Non-natural amino acids and their use as probes

Abstract

The present invention relates to non-natural amino acids and their use as probes. Specifically, the invention relates to a brand new non-natural amino acid, a non-natural polypeptide containing the non-natural amino acid, a method for preparing the non-natural polypeptide, and application of the non-natural polypeptide to detection of protein interaction or protein abnormal aggregation or detection of existence or amount of a target molecule entity. The invention also relates to an orthogonal aminoacyl-tRNA synthetase (O-RS), an orthogonal-tRNA (O-tRNA), a translation system, and a kit containing the non-natural amino acid and / or the orthogonal aminoacyl-tRNA synthetase (O-RS) and / or the orthogonal-tRNA (O-tRNA).

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a novel non-natural amino acid, a non-natural polypeptide comprising the non-natural amino acid, a method for preparing the non-natural polypeptide, and the use of the non-natural polypeptide for detecting protein interactions or abnormal protein aggregation or for detecting the presence or amount of a target molecular entity; and also relates to an orthogonal aminoacyl-tRNA synthetase (O-RS), an orthogonal-tRNA (O-tRNA), a translation system, and a kit comprising the above-mentioned non-natural amino acid and / or an orthogonal aminoacyl-tRNA synthetase (O-RS) and / or an orthogonal-tRNA (O-tRNA). Background Art

[0002] Fluorescent labeling of proteins is a crucial protein research method that enables researchers to explore dynamic changes in protein structure, localization, and biomolecular interactions in living cells, thereby gaining a deeper understanding of the key functions and roles of proteins in life activities. Currently commonly used protein fluorescent labeling methods, such as the fusion expression of fluorescent proteins and target proteins, and the fusion of protein tags that can be covalently coupled to fluorescent dyes with target proteins, have defects such as large protein volume and labeling sites limited to the ends of the target proteins, which may interfere with the normal structure and function of the target proteins. On the other hand, the genetic code expansion (GCE) technology that has emerged in recent years has shown unique advantages in the field of fluorescent labeling of proteins. GCE technology can accurately insert non-natural amino acids (ncAAs) at any site on the protein molecule through genetic coding, showing extremely high specificity and accuracy. More importantly, GCE technology usually only changes one amino acid, so the effect on protein structure and function is very small. However, the key to using GCE technology for protein fluorescent labeling is to develop better fluorescent non-natural amino acids (FL-ncAAs or FLAAs). Existing FLAAs are essentially traditional fluorescent dyes, which have limitations such as inherent fluorescence, fluorescence background problems, short wavelength, and easy photobleaching.

[0003] To address the above problems, it is necessary to develop a class of fluorescent probe molecules with low fluorescence background and high signal-to-noise ratio, as well as a new fluorescent labeling method with little interference to the target protein and strong applicability. Summary of the invention

[0004] The inventors of this application designed and synthesized for the first time a new type of fluorescent rotor noncanonical amino acids (FR-ncAAs or FRAAs). Different from the intrinsic fluorescence properties of traditional fluorescent small molecules and existing fluorescent ncAAs, the fluorescence of FRAAs is latent and activatable (fluorogenic), and can be turned on in specific chemical and biological environments. Therefore, this fluorescent "on-off" property provides many possibilities for various important applications of FRAAs.

[0005] Then, the inventors found aminoacyl-tRNA synthetases that can achieve gene codon expansion of FRAAs in prokaryotic cells and eukaryotic cells through screening methods, such as MjFmrRS, CC9 PylRS, AC2 PylRS, etc.

[0006] Then, the inventors introduced the fluorescent rotor non-natural amino acid into the protein, so that it can be used to respond to and detect changes in the protein microenvironment. On the one hand, the inventors developed a probe that can be used to detect protein interactions by using the fluorescent rotor of the fluorescent rotor non-natural amino acid to activate the fluorescence property. On the other hand, with the ability of the fluorescent rotor non-natural amino acid to respond to protein conformational changes, the inventors also developed a sensing allosteric protein with a high dynamic range, which can be used as a sensor for detecting target molecules, such as sensors for glucose, glutamine, GTP / GDP, and calcium ions.

[0007] Specifically, the present invention provides the following technical solutions.

[0008] In a first aspect, the present invention provides a non-natural amino acid having a structure of Formula I:

[0009]

[0010] Among them, X 1a , X 2a , X 3a and X 4a Independently selected from CH and N; preferably X 1a , X 2a , X 3a and X 4a 3 of them are CH; more preferably all 4 of them are CH;

[0011] R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b, R c , R d Independently selected from C1-C6 alkyl, preferably C1-C3 alkyl; preferably R1 is selected from: -NH2, -NHR a and-NR b R c , where R a , R b , R c Independently selected from C1-C6 alkyl, preferably C1-C3 alkyl;

[0012] R2 is selected from: cyano, sulfonic acid, nitro and trihalomethyl (e.g. trifluoromethyl, trichloromethyl); preferably R2 is selected from: cyano, sulfonic acid and trichloromethyl; more preferably R2 is selected from: cyano;

[0013] X 1b Selected from: N and CR3;

[0014] R3 is selected from: H, cyano, sulfonic acid, nitro and trihalomethyl (e.g. trifluoromethyl, trichloromethyl); preferably R3 is selected from: H, cyano, sulfonic acid and trichloromethyl; more preferably R3 is selected from: H and cyano;

[0015] X 2b Selected from CR 42 and N, preferably CR 42 ,

[0016] X 3b Selected from CR 43 and N, preferably CR 43 ,

[0017] X 4b Selected from CR 44 and N, preferably CR 44 ,and

[0018] X 5b Selected from CR 45 and N, preferably CR 45 ;

[0019] R 42 , R 43 , R 44 , R 45 Each independently selected from: H and -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3, and R 42 , R 43 , R 44 , R 45 At least one of is not H; preferably R 42 , R 43 , R 44 , R45 1 of the following (preferably R 42 or R 43 ) is selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1-3, and the rest are H.

[0020] In some embodiments, for the above-mentioned non-natural amino acids, they have the structure of Formula IA or Formula IB:

[0021]

[0022] Wherein, R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl;

[0023] Preferably, R1 is selected from: -NH2, -NHR a and-NR b R, where R a , R b , R c Independently selected from C1-C3 alkyl, preferably methyl, ethyl, propyl;

[0024] More preferably, R1 is selected from: -NH2 and -NR a R b , where R a , R b independently selected from methyl;

[0025] R3 is independently selected from: H, cyano, sulfonic acid, nitro and trihalomethyl (eg, trifluoromethyl and trichloromethyl);

[0026] Preferably, R3 is selected from: cyano, sulfonic acid and trichloromethyl;

[0027] More preferably, R3 is selected from cyano;

[0028] R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3;

[0029] Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH);

[0030] Preferably, Formula IA is selected from:

[0031]

[0032] R1, R3, and R4 in the above formula I-A1 and formula I-A2 are defined as the corresponding variables in the above formula IA;

[0033] or,

[0034]

[0035] Wherein, R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl;

[0036] Preferably, R1 is selected from: -NH2, -NHR a and-NR b R, where R a , R b , R c Independently selected from C1-C3 alkyl, preferably methyl, ethyl and propyl;

[0037] More preferably, R1 is selected from: -NH2, -NR a R b , where R a , R b independently selected from methyl;

[0038] R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3;

[0039] Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH);

[0040] Preferably, Formula IB is selected from:

[0041]

[0042]

[0043] R1 and R4 in the above formula I-B1 and formula I-B2 are defined as the corresponding variables in the above formula IB.

[0044] In some embodiments, the non-natural amino acid is selected from the following:

[0045] In a second aspect, the present invention provides an orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS is capable of recognizing the non-natural amino acid described in the first aspect, and aminoacylates the orthogonal tRNA (O-tRNA) with the non-natural amino acid;

[0046] Preferably, the O-RS is derived from an aminoacyl-tRNA synthetase of Methanococcus jannaschii or a Methanogenic archaeon, such as a Methanococcus jannaschii tyrosyl-tRNA synthetase or a Methanogenic archaeon pyrrolysyl-tRNA synthetase;

[0047] Preferably, the O-RS is a mutant of tyrosyl-tRNA synthetase (TyrRS); preferably, the TyrRS is derived from an archaea, such as Methanococcus jannaschii; preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO: 15, and retains the ability of SEQ ID NO: 15 to aminoacylate the orthogonal tRNA (O-tRNA) with the non-natural amino acid;

[0048] Preferably, the O-RS is a mutant of pyrrolysyl-tRNA synthetase (PylRS); preferably, the PylRS is derived from an archaeon, such as a methanogenic archaeon, Methanosarcina barkeri, Methanosarcina Mazei, or Methanomethylophilus Alvus; preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13, or has a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with SEQ ID NO: 12 or SEQ ID NO: 13. Compared to the amino acid sequence shown in NO:13, one or more amino acids are substituted, deleted or added (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are substituted, deleted or added), and the ability of SEQ ID NO:12 or SEQ ID NO:13 to aminoacylate an orthogonal tRNA (O-tRNA) with the non-natural amino acid is retained;

[0049] Preferably, the O-RS carries the non-natural amino acid described in the first aspect;

[0050] Preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, and is capable of aminoacylating an orthogonal tRNA (e.g., an O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 16 or encoded thereby) with a non-natural amino acid selected from the following:

[0051] Preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or a conservative variant thereof, and is capable of aminoacylating an orthogonal tRNA (e.g., an O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 14 or encoded thereby) with a non-natural amino acid selected from the following:

[0052] Preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 13 or a conservative variant thereof, and is capable of aminoacylating an orthogonal tRNA (e.g., an O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 14 or encoded thereby) with a non-natural amino acid selected from the following:

[0053] In a third aspect, the present invention provides an orthogonal-tRNA (O-tRNA), which is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell, and which is aminoacylated by the non-natural amino acid described in the first aspect;

[0054] Preferably, the O-tRNA is an amber suppressor tRNA;

[0055] Preferably, the O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, Methanogenic archaeon, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; Preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14;

[0056] Preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 16, and is aminoacylated with an unnatural amino acid selected from the group consisting of:

[0057] Preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 14, and is aminoacylated by an unnatural amino acid selected from the following:

[0058] In a fourth aspect, the present invention provides an isolated nucleic acid comprising a polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) of the second aspect.

[0059] In a fifth aspect, the present invention provides a vector comprising the isolated nucleic acid of the fourth aspect;

[0060] Preferably, the vector is a cloning vector or an expression vector;

[0061] Preferably, the vector further comprises a polynucleotide encoding an orthogonal tRNA (O-tRNA);

[0062] Preferably, the O-RS is capable of aminoacylating the O-tRNA with the non-natural amino acid described in the first aspect;

[0063] Preferably, the O-tRNA is an amber suppressor tRNA;

[0064] Preferably, the O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, Methanogenic archaeon, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; Preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14;

[0065] Preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, and the orthogonal tRNA comprises a polynucleotide sequence as shown in SEQ ID NO: 16 or an O-tRNA encoded thereby;

[0066] Preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or a conservative variant thereof, and the orthogonal tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 14;

[0067] Preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 13 or a conservative variant thereof, and the orthogonal tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 14.

[0068] In a sixth aspect, the present invention provides a host cell comprising the isolated nucleic acid of the fourth aspect or the vector of the fifth aspect;

[0069] Preferably, the host cell is a prokaryotic cell (such as E. coli) or a eukaryotic cell (such as a yeast cell, a fungal cell, a plant cell, an animal cell, such as a mammalian cell, such as a human cell);

[0070] Preferably, the host cell further contains a polynucleotide encoding an orthogonal tRNA (O-tRNA);

[0071] Preferably, the O-RS is capable of aminoacylating the O-tRNA with the non-natural amino acid described in the first aspect;

[0072] Preferably, the O-tRNA is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell; preferably, the O-tRNA is an amber suppressor tRNA; preferably, the O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, a methanogenic archaeon, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14;

[0073] Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

[0074] In a seventh aspect, the present invention provides a method for preparing the orthogonal aminoacyl-tRNA synthetase (O-RS) of the second aspect, comprising culturing the host cell of the sixth aspect under conditions allowing expression of the O-RS; and recovering the O-RS expressed by the host cell.

[0075] In an eighth aspect, the present invention provides a translation system, comprising:

[0076] (a) a first non-natural amino acid selected from the non-natural amino acids described in the first aspect;

[0077] (b) a first orthogonal aminoacyl-tRNA synthetase (O-RS); and

[0078] (c) a first orthogonal tRNA (O-tRNA);

[0079] wherein the first O-RS is capable of aminoacylating the first O-tRNA with the first unnatural amino acid;

[0080] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0081] Preferably, the first O-tRNA is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell; preferably, the O-tRNA is an amber suppressor tRNA; preferably, the O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, a methanogenic archaeon, Methanosarcina barkii, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14;

[0082] Preferably, the translation system further comprises a nucleic acid encoding a protein of interest, wherein the nucleic acid contains at least one selector codon, wherein the selector codon is recognized by the first O-tRNA;

[0083] Preferably, the first unnatural amino acid is selected from And, the O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof; and optionally, the O-tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 16;

[0084] Preferably, the first unnatural amino acid is selected from: And, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or a conservative variant thereof; and optionally, the O-tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 14;

[0085] Preferably, the first unnatural amino acid is selected from: And, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 13 or a conservative variant thereof; and optionally, the O-tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 14;

[0086] Preferably, the translation system further comprises a second O-RS and a second O-tRNA, wherein the second O-RS preferentially aminoacylates the second O-tRNA with a second unnatural amino acid different from the first unnatural amino acid, and wherein the second O-tRNA preferentially recognizes a selector codon different from the selector codon recognized by the first O-tRNA;

[0087] Preferably, the translation system comprises a host cell comprising the first unnatural amino acid, the first O-RS and the first O-tRNA; preferably, the host cell is a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., a yeast cell, a fungal cell, a plant cell, an animal cell, such as a mammalian cell, such as a human cell);

[0088] Preferably, the host cell contains a polynucleotide encoding the first O-RS, a polynucleotide encoding the first O-tRNA, or a combination thereof.

