising:
The method includes the steps of:
(1) adding the modified proximity-dependent modification enzyme-labeled analyte according to claim 12 or 13 to an immobilized substance immobilized directly or indirectly on a substrate in the presence of a label;
(2) detecting the labeling substance;
Evaluation method.
The evaluation method according to claim 14 , further comprising a step of cleaning the substrate between the step (1) and the step (2).
The method for evaluating the affinity of a modification proximity-dependent modification enzyme-labeled analyte according to claim 14 or 15, wherein the modification proximity-dependent modification enzyme-labeled analyte is the modification proximity-dependent modification enzyme-labeled analyte according to claim 12.
A method for evaluating a protein, which is an immobilized substance indirectly immobilized on an array via magnetic beads, and a modified proximity-dependent modifying enzyme-labeled analyte, comprising the steps of:
(1) adding the modified proximity-dependent modification enzyme-labeled analyte according to claim 12 or 13 in the presence of biotin to an immobilized substance indirectly immobilized on an array via magnetic beads;
(2) detecting the biotin;
Evaluation method.
The evaluation method according to claim 17 , further comprising a step of washing the array between the steps (1) and (2).
The method for evaluating the affinity of a modification proximity-dependent modification enzyme-labeled analyte according to claim 17 or 18, wherein the modification proximity-dependent modification enzyme-labeled analyte according to claim 13.
A method for introducing a modified proximity-dependent modification enzyme-labeled analyte into a cell, the method comprising introducing the modified proximity-dependent modification enzyme-labeled analyte according to claim 12 or 13 into the cell by electroporation.
The evaluation method according to claim 14 , wherein the substance to be analyzed maintains water solubility.
The evaluation method according to claim 17 , wherein the substance to be analyzed maintains water solubility.
The evaluation method according to claim 14 , wherein the target substance to be analyzed has an alkynyl group and the modified proximity-dependent modifying enzyme has an azide group.
The evaluation method according to claim 14, wherein the substance to be analyzed has an azide group and the modified proximity-dependent modification enzyme has an alkynyl group.