[0089] In a ninth aspect, the present invention provides a cell comprising the translation system described in the eighth aspect;

[0090] Preferably, the first O-RS is expressed from a nucleic acid integrated into the genome or expressed from a nucleic acid free from the genome, and the nucleic acid comprises a polynucleotide encoding the first O-RS;

[0091] Preferably, the first O-tRNA is expressed from a nucleic acid integrated into the genome or expressed from a nucleic acid free from the genome, and the nucleic acid comprises a polynucleotide encoding the first O-tRNA or a polynucleotide comprising the first O-tRNA;

[0092] Preferably, the cell further comprises a polynucleotide encoding a polypeptide of interest, wherein the polynucleotide comprises a selector codon recognized by the first O-tRNA; preferably, the polynucleotide is integrated into the genome or is free in the genome;

[0093] Preferably, the polypeptide of interest is selected from: GFP protein, ThRS protein, FNIII protein, Z protein, α or β tubulin, AAV virus capsid protein, VHH nanobody protein, Nanofitin protein, cytochrome b562 protein;

[0094] Preferably, the polypeptide of interest further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0095] In a tenth aspect, the present invention provides a kit comprising

[0096] (i): a first unnatural amino acid selected from the unnatural amino acids described in the first aspect; or

[0097] (ii): a first orthogonal aminoacyl-tRNA synthetase (O-RS), or a first polynucleotide encoding the first O-RS; or,

[0098] (iii) : a combination of (i) and (ii);

[0099] Preferably, the kit further comprises, (iv): a first orthogonal tRNA (O-tRNA), or, a second polynucleotide encoding the first O-tRNA;

[0100] Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the first unnatural amino acid;

[0101] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0102] Preferably, the first O-tRNA is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell; preferably, the O-tRNA is an amber suppressor tRNA; preferably, the O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, a methanogenic archaeon, Methanosarcina barkii, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the O-tRNA comprises or is encoded by the polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14;

[0103] Preferably, the kit further comprises the first polynucleotide and the second polynucleotide; preferably, the first polynucleotide and the second polynucleotide are contained together in the same nucleic acid molecule (e.g., a vector, such as an expression vector), or, are contained in separate nucleic acid molecules (e.g., the first and second vectors, such as the first and second expression vectors);

[0104] Preferably, the kit further comprises a nucleic acid vector for carrying or containing a nucleic acid encoding a protein of interest; preferably, the nucleic acid encoding a protein of interest contains at least one selector codon, wherein the selector codon is recognized by the first O-tRNA;

[0105] Preferably, the first unnatural amino acid is selected from: And, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof; and optionally, the O-tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 16;

[0106] Preferably, the first unnatural amino acid is selected from: And, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or a conservative variant thereof; and optionally, the O-tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 14;

[0107] Preferably, the first unnatural amino acid is selected from: And, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 13 or a conservative variant thereof; and optionally, the O-tRNA comprises or is encoded by a polynucleotide sequence as shown in SEQ ID NO: 14;

[0108] Preferably, the kit further comprises a second unnatural amino acid; further preferably, the kit further comprises:

[0109] (a): a second orthogonal aminoacyl-tRNA synthetase (O-RS), or a first polynucleotide encoding the second O-RS; or,

[0110] (b): a second orthogonal tRNA (O-tRNA), or a second polynucleotide encoding the second O-tRNA; or,

[0111] (c): a combination of (a) and (b);

[0112] Preferably, the second O-RS preferentially aminoacylates the second O-tRNA with a second unnatural amino acid different from the first unnatural amino acid; preferably, the second O-tRNA recognizes a selector codon different from the selector codon recognized by the first O-tRNA;

[0113] Preferably, the kit further comprises a reagent for delivering the nucleic acid molecule into a host cell; a culture medium for culturing the host cell; an enzyme (e.g., a restriction endonuclease) for manipulating the nucleic acid molecule; or any combination thereof;

[0114] Preferably, the kit is used to express a non-natural polypeptide containing the first non-natural amino acid outside a cell or in a host cell;

[0115] Preferably, the host cell is a prokaryotic cell (eg, E. coli) or a eukaryotic cell (eg, a yeast cell, a fungal cell, a plant cell, an animal cell, such as a mammalian cell, such as a human cell).

[0116] In an eleventh aspect, the present invention provides a method for modifying a polypeptide, comprising:

[0117] A non-natural amino acid, preferably the non-natural amino acid described in the first aspect, is introduced into the desired position of the polypeptide, or the amino acid at the desired position is replaced with a non-natural amino acid, preferably the non-natural amino acid described in the first aspect; optionally, the amino acid residues at positions adjacent to the desired position are mutated (e.g., point mutations), preferably, the mutations can enhance or weaken the restriction on the intramolecular rotation of the non-natural amino acid.

[0118] In a twelfth aspect, the present invention provides a non-natural polypeptide, wherein the non-natural polypeptide comprises the non-natural amino acid described in the first aspect above.

[0119] Specifically, the non-natural polypeptide comprises a non-natural amino acid of formula I:

[0120]

[0121] Among them, X 1a , X 2a , X 3a and X 4a Independently selected from CH and N; preferably X 1a , X 2a , X 3a and X 4a 3 of them are CH; more preferably all 4 of them are CH;

[0122] R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl, preferably C1-C3 alkyl; preferably R1 is selected from: -NH2, -NHRa and-NR b R c , where R a , R b , R c Independently selected from C1-C6 alkyl, preferably C1-C3 alkyl;

[0123] R2 is selected from: cyano, sulfonic acid, nitro and trihalomethyl (e.g. trifluoromethyl, trichloromethyl); preferably R2 is selected from: cyano, sulfonic acid and trichloromethyl; more preferably R2 is selected from: cyano;

[0124] X 1b Selected from: N and CR3;

[0125] R3 is selected from: H, cyano, sulfonic acid, nitro and trihalomethyl (e.g. trifluoromethyl, trichloromethyl); preferably R3 is selected from: H, cyano, sulfonic acid and trichloromethyl; more preferably R3 is selected from: H and cyano;

[0126] X 2b Selected from CR 42 and N, preferably CR 42 ,

[0127] X 3b Selected from CR 43 and N, preferably CR 43 ,

[0128] X 4b Selected from CR 44 and N, preferably CR 44 ,and

[0129] X 5b Selected from CR 45 and N, preferably CR 45 ;

[0130] R 42 , R 43 , R 44 , R 45 Each independently selected from: H and -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3, and R 42 , R 43 , R 44 , R 45 At least one of is not H; preferably R 42 , R 43 , R 44 , R 45 1 of the following (preferably R 42 or R 43 ) is selected from: -(CH2) nCH(NH2)(COOH), n is an integer selected from 1-3, and the rest are H.

[0131] In some embodiments, for the above non-natural polypeptide, wherein the non-natural amino acid has a structure of Formula IA or Formula IB:

[0132]

[0133] Wherein, R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl;

[0134] Preferably, R1 is selected from: -NH2, -NHR a and-NR b R, where R a , R b , R c Independently selected from C1-C3 alkyl, preferably methyl, ethyl, propyl;

[0135] More preferably, R1 is selected from: -NH2 and -NR a R b , where R a , R b independently selected from methyl;

[0136] R3 is independently selected from: H, cyano, sulfonic acid, nitro and trihalomethyl (eg, trifluoromethyl and trichloromethyl);

[0137] Preferably, R3 is selected from: cyano, sulfonic acid and trichloromethyl;

[0138] More preferably, R3 is selected from cyano;

[0139] R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3;

[0140] Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH);

[0141] Preferably, Formula IA is selected from:

[0142]

[0143] R1, R3, and R4 in the above formula I-A1 and formula I-A2 are defined as the corresponding variables in the above formula IA;

[0144] or,

[0145]

[0146] Wherein, R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl;

[0147] Preferably, R1 is selected from: -NH2, -NHR a and-NR b R, where R a , R b , R c Independently selected from C1-C3 alkyl, preferably methyl, ethyl and propyl;

[0148] More preferably, R1 is selected from: -NH2, -NR a R b , where R a , R b independently selected from methyl;

[0149] R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3;

[0150] Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH);

[0151] Preferably, Formula IB is selected from:

[0152]

[0153] R1 and R4 in the above formula I-B1 and formula I-B2 are defined as the corresponding variables in the above formula IB.

[0154] In some embodiments, for the above non-natural polypeptide, wherein the non-natural amino acid is selected from the following:

[0155]

[0156] In some embodiments, for the above-mentioned non-natural polypeptide, the intramolecular rotation of the non-natural amino acid in the spatial structure of the non-natural polypeptide is restricted, or the non-natural amino acid can freely perform intramolecular rotation in the spatial structure of the non-natural polypeptide.

[0157] In some embodiments, for the above-mentioned non-natural polypeptide, the intramolecular rotation state of the non-natural amino acid in the spatial structure of the non-natural polypeptide can change, for example, from free intramolecular rotation to restricted intramolecular rotation, or from restricted intramolecular rotation to free intramolecular rotation;

[0158] Preferably, the change in the intramolecular rotation state is caused by a change in the spatial conformation of the non-natural polypeptide, or by the binding of the non-natural polypeptide to a target molecular entity.

[0159] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide can specifically bind to a target molecular entity to form a complex;

[0160] Preferably, the non-natural amino acid has a first intramolecular rotation state in the spatial structure of the non-natural polypeptide not bound to the target molecular entity, and has a second intramolecular rotation state in the spatial structure of the complex, and the first intramolecular rotation state is different from the second intramolecular rotation state;

[0161] Preferably, the first intramolecular rotation state is free intramolecular rotation, and the second intramolecular rotation state is restricted intramolecular rotation; or, the first intramolecular rotation state is restricted intramolecular rotation, and the second intramolecular rotation state is free intramolecular rotation;

[0162] Preferably, the target molecular entity is another polypeptide, or a substrate of the non-natural polypeptide, or the non-natural polypeptide itself.

[0163] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the target molecular entity is another polypeptide, and the contact or binding between the non-natural polypeptide and the other polypeptide causes the intramolecular rotation state of the non-natural amino acid to change (for example, from free intramolecular rotation to restricted intramolecular rotation);

[0164] Preferably, the non-natural amino acid is located at or near the interface where the non-natural polypeptide and the other polypeptide contact or bind to each other;

[0165] Preferably, the non-natural polypeptide and the other polypeptide form a molecular pair that specifically binds to each other or interacts with each other;

[0166] Preferably, the non-natural polypeptide and the additional polypeptide form an antigen-antibody molecule pair that specifically binds to each other; for example, the non-natural polypeptide comprises or is an antibody, and the additional polypeptide comprises or is an antigen that the antibody specifically binds to; or, the non-natural polypeptide comprises or is an antigen, and the additional polypeptide comprises or is an antibody that specifically binds to the antigen;

[0167] Preferably, the non-natural polypeptide and the other polypeptide constitute a protein Z-Affibody molecule pair that specifically binds to each other; for example, the non-natural polypeptide comprises or is protein Z containing the non-natural amino acid, and the other polypeptide comprises or is Affibody; or, the non-natural polypeptide comprises or is Affibody containing the non-natural amino acid, and the other polypeptide comprises or is protein Z;

[0168] Preferably, the non-natural polypeptide and the additional polypeptide form a FKBP-FRB molecular pair that specifically binds to each other; for example, the non-natural polypeptide comprises or is a FRB containing the non-natural amino acid, and the additional polypeptide comprises or is FKBP; or, the non-natural polypeptide comprises or is a FKBP containing the non-natural amino acid, and the additional polypeptide comprises or is FRB;

[0169] Preferably, the non-natural polypeptide and the other polypeptide constitute a spike protein and an ACE2 receptor molecule pair that specifically bind to each other; for example, the non-natural polypeptide comprises or is a spike protein containing the non-natural amino acid, and the other polypeptide comprises or is an ACE2 receptor; or, the non-natural polypeptide comprises or is an ACE2 receptor containing the non-natural amino acid, and the other polypeptide comprises or is a spike protein; preferably, the spike protein is derived from a new coronavirus;

[0170] Preferably, the non-natural polypeptide and the additional polypeptide form a pair of α-tubulin and β-tubulin molecules that specifically bind to each other; for example, the non-natural polypeptide comprises or is α-tubulin containing the non-natural amino acid, and the additional polypeptide comprises or is β-tubulin; or, the non-natural polypeptide comprises or is β-tubulin containing the non-natural amino acid, and the additional polypeptide comprises or is α-tubulin.

[0171] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is a protein Z containing the non-natural amino acid;

[0172] Preferably, the protein Z containing the non-natural amino acid can interact with Affibody and change the intramolecular rotation state of the non-natural amino acid (for example, from free intramolecular rotation to restricted intramolecular rotation); preferably, the non-natural amino acid is located at the interface where protein Z interacts with Affibody or at a position adjacent thereto, for example, at the following sites of protein Z: sites corresponding to sites 10, 13, 14, 17, 28, 31 or 35 of SEQ ID NO:2;

[0173] Preferably, the unnatural amino acid is located at the following position of protein Z: the position corresponding to position 13 or 17 of SEQ ID NO: 2; preferably, the unnatural amino acid is pAPCF;

[0174] Preferably, the protein Z containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 13 or 17 of SEQ ID NO:2;

[0175] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0176] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is a FRB protein containing the non-natural amino acid;

[0177] Preferably, the FRB protein containing the non-natural amino acid can interact with FKBP in the presence of rapamycin, and changes the intramolecular rotation state of the non-natural amino acid (for example, from free intramolecular rotation to restricted intramolecular rotation); preferably, the non-natural amino acid is located at the interface where the FRB protein interacts with FKBP or at a position adjacent thereto, for example, at the following sites of the FRB protein: sites corresponding to sites 76, 80, 84, 87 or 91 of SEQ ID NO: 18;

[0178] Preferably, the unnatural amino acid is located at the following site of the FRB protein: a site corresponding to site 84 or 87 of SEQ ID NO: 18; preferably, the unnatural amino acid is pAPCF or PAPd;

[0179] Preferably, the FRB protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO: 18 or SEQ ID NO: 27 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 27, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 27; preferably, the non-natural amino acid (e.g., pAPCF or PAPd) is located at a position corresponding to position 84 or 87 of SEQ ID NO: 18;

[0180] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0181] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the target molecular entity is the non-natural polypeptide itself, and the binding or aggregation of the non-natural polypeptides causes the intramolecular rotation state of the non-natural amino acid to change (for example, from free intramolecular rotation to restricted intramolecular rotation);

[0182] Preferably, the non-natural amino acid is located in an exposed region of the non-natural polypeptide or in a proximal position thereof;

[0183] Preferably, the non-natural polypeptide comprises or is: superoxide dismutase 1 (SOD1) containing the non-natural amino acid, Huntingtin protein (see, e.g., GenBank: AAB38240.1) containing the non-natural amino acid, Nucleophosmin (see, e.g., GenBank: AAA36380.1) containing the non-natural amino acid, or Fibrillarin (see, e.g., GenBank: AAA52453.1) containing the non-natural amino acid.

[0184] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is superoxide dismutase 1 (SOD1) containing the non-natural amino acid;

[0185] Preferably, the SOD1 containing the non-natural amino acid can change the intramolecular rotation state of the non-natural amino acid when it aggregates (for example, from free intramolecular rotation to restricted intramolecular rotation);

[0186] Preferably, the unnatural amino acid is located in an exposed region of SOD1, for example, at the following sites of SOD1: a site corresponding to site 25, 40, 47 or 67 of SEQ ID NO:7;

[0187] Preferably, the non-natural amino acid is located at the following position of SOD1: the position corresponding to position 25 or 40 of SEQ ID NO:7; preferably, the non-natural amino acid is pAPCF; preferably, the SOD1 containing the non-natural amino acid further contains a mutation selected from the following: the amino acid residue at the position corresponding to position 4 of SEQ ID NO:7 is replaced by V, the amino acid residue at the position corresponding to position 31 of SEQ ID NO:7 is replaced by A, or a combination thereof;

[0188] Preferably, the SOD1 protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:11 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:11, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:11; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 25 or 40 of SEQ ID NO:7;

[0189] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0190] In embodiment A of the twelfth aspect, for the above-mentioned non-natural polypeptide, wherein the target molecular entity is a substrate of the non-natural polypeptide, and the contact or binding between the non-natural polypeptide and the substrate causes the intramolecular rotation state of the non-natural amino acid to change (for example, from free intramolecular rotation to restricted intramolecular rotation; or from restricted intramolecular rotation to free intramolecular rotation);

[0191] Preferably, the non-natural amino acid is located in the following region of the non-natural polypeptide or in a vicinity thereof, wherein the region has a different spatial conformation in the non-natural polypeptide when bound or not bound to the substrate;

[0192] Preferably, the non-natural polypeptide and the substrate form a molecular pair that specifically binds to each other or a molecular pair that interacts with each other;

[0193] Preferably, the non-natural polypeptide and the substrate form a glucose-binding protein-glucose molecule pair that specifically interacts with each other; for example, the non-natural polypeptide comprises or is a glucose-binding protein, and the substrate comprises or is glucose;

[0194] Preferably, the non-natural polypeptide and the substrate form a glutamine binding protein-glutamine molecule pair that interacts specifically with each other; for example, the non-natural polypeptide comprises or is a glutamine binding protein, and the substrate comprises or is glutamine;

[0195] Preferably, the non-natural polypeptide and the substrate form a G protein domain-GTP / GDP molecule pair that interacts specifically with each other; for example, the non-natural polypeptide comprises or is a G protein domain, and the substrate comprises or is GTP or GDP;

[0196] Preferably, the non-natural polypeptide and the substrate form a calmodulin-Ca 2+ Molecular pair; for example, the non-natural polypeptide comprises or is calmodulin, and the substrate comprises or is Ca 2+ ;

[0197] Preferably, the non-natural polypeptide and the substrate form a mineralocorticoid receptor-aldosterone molecular pair that interacts specifically with each other; for example, the non-natural polypeptide comprises or is a mineralocorticoid receptor, and the substrate comprises or is aldosterone.

[0198] In embodiment B of the twelfth aspect, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is a glucose binding protein containing the non-natural amino acid;

[0199] Preferably, the glucose-binding protein containing the non-natural amino acid is capable of undergoing a conformational change after binding to a glucose molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation); preferably, the non-natural amino acid is located in the following region of the glucose-binding protein or its adjacent position, and the region has different spatial conformations in the glucose-binding protein when it is bound or not bound to glucose; for example, the non-natural amino acid is located at the following sites of the glucose-binding protein: sites corresponding to sites 19, 149, 182, 187, 213 or 238 of SEQ ID NO:31;

[0200] Preferably, the non-natural amino acid is located at the following position of the glucose binding protein: the position corresponding to position 149 or 238 of SEQ ID NO: 31; preferably, the non-natural amino acid is pAPCF;

[0201] Preferably, the glucose binding protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:34 or SEQ ID NO:35 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:34 or SEQ ID NO:35, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO:34 or SEQ ID NO:35; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 149 or 238 of SEQ ID NO:31;

[0202] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0203] In embodiment C of the twelfth aspect, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is a glutamine binding protein containing the non-natural amino acid;

[0204] Preferably, the glutamine binding protein containing the non-natural amino acid is capable of undergoing a conformational change after binding to a glutamine molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation); preferably, the non-natural amino acid is located in the following region of the glutamine binding protein or its adjacent position, and the region has different spatial conformations in the glutamine binding protein when it is bound or not bound to glutamine; for example, the non-natural amino acid is located at the following sites of the glutamine binding protein: sites corresponding to sites 51, 74, 117, 118, 138 or 159 of SEQ ID NO:36;

[0205] Preferably, the non-natural amino acid is located at the following position of the glutamine binding protein: a position corresponding to position 51, 117 or 118 of SEQ ID NO: 36; preferably, the non-natural amino acid is pAPCF;

[0206] Preferably, the glutamine binding protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38; preferably, the non-natural amino acid (e.g., pAPCF) is located at the position of SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38. The site corresponding to position 51, 117 or 118 of IDNO:36;

[0207] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0208] In embodiment D of the twelfth aspect, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is a G protein domain containing the non-natural amino acid;

[0209] Preferably, the G protein domain containing the non-natural amino acid can undergo a conformational change after binding to a GTP or GDP molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation); preferably, the non-natural amino acid is located in the following region of the G protein domain or its adjacent position, and the region has different spatial conformations in the G protein domain when bound or not bound to GTP or GDP; for example, the non-natural amino acid is located at the following sites of the G protein domain: sites corresponding to sites 33, 36, 145, 146, 147 or 148 of SEQ ID NO:39;

[0210] Preferably, the unnatural amino acid is located at the following site of the G protein domain: the site corresponding to site 33 of SEQ ID NO: 39; preferably, the unnatural amino acid is PAPd;

[0211] Preferably, the G protein domain containing non-natural amino acids comprises the amino acid sequence as shown in SEQ ID NO:39 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:39, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:39; preferably, the non-natural amino acid (e.g., PAPd) is located at a position corresponding to position 33 of SEQ ID NO:39;

[0212] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

[0213] In embodiment E of the twelfth aspect, for the above-mentioned non-natural polypeptide, wherein the non-natural polypeptide comprises or is calmodulin containing the non-natural amino acid;

[0214] Preferably, the calmodulin containing unnatural amino acids is capable of binding to Ca 2+ The non-natural amino acid undergoes a conformational change after binding to the calmodulin molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation, or from free intramolecular rotation to restricted intramolecular rotation); preferably, the non-natural amino acid is located in the following region of calmodulin or its adjacent position, the region is bound or not bound to Calmodulin 2+In the case of the non-natural amino acid, the non-natural amino acid has a different spatial conformation in the calmodulin; for example, the non-natural amino acid is located at the following sites of calmodulin: sites corresponding to sites 19, 68, 72, 88, 91, 92, 108 or 141 of SEQ ID NO:40;

[0215] Preferably, the unnatural amino acid is located at the following position of calmodulin: a position corresponding to position 19, 68, 92 or 141 of SEQ ID NO: 40; preferably, the unnatural amino acid is PAPd;

[0216] Preferably, the calmodulin containing unnatural amino acids comprises an amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45; preferably, the unnatural amino acid (e.g., PAPd) is located at the position of SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45. The site corresponding to site 19, 68, 92 or 141 of ID NO:40;

[0217] Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, an M13 polypeptide or any combination thereof.

[0218] In a thirteenth aspect, the present invention provides an isolated nucleic acid comprising a polynucleotide encoding the non-natural polypeptide of the twelfth aspect, wherein the non-natural amino acid is encoded by a selector codon (eg, a stop codon, eg, an amber codon).

[0219] In a fourteenth aspect, the present invention provides a vector comprising the isolated nucleic acid of the thirteenth aspect;

[0220] Preferably, the vector is a cloning vector or an expression vector.

[0221] In a fifteenth aspect, the present invention provides a host cell comprising the isolated nucleic acid of the thirteenth aspect or the vector of the fourteenth aspect;

[0222] Preferably, the host cell is a prokaryotic cell (such as E. coli) or a eukaryotic cell (such as a yeast cell, a fungal cell, a plant cell, an animal cell, such as a mammalian cell, such as a human cell);

[0223] Preferably, the host cell further contains a polynucleotide encoding a first orthogonal aminoacyl-tRNA synthetase (O-RS), a polynucleotide encoding a first orthogonal tRNA (O-tRNA), or a combination thereof;

[0224] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0225] Preferably, the first orthogonal tRNA (O-tRNA) is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell;

[0226] Preferably, said first orthogonal-tRNA is capable of being aminoacylated by said first O-RS with said unnatural amino acid;

[0227] Preferably, the first O-tRNA is an amber suppressor tRNA;

[0228] Preferably, the first O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, Methanogenic archaeon, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof or is encoded by SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof;

[0229] Preferably, the first O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or a conservative variant thereof or is encoded by SEQ ID NO: 16 or a conservative variant thereof;

[0230] Preferably, the first O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 14 or a conservative variant thereof or is encoded by SEQ ID NO: 14 or a conservative variant thereof;

[0231] Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the unnatural amino acid;

[0232] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is capable of recognizing the unnatural amino acid described in the first aspect and aminoacylates the first orthogonal tRNA (O-tRNA) with the unnatural amino acid;

[0233] Preferably, the first O-RS is derived from an aminoacyl-tRNA synthetase of Methanococcus jannaschii or a Methanogenic archaeon, such as a Methanococcus jannaschii tyrosyl-tRNA synthetase or a Methanogenic archaeon pyrrolysyl-tRNA synthetase;

[0234] Preferably, the first O-RS is a mutant of a tyrosyl-tRNA synthetase (TyrRS); preferably, the TyrRS is derived from an archaea, such as Methanococcus jannaschii; preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO: 15, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO: 15, and retains the ability of SEQ ID NO: 15 to aminoacylate the first orthogonal tRNA (O-tRNA) with the non-natural amino acid; preferably, the orthogonal tRNA comprises SEQ ID NO: The polynucleotide sequence shown in SEQ ID NO: 16 or its conservative variants or the O-tRNA encoded by SEQ ID NO: 16 or its conservative variants; further preferably, the host cell is a prokaryotic cell, such as Escherichia coli;

[0235] Preferably, the first O-RS is a mutant of pyrrolysyl-tRNA synthetase (PylRS); preferably, the PylRS is derived from an archaeon, such as a methanogenic archaeon, Methanosarcina barkeri, Methanosarcina Mazei, or Methanomethylophilus Alvus; preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13, or has a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with SEQ ID NO: 12 or SEQ ID NO: 13. NO:13 has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions), and retains the ability of SEQ ID NO:12 or SEQ ID NO:13 to aminoacylate the first orthogonal tRNA (O-tRNA) with the non-natural amino acid; preferably, the orthogonal tRNA comprises a polynucleotide sequence shown in SEQ ID NO:14 or a conservative variant thereof or an O-tRNA encoded by SEQ ID NO:14 or a conservative variant thereof; further preferably, the host cell is a prokaryotic cell or a eukaryotic cell;

[0236] Preferably, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, and is capable of aminoacylating the first orthogonal tRNA (e.g., a first O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 16 or a conservative variant thereof or encoded by SEQ ID NO: 16 or a conservative variant thereof) with an unnatural amino acid selected from the following:

[0237] Preferably, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or a conservative variant thereof, and is capable of aminoacylating the first orthogonal tRNA (e.g., a first O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 14 or a conservative variant thereof or encoded by SEQ ID NO: 14 or a conservative variant thereof) with a non-natural amino acid selected from the following:

[0238] Preferably, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 13 or a conservative variant thereof, and is capable of aminoacylating the first orthogonal tRNA (e.g., a first O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 14 or a conservative variant thereof or encoded by SEQ ID NO: 14 or a conservative variant thereof) with a non-natural amino acid selected from the following:

[0239] Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

[0240] In a sixteenth aspect, the present invention provides a method for preparing the non-natural polypeptide of the twelfth aspect, comprising culturing the host cell of the fifteenth aspect in a culture medium containing the non-natural amino acid under conditions allowing expression of the non-natural polypeptide; and recovering the non-natural polypeptide expressed by the host cell;

[0241] Preferably, the host cell further expresses a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), or a combination thereof;

[0242] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0243] Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the unnatural amino acid;

[0244] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the first O-RS defined in the fifteenth aspect;

[0245] Preferably, the first O-tRNA is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell; preferably, the first O-tRNA is an amber suppressor tRNA; preferably, the first O-tRNA is derived from an archaea, such as Methanococcus jannaschii, a methanogenic archaeon, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the first O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof or is encoded by SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof.

[0246] In a seventeenth aspect, the present invention provides a use of the non-natural polypeptide of the twelfth aspect for detecting protein interactions or abnormal protein aggregation or for detecting the presence or amount of a target molecular entity;

[0247] Preferably, the target molecular entity is selected from glucose, glutamine, GTP, GDP or Ca 2+ .

[0248] In an eighteenth aspect, the present invention provides a use of the non-natural polypeptide according to embodiment A of the twelfth aspect for detecting the presence or amount of a target molecular entity; wherein the target molecular entity is a substrate of the non-natural polypeptide;

[0249] Preferably, the target molecular entity is selected from glucose, glutamine, GTP, GDP or Ca 2+ .

[0250] In the nineteenth aspect, the present invention provides a method for detecting the presence or amount of a target molecular entity in a sample to be tested, comprising contacting the non-natural polypeptide described in Implementation A of the twelfth aspect with the sample to be tested, and detecting changes in the fluorescent signal emitted by the non-natural amino acid; wherein the target molecular entity is a substrate of the non-natural polypeptide.

[0251] In the twentieth aspect, the present invention provides use of the non-natural polypeptide described in embodiment B of the twelfth aspect for detecting the presence or amount of glucose.

[0252] In the twenty-first aspect, the present invention provides a method for detecting the presence or amount of glucose in a sample to be tested, comprising contacting the non-natural polypeptide described in embodiment B of the twelfth aspect with the sample to be tested, and detecting changes in the fluorescent signal emitted by the non-natural amino acid.

[0253] In the twenty-second aspect, the present invention provides use of the non-natural polypeptide described in embodiment C of the twelfth aspect for detecting the presence or amount of glutamine.

[0254] In the twenty-third aspect, the present invention provides a method for detecting the presence or amount of glutamine in a sample to be tested, comprising contacting the non-natural polypeptide described in embodiment C of the twelfth aspect with the sample to be tested, and detecting the change in the fluorescent signal emitted by the non-natural amino acid.

[0255] In the twenty-fourth aspect, the present invention provides the use of the non-natural polypeptide described in embodiment D of the twelfth aspect for detecting the presence or amount of GTP or GDP.

[0256] In the twenty-fifth aspect, the present invention provides a method for detecting the presence or amount of GTP or GDP in a sample to be tested, comprising contacting the non-natural polypeptide described in embodiment D of the twelfth aspect with the sample to be tested, and detecting changes in the fluorescent signal emitted by the non-natural amino acid.

[0257] In the twenty-sixth aspect, the present invention provides the non-natural polypeptide according to embodiment E of the twelfth aspect for detecting Ca 2+ The existence or quantity of use.

[0258] In the twenty-seventh aspect, the present invention provides a method for detecting Ca 2+ A method for detecting the presence or amount of a non-natural amino acid in a sample to be tested, comprising contacting the non-natural polypeptide according to embodiment E of the twelfth aspect with the sample to be tested, and detecting changes in the fluorescent signal emitted by the non-natural amino acid.

[0259] Definition of terms

[0260] In this application, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the biochemistry, molecular biology, cytology, and immunology laboratory operation procedures used herein are conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0261] When the terms "for example," "such as," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0262] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0263] The terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments described in the present invention can be implemented in other sequences than those described or illustrated in the present invention.

[0264] As used herein, the term "natural amino acid" means the basic amino acid unit that constitutes a living protein or polypeptide, which includes the following 20 amino acids: K (Lys), R (Arg), H (His), A (Ala), V (Val), L (Leu), I (Ile), G (Gly), S (Ser), T (Thr), P (Pro), F (Phe), W (Trp), Y (Tyr), C (Cys), M (Met), D (Asp), E (Glu), N (Asn), Q (Gln). All natural amino acids (except Gly) are L-amino acids. The writing of the twenty kinds of natural amino acids involved in this article follows conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, ES Golub and DR Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. And in the present application, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0265] As used herein, the term "non-natural amino acid (ncAA)" refers to any other amino acid, modified amino acid and / or amino acid analog that is not among the 20 natural amino acids. Non-natural amino acids can be L-amino acids or D-amino acids. For example, the present application discloses a class of fluorescent rotor-type non-natural amino acids, which include but are not limited to pAPCF, PAPd, mPAPF, PAFCN, mDMPF, and DMPd. The non-natural amino acids of the present application have unique properties. As fluorescent molecular rotors, they do not show fluorescence after being excited by excitation light in a state of free intramolecular rotation (e.g., in a free state), but show fluorescence after being excited by excitation light in a state of restricted intramolecular rotation (e.g., when subject to steric hindrance). Therefore, the non-natural amino acids of the present application are also referred to as fluorescent rotor-type amino acids (FRAA) or fluorescent rotor-type non-natural amino acids.

[0266] As used herein, the term "polypeptide" refers to any oligomer or polymer of any length, usually but not absolutely amino acid residues (natural or non-natural, or a combination thereof) linked together by covalent peptide bonds. The polypeptide can be of any origin, for example, a natural polypeptide, a polypeptide prepared by chemical synthesis or genetic engineering technology, a polypeptide expressed by a cell or translation system, or a polypeptide prepared in a cell-free synthesis manner. The characteristics of a polypeptide are determined by its amino acid sequence, for example, the amino acid sequence constitutes the primary structure of the polypeptide. In this article, the amino acid sequence of a polypeptide is not limited to the full-length sequence, but may also be a partial sequence or sequence fragment. In addition, this does not mean that the polypeptide is limited according to whether it has any specific biological activity. In this application, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably.

[0267] As used herein, the term "non-natural polypeptide" means a polypeptide comprising a non-natural amino acid in an amino acid sequence. "Comprising a non-natural amino acid" is also referred to as "comprising a non-natural amino acid residue", which has the same meaning and can be used interchangeably. Non-natural amino acids, such as the non-natural amino acids of the present application, can be incorporated into proteins or polypeptides by various means and methods (e.g., gene codon expansion technology). In an exemplary embodiment, a selector codon can be inserted at a specified position in a polynucleotide fragment encoding a protein or polypeptide, or the codon nucleotide at a specified position can be replaced with a selector codon, and then the obtained polynucleotide fragment containing a selector codon is introduced into a host cell (which is usually cultured in a culture medium containing the non-natural amino acid) expressing an orthogonal aminoacyl tRNA synthetase / t-RNA pair (the aminoacyl tRNA synthetase can preferentially aminoacylate a t-RNA that specifically recognizes a selector codon with a non-natural amino acid) and expressed, thereby obtaining a protein or polypeptide comprising a non-natural amino acid. In addition, a protein or polypeptide comprising a non-natural amino acid can also be prepared or produced using chemical synthesis techniques.

[0268] As used herein, the term "C1-C6 alkyl" refers to a group obtained by removing a hydrogen atom from a straight or branched saturated hydrocarbon group containing 1-6 (such as 1, 2, 3, 4, 5 or 6) carbon atoms, and specific examples thereof include, but are not limited to, methyl, ethyl, propyl, n-butyl, isobutyl, isopropyl, tert-butyl, n-pentyl, and n-hexyl. Similarly, the term "C1-C3 alkyl" refers to a group obtained by removing a hydrogen atom from a straight or branched saturated hydrocarbon group containing 1-3 (such as 1, 2 or 3) carbon atoms, and specific examples thereof include, but are not limited to, methyl, ethyl, propyl, and isopropyl.

[0269] As used herein, the term "halogenated" refers to a group or compound in which hydrogen is replaced by one or more halogen atoms, including full halogenation and partial halogenation. For example, trihalogenated methyl may include trifluoromethyl, trichloromethyl, etc.

[0270] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0271] As used herein, the term "intramolecular rotation" means that for the non-natural amino acids of the present invention, whether they are in a free state or introduced into a polypeptide, there are electron donor units, electron acceptor units and electron-rich spacer units in the molecular structure, wherein the electron-rich spacer unit is composed of a network of alternating single bonds and double bonds, which puts the electron donor unit and the electron acceptor unit in an electronic conjugated state, thereby promoting the movement of electrons between the electron donor unit and the electron acceptor unit; and the rotation of the alternating single bonds and double bonds in the electron-rich spacer unit can be carried out freely or subject to different degrees of restriction. Correspondingly, the fluorescence intensity of the non-natural amino acids of the present invention also varies with whether the intramolecular rotation of the electron donor unit-electron acceptor unit bond is restricted. For example, in the molecule The electron donor unit is The electron acceptor unit is The electron-rich spacer unit is The electron-rich spacer unit in the molecule pAPCF The rotation of the molecule can be free or restricted to varying degrees, which will have a direct impact on the degree of fluorescence activation of the molecule.

[0272] As used herein, the term "free intramolecular rotation" means a state (e.g., a free state) of the non-natural amino acid of the present invention in which the alternating single bonds and double bonds in the electron-rich spacer unit in the non-natural amino acid molecule can rotate freely and therefore, no fluorescence is exhibited.

[0273] As used herein, the term "restricted intramolecular rotation" means: a state (e.g., a sterically hindered state) of the non-natural amino acid of the present invention in which the rotation of alternating single bonds and double bonds in an electron-rich spacer unit in the non-natural amino acid molecule is restricted, thereby exhibiting fluorescence; and the degree of fluorescence exhibited is related to the degree to which the rotation of alternating single bonds and double bonds in the electron-rich spacer unit in the molecule is restricted.

[0274] It is easy to understand that "free intramolecular rotation" and "restricted intramolecular rotation" represent different states of non-natural amino acids and are relative. Generally, under the condition of receiving excitation light, when the non-natural amino acid cannot exhibit detectable fluorescence, the non-natural amino acid is considered to be in a free intramolecular rotation state; conversely, when the non-natural amino acid exhibits detectable fluorescence, the non-natural amino acid is considered to be in a restricted intramolecular rotation state. The intramolecular rotation state of non-natural amino acids can be affected by many factors, such as the viscosity of the solvent containing it, the steric hindrance of its adjacent positions, etc.

[0275] As used herein, the term "aminoacyl-tRNA synthetase (aaRS or RS)" is a class of enzymes that can specifically recognize the side chain of a specific amino acid and a specific tRNA, catalyze the binding of the specific amino acid to a specific tRNA, and cause an aminoacylation reaction, thereby covalently linking the two. Aminoacyl-tRNA synthetase is a key enzyme that enables the genetic information of mRNA to be accurately reflected in the amino acid sequence of the protein.

[0276] As used herein, the term "orthogonal" refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and / or an orthogonal aminoacyl tRNA synthetase (O-RS)) that functions with endogenous components of a cell or translation system at reduced efficiency or that is unable to function with endogenous components of the cell compared to the corresponding molecule endogenous to the cell or translation system. With respect to tRNA and aminoacyl-tRNA synthetase, the term "orthogonal" refers to an orthogonal tRNA that cannot function with an endogenous tRNA synthetase or functions with reduced efficiency compared to an endogenous tRNA that functions with an endogenous tRNA synthetase; or an orthogonal aminoacyl tRNA synthetase that does not function with an endogenous tRNA or functions with reduced efficiency compared to an endogenous tRNA synthetase that functions with an endogenous tRNA, the efficiency reduction being, for example, less than 20% efficiency, less than 10% efficiency, less than 5% efficiency, or less than 1% efficiency. An orthogonal molecule lacks a functional endogenous complementary molecule in a cell. For example, compared with the endogenous RS aminoacylation of endogenous tRNA, the efficiency of any endogenous RS aminoacylation of the orthogonal tRNA in the cell is reduced or even 0. In another example, compared with the endogenous RS aminoacylation of endogenous tRNA, the efficiency of any endogenous tRNA in the cell of interest is reduced or even 0. The second orthogonal molecule that can work with the first orthogonal molecule can be introduced into the cell. For example, the orthogonal tRNA / RS pairing includes, working together in the cell and compared with the control (e.g., corresponding tRNA / RS endogenous pairing), its efficiency is, for example, 45% efficiency, 50% efficiency, 60% efficiency, 70% efficiency, 75% efficiency, 80% efficiency, 90% efficiency, 95% efficiency or 99% efficiency, or higher introduced supplementary components, or active orthogonal pairing.

[0277] As used herein, orthogonal aminoacyl tRNA synthetase (O-RS) is an enzyme that preferentially aminoacylates an orthogonal tRNA (O-tRNA) with an amino acid in a cell or translation system of interest. The amino acid that O-RS is loaded onto O-tRNA can be any amino acid, whether natural, non-natural or artificial, and is not limited to non-natural amino acids as described herein. In some exemplary embodiments, the aminoacyl tRNA synthetase is optionally identical or homologous to a natural tyrosyl-tRNA synthetase, or identical or homologous to a natural pyrrolysyl-tRNA synthetase.

[0278] As used herein, the term "pyrrolysyl-tRNA synthetase" refers to an enzyme that catalyzes the activation of pyrrolysine and covalently binds to the 3' end of the corresponding tRNA molecule. Pyrrolysine (Pyl) is found in the methylamine methyltransferase of methanogens and is the 22nd amino acid known to participate in protein biosynthesis. Unlike natural amino acids, pyrrolysine (Pyl) is formed by the sense coding of the stop codon UAG. Correspondingly, methanogens also contain specific pyrrolysyl-tRNA synthetases (PylRS) and pyrrolysine tRNA (tRNA Pyl ), which has a special structure different from classical tRNA. Methanogens generate pyrrolysyl-tRNA through direct and indirect pathways Pyl (Pyl-tRNA Pyl ), it may control the UAG coding on mRNA to become a stop codon or pyrrolysine through a special structure on mRNA and other mechanisms that have not yet been discovered. Wild-type pyrrolysyl-tRNA synthetase (PylRS) can be obtained from, for example, Methanosarcina mazei, Methanosarcina barkeri, and Methanosarcina acetivorans, which are methanogenic archaea, but are not limited to these.

[0279] As used herein, the term "pyrrolysyl-tRNA synthetase mutant" refers to a mutant obtained by introducing various mutations (e.g., amino acid addition, deletion, substitution, or any combination thereof) based on wild-type pyrrolysyl-tRNA synthetase (PylRS). The types of lysine derivatives that can be activated by wild-type PylRS are limited, and usually only pyrrolysine can be activated. The aminoacyl-tRNA synthetases C-C9 PylRS and AC2 PylRS disclosed herein are a kind of "pyrrolysyl-tRNA synthetase mutant", which can be obtained by mutating wild-type PylRS derived from archaea (e.g., methanogenic archaea). Through the PylRS mutant disclosed herein, even for non-natural amino acids (e.g., various non-natural amino acids listed above) that are inactive when using wild-type PylRS, proteins can be efficiently introduced. In some exemplary embodiments, the C-C9 PylRS disclosed herein (e.g., RS as shown in SEQ ID NO: 12 or a conservative variant thereof) can efficiently introduce non-natural amino acids pAPCF, PAPd, PAFCN, and DMPd into proteins using corresponding tRNAs (e.g., tRNAs as shown in SEQ ID NO: 14 or a conservative variant thereof). In some exemplary embodiments, the AC2 PylRS disclosed herein (e.g., RS as shown in SEQ ID NO: 13 or a conservative variant thereof) can efficiently introduce non-natural amino acids mPAPF, mDMPF, and DMPd into proteins using corresponding tRNAs (e.g., tRNAs as shown in SEQ ID NO: 14 or a conservative variant thereof). The C-C9 PylRS / tRNA and AC2 PylRS / tRNA disclosed herein are orthogonal to both prokaryotic cells and eukaryotic cells and can be used to express non-natural polypeptides in prokaryotic cells and eukaryotic cells.

[0280] As used herein, the term "tyrosyl-tRNA synthetase" refers to an enzyme that catalyzes the activation of tyrosine and covalently binds to the 3' end of the corresponding tRNA molecule. As used herein, the term "tyrosyl-tRNA synthetase mutant" refers to a mutant obtained by introducing various mutations (e.g., amino acid addition, deletion, substitution, or any combination thereof) based on wild-type tyrosyl-tRNA synthetase (TyrRS). The aminoacyl-tRNA synthetase MjFmrRS disclosed herein belongs to a "tyrosyl-tRNA synthetase mutant", which can be obtained by mutating wild-type TyrRS derived from archaea (e.g., Methanococcus jannaschii). Through the TyrRS mutant disclosed herein, even for non-natural amino acids (e.g., various non-natural amino acids listed above) that are inactive when using wild-type TyrRS, proteins can be efficiently introduced. In some exemplary embodiments, the MjFmrRS disclosed herein (e.g., RS as shown in SEQ ID NO: 15 or a conservative variant thereof) can efficiently introduce the non-natural amino acid pAPCF into protein using the corresponding tRNA (e.g., tRNA as shown in SEQ ID NO: 16 or a conservative variant thereof). The MjFmrRS / tRNA disclosed herein is orthogonal to prokaryotes and can be used to express non-natural polypeptides in prokaryotes.

[0281] As used herein, an orthogonal tRNA (O-tRNA) is a tRNA that is orthogonal to a cell or translation system of interest, wherein the tRNA is, for example: (1) identical or substantially similar to a naturally occurring tRNA; (2) derived from a naturally occurring tRNA by natural or artificial mutagenesis; (3) produced by any method that takes into account the wild-type or mutant tRNA sequence of (1) or (2); (4) homologous to a wild-type or mutant tRNA; (5) homologous to any exemplary tRNA known as an orthogonal tRNA synthetase substrate; or (6) a conservative variant of any exemplary tRNA known as an orthogonal tRNA synthetase substrate. The O-tRNA may be charged with an amino acid, or in an uncharged state. In addition, it should be understood that the "O-tRNA" may be optionally charged (aminoacylated) with a non-natural amino acid by a related synthetase. It is easy to understand that in the present application, it is preferred to utilize the O-tRNA disclosed herein to react to a selector codon during translation to incorporate substantially any non-natural amino acid (preferably a non-natural amino acid disclosed herein) into an extended polypeptide.

[0282] As used herein, the term "amber suppressor tRNA" refers to a tRNA that recognizes an amber codon or responds to an amber codon during translation.

[0283] As used herein, the term "genetic coding" refers to: living cells translate the genetic material information encoded in DNA or mRNA sequences into proteins. In some aspects, the term "coding" is used to describe the process of semi-conservative DNA replication, in which one strand of a double-stranded DNA molecule is used as a template and a new complementary sister strand is encoded and synthesized by a DNA-dependent DNA polymerase. On the other hand, the term "coding" refers to any procedure by which the information within a molecule is used to direct the synthesis of a second molecule with chemical properties different from the first molecule. For example, a DNA molecule can encode an RNA molecule, for example, through a transcription process involving a DNA-dependent RNA polymerase. In addition, an RNA molecule can encode a polypeptide, such as a translation process. When the term "coding" is used to describe the translation process, the term also refers to a triplet codon encoding an amino acid. In some aspects, an RNA molecule can also encode a DNA molecule, for example, through a reverse transcription process involving an RNA-dependent DNA polymerase. On the other hand, a DNA molecule can encode a polypeptide, and at this time, "coding" should be understood as a situation that includes both a transcription process and a translation process.

[0284] As used herein, the term "selector codon" refers to a codon that is recognized by O-tRNA during translation and is preferably not recognized by endogenous tRNA. The O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates the amino acid it carries, such as an unnatural amino acid, at this position in the polypeptide. Selector codons may include, for example, nonsense codons or stop codons (e.g., amber, ochre, and opal codons); four-base or more codons; rare codons; codons derived from natural or unnatural base pairs, and the like. As used herein, the terms "nonsense codon" and "stop codon" have the same meaning and are used interchangeably, including UAA / TAA (ochre codons), UAG / TAG (amber codons), and UGA / TGA (opal codons).

[0285] As used herein, the terms "amber codon", "amber stop codon", and "TAG codon" have the same meaning and are used interchangeably, and refer to: a triplet polynucleotide corresponding to the stop codon UAG on mRNA transcribed from protein gene DNA, i.e., TAG. It is easy to understand that since the base T in DNA corresponds to the base U in RNA, the terms "TAG codon" and "UAG codon" have the same meaning and are used interchangeably.

[0286] As used herein, the term "translation system" refers to the components that incorporate amino acids into an extending polypeptide chain (protein). The components of the translation system may include, for example, ribosomes, tRNAs, aminoacyl-tRNA synthetases, mRNAs, etc. The O-tRNA and / or O-RS of the present invention may be added to or part of an in vitro or in vivo translation system, for example, in non-eukaryotic cells, such as bacteria (e.g., E. coli), or in eukaryotic cells, such as yeast, mammalian cells, plant cells, algae cells, fungal cells, insect cells, etc., or in a cell-free in vitro translation system.

[0287] As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain Eukaryota, e.g., animals (e.g., mammals (e.g., humans), insects, reptiles, birds, etc.), plants (e.g., monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, ciliates, protists, etc. As used herein, the term "eukaryotic cell" refers to a cell of a eukaryotic organism.

[0288] As used herein, the term "non-eukaryote" refers to an organism belonging to the prokaryotic kingdom (also referred to as Prokaryotes). Non-eukaryotic organisms, such as prokaryotes, are generally distinguished from eukaryotic organisms by their following characteristics: unicellular organization, asexual reproduction by budding or fission, lack of membrane-bound nuclei and other membrane-bound organelles, circular chromosomes, the presence of operons, the absence of introns, messenger (RNA) capping and poly-AmRNA, and other biochemical characteristics, such as different ribosome structures. Prokaryotes include true bacteria and Archaea (sometimes referred to as "Archaebacteria") subkingdoms. In the prokaryotic kingdom, cyanobacteria (blue-green algae) and mycoplasmas are sometimes classified into different categories. As used herein, the term "prokaryotic cell" refers to a prokaryotic cell.

[0289] As used herein, the term "conservative variant" refers to a derivative obtained by various changes, mutations or modifications to a molecular entity, which has a structure different from the molecular entity, but retains the same or similar function as the molecular entity. For example, in the present application, the conservative variant of O-tRNA has the same or similar function as the O-tRNA derived from the conservative variant, but has a change in sequence. Similarly, in the present application, the conservative variant of O-RS has the same or similar function as the O-RS derived from the conservative variant, but has a change in sequence. Thus, it is easy to understand that the O-RS or its conservative variant of the present application will be able to aminoacylate the complementary O-tRNA or its conservative variant with a non-natural amino acid, although the O-RS and its conservative variant do not have the same amino acid sequence, and the O-tRNA and its conservative variant do not have the same nucleotide sequence. For example, the O-RS of the present application or its conservative variant can aminoacylate complementary O-tRNA with non-natural amino acids, although O-RS and its conservative variant do not have the same amino acid sequence; or, the O-RS of the present application can aminoacylate complementary O-tRNA or its conservative variant with non-natural amino acids, although O-tRNA and its conservative variant do not have the same nucleotide sequence. O-RS or O-tRNA conservative variants may have, for example, one change, two changes, three changes, four changes, or five or more changes in the sequence, as long as the conservative variant is complementary to the corresponding O-tRNA or O-RS.

[0290] In some cases, a conservative variant may have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence of the protein, polypeptide, or nucleic acid from which it is derived. For example, in some cases, an O-RS conservative variant may have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of the O-RS from which it is derived. In some cases, a conservative O-tRNA variant can have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleotide sequence of the O-tRNA from which it is derived.

[0291] In some cases, a conservative variant of a protein or polypeptide (e.g., O-RS) can be obtained by substituting, deleting or adding one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) to the amino acid sequence of a protein or polypeptide (e.g., O-RS), and the function or property of the protein or polypeptide (e.g., O-RS) is retained (e.g., the ability to aminoacylate complementary O-tRNA with non-natural amino acids). In some preferred cases, a conservative variant of a protein or polypeptide (e.g., O-RS) can be obtained by substituting one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) to the amino acid sequence of a protein or polypeptide (e.g., O-RS), and the function or property of the protein or polypeptide (e.g., O-RS) is retained (e.g., the ability to aminoacylate complementary O-tRNA with non-natural amino acids). In some preferred cases, one or more conservative substitutions of amino acids (e.g., 1, 2, 3, 4, or 5 conservative substitutions of amino acids) can be made to the amino acid sequence of a protein or polypeptide (e.g., O-RS) to obtain a conservative variant of the protein or polypeptide (e.g., O-RS) while retaining the function or property of the protein or polypeptide (e.g., O-RS) (e.g., the ability to aminoacylate complementary O-tRNA with a non-natural amino acid).

[0292] As used herein, the term "derived from" refers to a component separated from a specific molecule or organism, or based on the sequence information of the specific molecule or organism, or a component prepared using the specific molecule or organism or sequence information. For example, a polypeptide derived from a first polypeptide may include an amino acid sequence identical or substantially similar to the amino acid sequence of the first polypeptide. Taking a polypeptide as an example, a derived species may be obtained by, for example, naturally occurring mutagenesis, artificial site-directed mutagenesis, or artificial random mutagenesis. The mutagenesis used to derive a polypeptide may be intentionally site-directed or intentionally random, or a mixture of the two. Mutagenesis of a polypeptide to produce a different polypeptide derived from a first polypeptide may be random (for example, caused by polymerase infidelity), and the identification of the derived polypeptide may be accomplished by, for example, a suitable screening method described in the references cited herein. Mutagenesis of a polypeptide generally requires manipulation of a polynucleotide encoding the polypeptide. As used herein, when a molecule (for example, a nucleic acid or a polypeptide) is derived from another molecule (for example, a nucleic acid or a polypeptide), the two molecules are referred to as "homologous".

[0293] In the context of "non-natural polypeptides" of the present application, when referring to non-natural polypeptides derived from a certain protein or polypeptide, it refers to a polypeptide derived by incorporating the non-natural amino acid of the present application into the protein or polypeptide. For example, such non-natural polypeptides can be obtained by inserting the non-natural amino acid of the present application into the desired site of the protein or polypeptide, or replacing the amino acid residue of the desired site of the protein or polypeptide with the non-natural amino acid of the present application. However, it should be understood that, in addition to the incorporation of non-natural amino acids, non-natural polypeptides can also contain additional mutations compared to the protein or polypeptide on which they are based, such as additional amino acid additions, deletions, substitutions, or any combination thereof.

[0294] As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, the comparison is made when the two sequences are aligned to produce maximum identity. Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol. Biol. 48: 444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0295] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the essential properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10): 879-884 (1999); and Burks et al. Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0296] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0297] As used herein, the term "wild type" refers to a certain molecular entity (e.g., a nucleic acid or a polypeptide) that occurs naturally. It is easy to understand that for a certain molecular entity (e.g., a nucleic acid or a polypeptide), there may be a variety of wild-type forms that exist naturally, which have slight differences in structure (e.g., sequence) from each other, but have substantially the same function or property. It should also be understood that the wild-type molecular entity is not limited to its acquisition method, for example, it can be isolated from a natural environment, or it can be artificially synthesized. As used herein, the term "mutant" or "mutant" is relative to the wild type. Typically, a mutant or mutant has differences (or mutations) in structure (e.g., sequence) relative to a wild-type molecular entity (e.g., a nucleic acid or a polypeptide), and this difference (or mutation) can be naturally occurring or artificially introduced.

[0298] As used herein, the term "adjacent position" refers to a position adjacent to a target site or region in the spatial conformation of a protein or polypeptide, such as a position at a distance of no more than 20 (e.g., no more than 15, preferably no more than 10, 9, 8, 7, 6, 5, 4 or 3) angstroms (10 angstroms, i.e., 1 nm) from the target site or region. In general, a protein or polypeptide expressed by a translation system undergoes spatial folding to form a higher order structure with a specific spatial conformation, thereby exerting its function or activity. Therefore, it is readily understood that a position adjacent to a target site or region may be a position adjacent to the target site or region on a primary structure (i.e., an amino acid sequence) (e.g., a position directly adjacent or spaced by 1, 2 or 3 amino acid residues), or may be a position that is not adjacent on a primary structure (i.e., an amino acid sequence) but is adjacent to the target site or region in the spatial conformation of a protein or polypeptide due to the spatial folding of a protein or polypeptide. The spatial conformation of a protein or polypeptide can be determined by various methods known in the art, such as X-ray diffraction analysis, nuclear magnetic resonance technology, cryo-electron microscopy, analysis technology using AI algorithms (such as the AlphaFold algorithm), etc. In addition, the spatial conformation of a protein or polypeptide can also be obtained in a protein database (such as the PDB database) that stores protein structures.

[0299] As used herein, the term "corresponding site" or "corresponding site" refers to a site in the compared sequences that is located at an equivalent position when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity. Thus, the expression "a site corresponding to site M of SEQ ID NO:X" refers to a site in the target sequence that is located at an equivalent position to site M of SEQ ID NO:X when the target sequence is optimally aligned with SEQ ID NO:X, i.e., when the target sequence is aligned with SEQ ID NO:X to obtain the highest percentage identity. For example, for protein Z, the expression "a site corresponding to site 13 or 17 of SEQ ID NO:2" refers to a site in the amino acid sequence of protein Z that is located at an equivalent position to site 13 or 17 of SEQ ID NO:2 when protein Z is aligned with SEQ ID NO:2 to obtain the highest percentage identity.

[0300] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0301] Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transformed, the desired expression level, etc. A vector may be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including those derived from non-natural polypeptides as described herein, etc.

[0302] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0303] As used herein, the term "antigen-antibody molecule pair" refers to an antigen and an antibody that can specifically bind to each other.

[0304] As used herein, the term "antibody" refers to an immunoglobulin molecule, typically composed of two pairs of polypeptide chains (each pair having a "light" (L) chain and a "heavy" (H) chain), that specifically binds and recognizes an analyte (antigen). Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region consists of three domains (C H 1. C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the light chain constant region (C L ). The light chain constant region consists of a domain C L The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. H and V L The V region can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region (V H and V L) form antibody binding sites respectively. The allocation of amino acids to each region or domain follows the Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987and1991)), or the definition of Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883. The term "antibody" is not limited by any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be antibodies of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0305] As used herein, the term "antibody" is also intended to include its antigen binding fragment. The term "antigen binding fragment" refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". Generally, see Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen binding fragments of antibodies can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of complete antibodies. In some cases, antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, double antibodies (diabody) and such polypeptides, which include at least a portion of an antibody sufficient to confer polypeptide-specific antigen binding ability.

[0306] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. In certain embodiments, a second molecule (such as a polypeptide or antibody) that specifically binds to a first molecule (such as a substrate or antigen) refers to a second molecule (such as a polypeptide or antibody) that binds to the first molecule (such as a substrate or antigen) with a specific binding affinity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less affinity (K D) binds to a first molecule (eg, a substrate or antigen).

[0307] Beneficial Effects

[0308] The present invention develops genetically encodable fluorescent rotor non-natural amino acids (FR-ncAAs) for the first time; and by directly and specifically introducing the non-natural amino acids into specific sites of the target protein, accurate, low-interference, background-free fluorescent labeling of proteins and the development of high signal-to-noise ratio, highly versatile fluorescent probes are achieved, thereby realizing the detection of protein interactions and the development of biosensors. BRIEF DESCRIPTION OF THE DRAWINGS

[0309] Figure 1 Shown are the (a) absorption spectra and (b) fluorescence emission spectra of pAPCF in different solvents.

[0310] Figure 2 The fluorescence excitation spectra (left) and fluorescence emission spectra (right) of various unnatural amino acids of the present application in glycerol are shown.

[0311] Figure 3 Shows the fluorescence intensity changes of pAPCF in glycerol / methanol mixed systems with different viscosities.

[0312] Figure 4 The fluorescence intensity analysis of various unnatural amino acids of the present application in glycerol / methanol mixed systems of different viscosities is shown.

[0313] Figure 5 Shown are: (a) fluorescence measurement of sfGFP fluorescent protein reporter element; (b) mass spectrometry identification results of sfGFP (151pAPCF) expressed using MjFmrRS.

[0314] Figure 6 Fluorescence flow cytometry screening of the PylRS library: (a) positive screening flow cytometry analysis; (b) negative screening flow cytometry analysis.

[0315] Figure 7 The screened MaPylRS mutants - CC9 RS and AC2 RS - are shown to efficiently recognize and catalyze a variety of fluorescent rotor-type unnatural amino acids: (a) Characterization of the introduction efficiency of CC9 RS for various unnatural amino acids; (b) Characterization of the introduction efficiency of AC2 RS for various unnatural amino acids.

[0316] Figure 8 The application of pAPCF as a probe to detect the interaction between protein Z and Affibody is shown. Figure 8 a Schematic diagram showing the principle of using pAPCF as a probe to detect the interaction between protein Z and Affibody; Figure 8b shows the fluorescence quantitative detection results after Z-17 binds to Affibody with gradient concentrations; Figure 8 c shows the fluorescence images of Z-17 binding to Affibody with gradient concentrations.

[0317] Fig. 9 It shows that Z-wt can compete with Z-17 for the binding of Affibody and reduce the fluorescence of the system. Fig. 9 a shows the fluorescence quantitative detection results of Z-17 binding to Affibody in the presence of different concentrations of Z-wt; Fig. 9 b shows the fluorescence images of Z-17 after binding to Affibody in the presence of different concentrations of Z-wt. Statistical analysis was performed using a two-tailed t-test, ***P<0.001; ****P<0.0001.

[0318] Fig.10 The results of the binding detection experiment between Z-13 and Affibody are shown. Fig.10 a shows the fluorescence quantitative detection results after Z-13 binds to Affibody with gradient concentrations; Fig.10 b shows the fluorescence quantitative detection results after Z-13 binds to Affibody in the presence of different concentrations of Z-wt.

[0319] Fig.11 The experimental results show that temperature increase induces SOD1(V31A)-25pAPCF aggregation and increases fluorescence. Fig.11 a shows that as the temperature increases, the protein SOD1(V31A)-25pAPCF aggregates, resulting in increased turbidity of the protein solution. Fig.11 b shows that the fluorescence intensity of the protein solution increases with increasing temperature. Fig.11 c shows the fluorescence photograph of the protein solution before and after the heating experiment.

[0320] Fig.12 It shows that the detection of SOD1(V31A)-25pAPCF ( Fig.12 a) and SOD1(A4V)-40pAPCF( Fig.12 b) Abnormal aggregation phenomenon in cells, where the red fluorescence comes from mCherry fused with the SOD1 mutant; the green fluorescence comes from pAPCF incorporated into the SOD1 mutant, which is generated due to the abnormal aggregation of SOD1 in cells.

[0321] Fig.13 The graph shows the fluorescence comparison before and 10 min after the addition of 100 nM rapamycin in HEK293T cells expressing FRB-87PAPd-mCherry and FKBP.

[0322] Fig.14 The graph shows the fluorescence comparison before and after the addition of 100 nM rapamycin in HEK293T cells expressing mCherry-FKBP (membrane localized) and FRB-87PAPd (scale bar: 10 μm).

[0323] Fig.15 The results of experiments to determine the selectivity of GBP-238 for various sugar molecules are shown ( Fig.15 a), and the affinity of GBP-238 for glucose measured by SPR technology ( Fig.15 b).

[0324] Fig.16 The results of the experiment to measure the fluorescence responsiveness of glnH-51 to glutamine are shown ( Fig.16 a), and the experimental results of determining the selectivity of glnH-51 for various substrate molecules ( Fig.16 b).

[0325] Fig.17 Shown are the results of experiments to measure the fluorescence responsiveness of MjFeoB-33PAPd to GTP or GDP.

[0326] Fig.18 The results show that the four CaM-M13 mutants were tested for Ca 2+ The experimental results of fluorescence responsiveness.

[0327] Fig.19 The quantitative determination of CaM-92PAPd-M13 calcium sensor for Ca 2+ The experimental results of fluorescence responsiveness.

[0328] Fig. 20 The CaM-92PAPd-M13 calcium probe was used to monitor Ca in HEK293T cells. 2+ The experimental results of concentration changes, where Fig. 20 a shows the fluorescence images of cells before administration, after administration of carbachol, and after administration of carbachol + ionomycin calcium salt (scale bar: 20 μm); Fig. 20 b shows the statistical analysis results of the changes in the mean fluorescence intensity of cells after different drug treatments, wherein the mean fluorescence intensity of cells in each treatment group was corrected with the baseline fluorescence intensity before drug addition (Mean±sem; n=16).

[0329] Fig.21 The quantitative determination of CaM-92PAPd-F141L-M13 calcium sensor for Ca 2+ The experimental results of fluorescence responsiveness. DETAILED DESCRIPTION

[0330] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention (but not to limit the present invention), but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the drawings and preferred embodiments.

[0331] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this application are basically based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compiled Guide to Molecular Biology Experiments, 3rd edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturer. If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially. Those skilled in the art will appreciate that the examples describe the present invention by way of example and are not intended to limit the scope of protection claimed in this application.

[0332] Sequence information

[0333] The following provides information on some of the sequences involved in this application.

[0334] SEQ ID NO: 1 (nucleotide sequence of the T7 RNA polymerase gene that controls RFP transcription)

[0335] atgaccatgattaccgtgcactagaataccattaacattgctaagaacgacttctctgacatcgaactggctgctatcccgttcaacactctggctgacca

[0336] ttacggtgagcgtttagctcgcgaacagttggcccttgagcatgagtcttacgagatgggtgaagcacgcttccgcaagatgtttgagcgtcaacttaaa

[0337] gctggtgaggttgcggataacgctgccgccaagcctctcatcactaccctactccctaagatgattgcacgcatcaacgactggtttgaggaagtgaaag

[0338] ctaagcgcggcaagcgcccgacagccttccagttcctgtaggaaatcaagccggaagccgtagcgtacatcaccattaagaccactctggcttgcctaa

[0339] ccagtgctgacaatacaaccgttcaggctgtagcaagcgcaatcggtcgggccattgaggacgaggctcgcttcggtcgtatccgtgaccttgaagcta

[0340] agcacttcaagaaaaacgttgaggaacaactcaacaagcgcgtagggcacgtctacaagaaagcatttatgcaagttgtcgaggctgacatgctctct

[0341] aagggtctactcggtggcgaggcgtggtcttcgtggcataaggaagactctattcatgtaggagtacgctgcatcgagatgctcattgagtcaaccgga

[0342] atggttagcttacaccgccaaaatgctggcgtagtaggtcaagactctgagactatcgaactcgcacctgaatacgctgaggctatcgcaacccgtgca

[0343] ggtgcgctggctggcatctctccgatgttccaaccttgcgtagttcctcctaagccgtggactggcattactggtggtggctattgggctaacggtcgtcgt

[0344] cctctggcgctggtgcgtactcacagtaagaaagcactgatgcgctacgaagacgtttacatgcctgaggtgtacaaagcgattaacattgcgcaaaac

[0345] accgcatggaaaatcaacaagaaagtcctagcggtcgccaacgtaatcaccaagtggaagcattgtccggtcgaggacatccctgcgattgagcgtga

[0346] agaactcccgatgaaaccggaagacatcgacatgaatcctgaggctctcaccgcgtggaaacgtgctgccgctgctgtgtaccgcaaggacaaggctc

[0347] gcaagtctcgccgtatcagccttgagttcatgcttgagcaagccaataagtttgctaaccataaggccatctggttcccttacaacatggactggcgcggt

[0348] cgtgtttacgctgtgtcaatgttcaacccgcaaggtaacgatatgaccaaaggactgcttacgctggcgaaaggtaaaccaatcggtaaggaaggttac

[0349] tactggctgaaaatccacggtgcaaactgtgcgggtgtcgataaggttccgttccctgagcgcatcaagttcattgaggaaaaccacgagaacatcatg

[0350] gcttgcgctaagtctccactggagaacacttggtgggctgagcaagattctccgttctgcttccttgcgttctgctttgagtacgctggggtacagcaccac

[0351] ggcctgagctataactgctcccttccgctggcgtttgacgggtcttgctctggcatccagcacttctccgcgatgctccgagatgaggtaggtggtcgcgc

[0352] ggttaacttgcttcctagtgaaaccgttcaggacatctacgggattgttgctaagaaagtcaacgagattctacaagcagacgcaatcaatgggaccgat

[0353] aacgaagtagttaccgtgaccgatgagaacactggtgaaatctctgagaaagtcaagctgggcactaaggcactggctggtcaatggctggcttacgg

[0354] tgttactcgcagtgtgactaagcgttcagtcatgacgctggcttacgggtccaaagagttcggcttccgtcaacaagtgctggaagataccattcagcca

[0355] gctattgattccggcaagggtctgatgttcactcagccgaatcaggctgctggatacatggctaagctgatttgggaatctgtgagcgtgacggtggtag

[0356] ctgcggttgaagcaatgaactggcttaagtctgctgctaagctgctggctgctgaggtcaaagataagaagactggagagattcttcgcaagcgttgcg

[0357] ctgtgcattgggtaactcctgatggtttccctgtgtggcaggaatacaagaagcctattcagacgcgcttgaacctgatgttcctcggtcagttccgcttac

[0358] agcctaccattaacaccaacaaagatagcgagattgatgcacacaaacaggagtctggtatcgctcctaactttgtacacagccaagacggtagccac

[0359] cttcgtaagactgtagtgtgggcacacgagaagtacggaatcgaatcttttgcactgattcacgactccttcggtaccattccggctgacgctgcgaacct

[0360] gttcaaagcagtgcgcgaaactatggttgacacatatgagtcttgtgatgtactggctgatttctacgaccagttcgctgaccagttgcacgagtctcaatt

[0361] ggacaaaatgccagcacttccggctaaaggtaacttgaacctccgtgacatcttagagtcggacttcgccttcgcctaa

[0362] SEQ ID NO:2 (amino acid sequence of wild-type protein Z)

[0363] MTSKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0364] SEQ ID NO:3 (amino acid sequence of Z-17)

[0365] MTSKFNKEQQNAAYEIXHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK X stands for pAPCF

[0366] SEQ ID NO:4 (amino acid sequence of Z-13)

[0367] MTSKFNKEQQNAXYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK X stands for pAPCF

[0368] SEQ ID NO:5 (6*His tag amino acid sequence)

[0369] LEHHHHHH

[0370] SEQ ID NO:6 (amino acid sequence of MBP-Affibody)

[0371] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWA

[0372] HDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKT

[0373] WEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAG

[0374] LTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSK

[0375] PFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATM

[0376] ENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGR

[0377] ISHMTSKFNKELSVAGREIVTLPNLNDPQKKAFIFSLWDDPSQSANLLAEAKKLNDAQAPKLEHH

[0378] HHHH

[0379] SEQ ID NO:7 (Amino acid sequence of SOD1(A4V)-40pAPCF)

[0380] ATKVVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTXGLHGFHVHEFGDNTAGCTSAGPHF

[0381] NPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKG

[0382] GNEESTKTGNAGSRLACGVIGIAQ

[0383] X represents pAPCF

[0384] SEQ ID NO:8 (Amino acid sequence of the linker)

[0385] GSGSGSGS

[0386] SEQ ID NO:9 (Amino acid sequence of mCherry)

[0387] VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDI

[0388] LSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKL

[0389] RGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKP

[0390] VQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK

[0391] SEQ ID NO:10 (Nucleotide sequence encoding the fusion protein SOD1(A4V)-40pAPCF-mCherry)

[0392] atggcaaccaaagtggtttgcgttctgaaaggtgacggtccggttcagggcattattaattttgaacagaaagaaagcaacggcccggtgaaagtgtgg

[0393] ggtagcattaagggcctgacctagggtctgcatggttttcatgttcatgaatttggcgataataccgccggttgcaccagcgcaggcccgcattttaatcc

[0394] gctgagtcgtaaacatggtggtccgaaagatgaagaacgtcatgttggtgacctgggcaatgtgaccgcagataaagatggtgtggcagatgtgagta

[0395] ttgaagatagtgttattagcctgagcggtgaccattgtattattggtcgcaccctggttgttcatgaaaaagcagatgatctgggcaaaggtggcaatga

[0396] agaaagcaccaaaaccggtaatgccggcagccgtctggcctgcggcgtgattggcattgcacagggaagcggatccggttcaggtagtgtgagcaag

[0397] ggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcg

[0398] agggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctca

[0399] gttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatg

[0400] aacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttccc

[0401] ctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaa

[0402] gcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaa

[0403] cgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatg

[0404] gacgagctgtacaag

[0405] SEQ ID NO:11 (Amino acid sequence of SOD1(V31A)-25pAPCF)

[0406] ATKAVCVLKGDGPVQGIINFEQKEXNGPVKAWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHF

[0407] NPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKG

[0408] GNEESTKTGNAGSRLACGVIGIAQ

[0409] X represents pAPCF

[0410] SEQ ID NO:12 (Amino acid sequence of C-C9 PylRS)

[0411] MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIANPRNDLTD

[0412] LENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALRPMHAMNVIKVSR

[0413] ELRDHTKGPVKIFEIGSCFRKESKSSTHLEEFTMLALAEMGPDGDPMEHLKMYIGDIMDAVGVE

[0414] YTTSREESDVWVETLDVEINGTEVASGAVGPHKLDPAHDVHEPWAGIGFGLERLLMLKNGKSNA

[0415] RKTGKSITYLNGYKLD

[0416] SEQ ID NO:13 (Amino acid sequence of AC2 PylRS)

[0417] MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIANPRNDLTD

[0418] LENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALRPMHAMNLKKVG

[0419] RELRDHTKGPVKIFEIGSCFRKESKSSAHLEEFTMLALAEMGPDGDPMEHLKMYIGDIMDAVGV

[0420] EYTTSREESDVYVETLDVEINGTEVASGAVGPHKLDPAHDVHEPWAGIGFGLERLLMLKNGKSN

[0421] ARKTGKSITYLNGYKLD

[0422] SEQ ID NO:14 (Nucleotide sequence of tRNA pyl (end)

[0423] GGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCCAGCGGGGTTCGACGCCCCG

[0424] GTCTCTCGCCA

[0425] SEQ ID NO:15 (Amino acid sequence of MjFmrRS)

[0426] MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIIYLA

[0427] DLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEHGLDKDYTLNVYRLALKTTLKRA

[0428] RRSMELIAREDENPKVAEVIYPIMQVNGIHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNP

[0429] VLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEK

[0430] FGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL

[0431] SEQ ID NO:16 (Nucleotide sequence of tRNA tyr (

[0432] CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCAGGGGTTCAAAT

[0433] CCCCTCCGCCGGACCA

[0434] SEQ ID NO:17 (Amino acid sequence of mCherry-GFP fusion protein)

[0435] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAW

[0436] DILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKV

[0437] KLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKA

[0438] KKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHRSQPWVPRARDPPVA

[0439] TLVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTLVTT

[0440] LTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKG

[0441] IDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGP

[0442] VLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0443] X represents pAPCF or PAPd

[0444] SEQ ID NO:18 (Amino acid sequence of FRB - 84PAPd)

[0445] GSILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLME

[0446] AQEWCRKYMKSGNVKDLTQAWXLYYHVFRRISK

[0447] X represents PAPd

[0448] SEQ ID NO:19 (Amino acid sequence of sfGFP)

[0449] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTL

[0450] TYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGI

[0451] DFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGP

[0452] VLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0453] SEQ ID NO:20 (Nucleotide sequence of GFP)

[0454] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGA

[0455] TGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAAC

[0456] TCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTA

[0457] CTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTT

[0458] TCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGG

[0459] ACCTACAAGACGCGTTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAA

[0460] GGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAACTC

[0461] ACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCG

[0462] CCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGG

[0463] CGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGA

[0464] TCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACAC

[0465] ATGGCATGGATGAGCTCTACAAATAA

[0466] SEQ ID NO:21(Nucleotide sequence of RFP)

[0467] ATGGTGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGTGA

[0468] ACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAGAC

[0469] CATGAGAATCAAGGCGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTACCA

[0470] GCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTAAG

[0471] CAGTCCTTCCCCGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGTGC

[0472] TGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCAGA

[0473] GGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGCCT

[0474] CCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTGAA

[0475] GCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCCAAGAAACCC

[0476] GCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGGAAAGAATCAAGGA

[0477] GGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATACTGCGACCTC

[0478] CCTAGCAAACTGGGGCACAGATAA

[0479] SEQ ID NO:22 (Nucleotide sequence of sfGFP(151TAG) reporter gene)

[0480] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGA

[0481] TGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAAC

[0482] TCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTA

[0483] CTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTT

[0484] TCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGG

[0485] ACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTA

[0486] AAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTAAC

[0487] TCACACAATGTATAGATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATT

[0488] CGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATT

[0489] GGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAA

[0490] GATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTAC

[0491] ACATGGCATGGATGAGCTCTACAAAGGATCCCACCACCACCACCACCACTAA

[0492] SEQ ID NO:23 (Amino acid sequence of G1pylRS)

[0493] MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIANPRNDLTD

[0494] LENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALRPMHAMNLYKVM

[0495] RELRDHTKGPVKIFEIGSCFRKESKSSTHLEEFTMLNLVEMGPDGDPMEHLKMYIGDIMDAVGV

[0496] EYTTSREESDVYVETLDVEINGTEVASGAVGPHKLDPAHDVHEPWAGIGFGLERLLMLKNGKSN

[0497] ARKTGKSITYLNGYKLD

[0498] SEQ ID NO:24 (Nucleotide sequence encoding the fusion protein SOD1(V31A)-25pAPCF-mCherry)

[0499] atggcaaccaaagcagtttgcgttctgaaaggtgacggtccggttcagggcattattaattttgaacagaaagaatagaacggcccggtgaaagtgtgg

[0500] ggtagcattaagggcctgaccgaaggtctgcatggttttcatgttcatgaatttggcgataataccgccggttgcaccagcgcaggcccgcattttaatcc

[0501] gctgagtcgtaaacatggtggtccgaaagatgaagaacgtcatgttggtgacctgggcaatgtgaccgcagataaagatggtgtggcagatgtgagta

[0502] ttgaagatagtgttattagcctgagcggtgaccattgtattattggtcgcaccctggttgttcatgaaaaagcagatgatctgggcaaaggtggcaatga

[0503] agaaagcaccaaaaccggtaatgccggcagccgtctggcctgcggcgtgattggcattgcacagggaagcggatccggttcaggtagtgtgagcaag

[0504] ggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggagggctccgtgaacggccacgagttcgagatcgagggcg

[0505] agggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaagggtggccccctgcccttcgcctgggacatcctgtcccctca

[0506] gttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagctgtccttccccgagggcttcaagtgggagcgcgtgatg

[0507] aacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgagttcatctacaaggtgaagctgcgcggcaccaacttccc

[0508] ctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggatgtaccccgaggacggcgccctgaagggcgagatcaa

[0509] gcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctacaaggccaagaagcccgtgcagctgcccggcgcctacaa

[0510] cgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagtacgaacgcgccgagggccgccactccaccggcggcatg

[0511] gacgagctgtacaag

[0512] SEQ ID NO:25 (Amino acid sequence of the linker)

[0513] GGGGSGGGGS

[0514] SEQ ID NO:26 (Nucleotide sequence encoding the FRB-84PAPd-mCherry fusion protein)

[0515] atgggaagcatcctgtggcacgagatgtggcacgagggcctggaggaggcctccaggctgtacttcggcgagagaaacgtgaagggcatgttcgagg

[0516] tgctggagcccctgcacgccatgatggagagaggcccccagacactgaaggagacatccttcaatcaggcctacggcagagacctgatggaggccca

[0517] ggagtggtgcaggaagtacatgaagagcggcaatgtgaaggatctgacacaggcctggtagctgtactaccacgtgtttaggagaatctccaaggga

[0518] ggcggcggaagcggcggtggcggaagcgtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggag

[0519] ggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaaggg

[0520] tggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagc

[0521] tgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgag

[0522] ttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggat

[0523] gtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctaca

[0524] aggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagta

[0525] cgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaag

[0526] SEQ ID NO:27 (Amino acid sequence of FRB-87PAPd)

[0527] GSILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLME

[0528] AQEWCRKYMKSGNVKDLTQAWDLYXHVFRRISK

[0529] X represents PAPd

[0530] SEQ ID NO:28 (Nucleotide sequence encoding the FRB-87PAPd-mCherry fusion protein)

[0531] atgggaagcatcctgtggcacgagatgtggcacgagggcctggaggaggcctccaggctgtacttcggcgagagaaacgtgaagggcatgttcgagg

[0532] tgctggagcccctgcacgccatgatggagagaggcccccagacactgaaggagacatccttcaatcaggcctacggcagagacctgatggaggccca

[0533] ggagtggtgcaggaagtacatgaagagcggcaatgtgaaggatctgacacaggcctgggacctgtactagcacgtgtttaggagaatctccaaggga

[0534] ggcggcggaagcggcggtggcggaagcgtgagcaagggcgaggaggataacatggccatcatcaaggagttcatgcgcttcaaggtgcacatggag

[0535] ggctccgtgaacggccacgagttcgagatcgagggcgagggcgagggccgcccctacgagggcacccagaccgccaagctgaaggtgaccaaggg

[0536] tggccccctgcccttcgcctgggacatcctgtcccctcagttcatgtacggctccaaggcctacgtgaagcaccccgccgacatccccgactacttgaagc

[0537] tgtccttccccgagggcttcaagtgggagcgcgtgatgaacttcgaggacggcggcgtggtgaccgtgacccaggactcctccctgcaggacggcgag

[0538] ttcatctacaaggtgaagctgcgcggcaccaacttcccctccgacggccccgtaatgcagaagaagaccatgggctgggaggcctcctccgagcggat

[0539] gtaccccgaggacggcgccctgaagggcgagatcaagcagaggctgaagctgaaggacggcggccactacgacgctgaggtcaagaccacctaca

[0540] aggccaagaagcccgtgcagctgcccggcgcctacaacgtcaacatcaagttggacatcacctcccacaacgaggactacaccatcgtggaacagta

[0541] cgaacgcgccgagggccgccactccaccggcggcatggacgagctgtacaag

[0542] SEQ ID NO:29 (Amino acid sequence of mCherry-FKBP fusion protein)

[0543] MRLPGAMPALALKGELLLLSLLLLLEPQISQGVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFE

[0544] IEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFK

[0545] WERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPED

[0546] GALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE

[0547] GRHSTGGMDELYKGSGSGSGSGSVVISAILALVVLTIISLIILIMLWQKKPRYEIRWKVIESVSSDG

[0548] HEYISGSGSGSGSGSMGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKF

[0549] MLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0550] SEQ ID NO:30 (Amino acid sequence of StII signal peptide)

[0551] MKKNIAFLLASMFVFSIATNAYA

[0552] SEQ ID NO:31 (Amino acid sequence of GBP protein)

[0553] ADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKAL

[0554] AINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWA

[0555] ANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKD

[0556] KMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTV

[0557] LNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK

[0558] SEQ ID NO:32 (Amino acid sequence of 9x his tag)

[0559] LEHHHHHHHHH

[0560] SEQ ID NO:33 (Nucleotide sequence encoding StII-GBP-9his fusion protein)

[0561] atgaaaaagaatatcgcatttcttcttgctagcatgttcgttttttctattgctacaaacgcatacgctgctgatactcgcattggtgtaacaatctataagta

[0562] cgacgataactttatgtctgtagtgcgcaaggctattgagcaagatgcgaaagccgcgccagatgttcagctgctgatgaatgattctcagaatgaccag

[0563] tccaagcagaacgatcagatcgacgtattgctggcgaaaggggtgaaggcactggcaatcaacctggttgacccggcagctgcgggtacggtgattga

[0564] gaaagcgcgtgggcaaaacgtgccggtggttttcttcaacaaagaaccgtctcgtaaggcgctggatagctacgacaaagcctactacgttggcactga

[0565] ctccaaagagtccggcattattcaaggcgatttgattgctaaacactgggcggcgaatcagggttgggatctgaacaaagacggtcagattcagttcgt

[0566] actgctgaaaggtgaaccgggccatccggatgcagaagcacgtaccacttacgtgattaaagaattgaacgataaaggcatcaaaactgaacagtta

[0567] cagttagataccgcaatgtgggacaccgctcaggcgaaagataagatggacgcctggctgtctggcccgaacgccaacaaaatcgaagtggttatcgc

[0568] caacaacgatgcgatggcaatgggcgcggttgaagcgctgaaagcacacaacaagtccagcattccggtgtttggcgtcgatgcgctgccagaagcg

[0569] ctggcgctggtgaaatccggtgcactggcgggcaccgtactgaacgatgctaacaaccaggcgaaagcgacctttgatctggcgaaaaacctggccga

[0570] tggtaaaggtgcggctgatggcaccaactggaaaatcgacaacaaagtggtccgcgtaccttatgttggcgtagataaagacaacctggctgaattca

[0571] gcaagaaactcgagcaccaccaccaccaccaccaccaccac

[0572] SEQ ID NO:34 (Amino acid sequence of GBP-149)

[0573] ADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKAL

[0574] AINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWA

[0575] ANQGWDLNKDGQIQFVLLKGXPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKD

[0576] KMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDALPEALALVKSGALAGTV

[0577] LNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK

[0578] X represents pAPCF

[0579] SEQ ID NO:35 (Amino acid sequence of GBP-238)

[0580] ADTRIGVTIYKYDDNFMSVVRKAIEQDAKAAPDVQLLMNDSQNDQSKQNDQIDVLLAKGVKAL

[0581] AINLVDPAAAGTVIEKARGQNVPVVFFNKEPSRKALDSYDKAYYVGTDSKESGIIQGDLIAKHWA

[0582] ANQGWDLNKDGQIQFVLLKGEPGHPDAEARTTYVIKELNDKGIKTEQLQLDTAMWDTAQAKD

[0583] KMDAWLSGPNANKIEVVIANNDAMAMGAVEALKAHNKSSIPVFGVDAXPEALALVKSGALAGT

[0584] VLNDANNQAKATFDLAKNLADGKGAADGTNWKIDNKVVRVPYVGVDKDNLAEFSKK

[0585] X represents pAPCF

[0586] SEQ ID NO:36 (Amino acid sequence of gH-51)

[0587] ADKKLVVATDTAFVPFEFKQGDKYVGFDVDLWAAIAKELKLDYELKPMDFXGIIPALQTKNVDL

[0588] ALAGITITDEMKKAIDFSDGYAKSGLLVMVKANNNDVKSVKDLDGKVVAVKSGTGSVDYAKANI

[0589] KTKDLRQFPNIDNAYMELGTNRADAVLHDTPNILYFIKTAGNGQFKAVGDSLEAQQYGIAFPKGS

[0590] DELRDKVNGALKTLRENGTYNEIYKKWFGTEPK

[0591] X represents pAPCF

[0592] SEQ ID NO:37 (Amino acid sequence of gH-116)

[0593] ADKKLVVATDTAFVPFEFKQGDKYVGFDVDLWAAIAKELKLDYELKPMDFSGIIPALQTKNVDLA

[0594] LAGITITDEMKKAIDFSDGYAKSGLLVMVKANNNDVKSVKDLDGKVVAVKXGTGSVDYAKANIK

[0595] TKDLRQFPNIDNAYMELGTNRADAVLHDTPNILYFIKTAGNGQFKAVGDSLEAQQYGIAFPKGSD

[0596] ELRDKVNGALKTLRENGTYNEIYKKWFGTEPK

[0597] X represents pAPCF

[0598] SEQ ID NO:38 (Amino acid sequence of gH-117)

[0599] ADKKLVVATDTAFVPFEFKQGDKYVGFDVDLWAAIAKELKLDYELKPMDFSGIIPALQTKNVDLA

[0600] LAGITITDEMKKAIDFSDGYAKSGLLVMVKANNNDVKSVKDLDGKVVAVKSXTGSVDYAKANIK

[0601] TKDLRQFPNIDNAYMELGTNRADAVLHDTPNILYFIKTAGNGQFKAVGDSLEAQQYGIAFPKGSD

[0602] ELRDKVNGALKTLRENGTYNEIYKKWFGTEPK

[0603] X represents pAPCF

[0604] SEQ ID NO:39 (amino acid sequence of MjFeoB-33PAPd)

[0605] MKSYEIALIGNPNVGKSTIFNALTGENVYIGNXPGVTVEKKEGEFEYNGEKFKVVDLPGVYSLTA

[0606] NSIDEIIARDYIINEKPDLVVNIVDATALERNLYLTLQLMEMGANLLLALNKMDLAKSLGIEIDVD

[0607] KLEKILGVKVVPLSAAKKMGIEDLKKAISIAVKDKKTAEIKYPNFEPYIKKIT

[0608] X stands for PAPd

[0609] SEQ ID NO:40 (amino acid sequence of CaM-19PAPd)

[0610] ADQLTEEQIAEFKEAFSLXDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDF

[0611] PEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREAD

[0612] IDGDGQVNYEEFVQMMTAK

[0613] X stands for PAPd

[0614] SEQ ID NO:41 (amino acid sequence of linker)

[0615] GSGS

[0616] SEQ ID NO:42 (amino acid sequence of M13 short peptide)

[0617] RRKWQKTGHAVRAIGRLSS

[0618] SEQ ID NO:43 (amino acid sequence of CaM-68PAPd)

[0619] ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDF

[0620] PEXLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA

[0621] DIDGDGQVNYEEFVQMMTAK

[0622] X stands for PAPd

[0623] SEQ ID NO:44 (amino acid sequence of CaM-92PAPd)

[0624] ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDF

[0625] PEFLTMMARKMKDTDSEEEIREAFRVXDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA

[0626] DIDGDGQVNYEEFVQMMTAK

[0627] X stands for PAPd

[0628] SEQ ID NO:45 (amino acid sequence of CaM-141PAPd)

[0629] ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDF

[0630] PEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA

[0631] DIDGDGQVNYEEXVQMMTAK

[0632] X stands for PAPd

[0633] SEQ ID NO:46 (amino acid sequence of CaM-92PAPd-F141L)

[0634] ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDF

[0635] PEFLTMMARKMKDTDSEEEIREAFRVXDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREA

[0636] DIDGDGQVNYEELVQMMTAK

[0637] X stands for PAPd

[0638] Example 1: Synthesis of unnatural amino acid pAPCF

[0639] The synthetic route of the unnatural amino acid pAPCF, (S,Z)-2-amino-3-(4-(2-(4-aminophenyl)-1-cyanovinyl)phenyl)propanoic acid is shown below.

[0640]

[0641] (1) tert-butyl(S)-3-(4-(bromomethyl)phenyl)-2-((diphenylmethylene)amino)propanoate

[0642] First, 8 g (27.1 mmol, 1 eq) of the raw material N-dibenzylidene-glycine tert-butyl ester and 21.4 g (81.3 mmol, 3 eq) of the raw material 1,4-di(bromomethyl)benzene were mixed together, dissolved in 200 mL of anhydrous acetonitrile, and then 22.0 g (67.5 mmol, 2.5 eq) of cesium carbonate was added, and stirred overnight at 50 degrees. After about 15 hours of reaction, the solid residue was filtered out, and the filtered organic phase was separated by silica gel column chromatography. The eluent was petroleum ether (containing 0.5% triethylamine). After the excess raw material was eluted, the polarity was increased to ethyl acetate / petroleum ether = 1 / 100 (containing 0.5% triethylamine), and compound (1) was eluted. After the solvent was spin-dried, 4.1 g of white solid (Mw = 478, 8.58 mmol) was obtained, with a yield of 31.7%.

[0643] H NMR characterization:

[0644] 1H NMR (400MHz, DMSO): δ7.49-7.46(m,2H),7.41-7.34(m,6H),7.27(d,J=8.0Hz,2H),7.00(d,J=8.0Hz,2H),6.59(d, J=7.1Hz,2H),4.66(s,2H),3.95(dd,J=8.8,4.4Hz,1H),3.11(dd,1H),3.00(dd,J=13.2,8.8Hz,1H),1.36(s,9H).

[0645] C NMR characterization:

[0646] 13 C NMR (101MHz, DMSO): δ169.83,169.52,138.92,138.25,136.14,135.52,130.45,129.93 ,129.62,129.03,128.33,128.26,128.16,127.14,80.66,67.59,38.56,34.62,27.61.

[0647] Mass spectrometry characterization:

[0648] LC-MS(ESI)calcd for C 27 H 28 BrNO2(M+1):478.1;obsd:478.1

[0649] (2) tert-butyl(S)-3-(4-(cyanomethyl)phenyl)-2-((diphenylmethylene)amino)propanoate

[0650] The product (1) from the previous step was dissolved in 60 mL of anhydrous acetonitrile, and 2 eq of anhydrous potassium carbonate (2.37 g, 17.2 mM) was added, followed by 2 eq of trimethylsilyl cyanide (1.71 g, 17.2 mmol), and finally heated under reflux at 85 degrees overnight. The next day, the raw material basically disappeared, and a new point with significantly increased polarity appeared. After the reaction was completed, the solid residue was filtered out and oxidized with sodium hypochlorite. The filtered organic phase was separated by silica gel column chromatography, and the eluent composition was ethyl acetate / petroleum ether = 1 / 10 (containing 0.5% triethylamine). Compound (2) was eluted and dried to obtain a white solid, 2.86 g, (Mw = 424, 6.75 mmol), with a yield of 78.6%.

[0651] H NMR characterization:

[0652] 1 H NMR (400MHz, DMSO): δ7.49-7.35(m,8H),7.18(d,J=8.0Hz,2H),7.04(d,J=8.1Hz,2H),6.64(d,J=7.0Hz,2H),3. 97(dd,J=8.6,4.8Hz,1H),3.96(s,2H),3.13(dd,J=13.2,4.7Hz,1H),3.00(dd,J=13.2,8.6Hz,1H),1.36(s,9H).

[0653] C NMR characterization:

[0654] 13 C NMR (101MHz, DMSO): δ169.83,169.49,138.94,137.40,135.57,130.42,130.15,129.29,12 8.53,128.32,128.22,128.17,127.80,127.15,119.37,80.63,67.53,38.47,27.59,22.00.

[0655] Mass spectrometry characterization:

[0656] LC-MS(ESI)calcd for C 28 H 28 N2O2(M+1):425.2; obsd:425.2

[0657] (3) tert-butyl(S,Z)-3-(4-(2-(4-((tert-butoxycarbonyl)amino)phenyl)-1-cyanovinyl)phenyl)-2-((diphenylmethylene)amino)propanoate

[0658] The product (2) (1 eq) of the previous step was dissolved in 80 mL of ultra-dry ethanol, stirred in an ice bath, and then 2.1 eq of KOH (0.80 g, 14.2 mmol) was added. Finally, 1.05 eq of the raw material 4-(Boc-amino)benzaldehyde (1.57 g, 7.10 mmol) dissolved in an appropriate amount of ultra-dry ethanol was slowly added dropwise into the reaction system. After the ice bath system naturally returned to room temperature, the reaction was stirred overnight. The next day, the reaction system changed from colorless to slightly yellow. During post-treatment, the reaction solution was directly mixed with an appropriate amount of silica gel and then mixed. The sample was separated by silica gel column chromatography. The eluent composition was ethyl acetate / petroleum ether = 1 / 20 to 1 / 10 (containing 0.5% triethylamine). Compound (3) was eluted. The product obtained after the solvent was dried was a light yellow solid with a weight of 3.40 g (Mw = 627, 5.42 mmol) and a yield of 80.3%.

[0659] H NMR characterization:

[0660] 1 H NMR (400MHz, MeOD): δ7.86(d,J=8.9Hz,2H),7.66(s,1H),7.56-7.49(m,6H),7.42-7.33(m,6H),7.14(d,J=8.3Hz,2H),6.68(d,J =7.3Hz,2H),4.17(dd,J=9.3,4.3Hz,1H),3.25(dd,J=13.4,4.3Hz,1H),3.13(dd,J=13.4,9.3Hz,1H),1.53(s,9H),1.46(s,9H).

[0661] C NMR characterization:

[0662] 13 C NMR (101MHz, MeOD): δ173.47,172.18,154.78,143.12,142.91,140.59,140.44,137.38,134.42,131.88,131.69,131.66,131.4 1,129.94,129.82,129.46,129.37,129.19,128.73,126.60,119.49,119.20,109.36,82.85,81.29,69.06,40.14,28.64,28.29.

[0663] Mass spectrometry characterization:

[0664] LC-MS(ESI)calcd for C 40 H 41 N3O4(M+1):628.3; obsd:628.3

[0665] (4) pAPCF amino acid

[0666] The product (3) in the previous step was first dissolved in 20 mL of ultra-dry dichloromethane, and then 20 mL of trifluoroacetic acid was added to remove the protecting group. The mixture was stirred at room temperature. After a period of time, a brown-red solid was precipitated in the system. The reaction was continued for several hours. Finally, the solid product was filtered to obtain a solid product, which was rinsed several times with dichloromethane. The product was then dissolved in 3 mL of methanol and added dropwise to 150 mL of ether while stirring vigorously. After several hours, an orange-red solid was filtered to obtain a solid, which was rinsed several times with ether. Finally, the solid was vacuum dried to obtain an orange-red powder, which was the final product pAPCF, (S,Z)-2-amino-3-(4-(2-(4-aminophenyl)-1-cyanovinyl)phenyl)propanoic acid, 1.54 g (5.02 mmol), with a yield of 92.6%.

[0667] H NMR characterization:

[0668] 1 H NMR (600MHz, MeOD): δ7.80(d,J=8.7Hz,2H),7.68(d,J=8.3Hz,2H),7.64(s,1H),7.38(d,J=8.3Hz,2H),6.8 3(d,J=8.7Hz,2H), 4.28(dd,J=7.8,5.6Hz,1H), 3.35(dd,J=14.6,5.6Hz,1H), 3.19(dd,J=14.5,7.8Hz,1H).

[0669] C NMR characterization:

[0670] 13 C NMR (151MHz, MeOD): δ171.16,144.23,136.25,135.79,132.58,131.18,127.09,120.65,120.04,116.56,105.55,64.31,54.99,36.96.

[0671] Mass spectrometry characterization:

[0672] LC-MS(ESI)calcd for C 18 H 17 N3O2(M+1):308.14; obsd:308.14

[0673] Example 2 Synthesis of unnatural amino acids mPAPF and mDMPF

[0674] Referring to the synthesis method of Example 1, the initial raw material was changed from 1,4-di(bromomethyl)benzene to 2,6-di(bromomethyl)benzene to obtain mPAPF, (S,Z)-2-amino-3-(3-(2-(4-aminophenyl)-1-cyanovinyl)phenyl)propanoic acid.

[0675] Based on the above-mentioned route for synthesizing mPAPF, the original raw material 4-(boc-amino)benzaldehyde is replaced with p-dimethylaminobenzaldehyde to finally synthesize mDMPF, (S,Z)-2-amino-3-(3-(1-cyano-2-(4-(dimethylamino)phenyl)vinyl)phenyl)propanoic acid. The synthetic route is shown below.

[0676]

[0677] The comprehensive yield of mPAPF was 29.2%.

[0678] Mass spectrometry characterization: (M+1) + calcd:308.14;obsd:308.15

[0679] NMR characterization of mPAPF:

[0680] 1 H NMR (400MHz, MeOD): δ7.76(d,J=8.3Hz,2H),7.60(s,2H),7.57(d,J=7.9Hz,1H),7.42(t,J=7.8Hz,1H), 7.30(d,J=7.7Hz,1H),6.72(d,J=8.5Hz,2H),3.92(s,1H),3.38(dd,1H),3.10(dd,J=11.7,11.7Hz,1H).

[0681] 13C NMR (101MHz, MeOD): δ152.88,144.91,138.03,137.52,132.69,130.64,130.06,127.37,125.55,123.71,120.42,115.17,104.35,57.00,38.09.

[0682] The comprehensive yield of mDMPF was 31.9%.

[0683] Mass spectrometry characterization: (M+1) + calcd:336.17;obsd:336.18

[0684] NMR characterization of mDMPF:

[0685] 1 H NMR (400MHz, DMSO): δ7.87(d,J=8.6Hz,2H),7.76(s,1H),7.52(s,1H),7.44(d,J=7.8Hz,1H),7.32(t,J=7 .3Hz,1H),7.21(d,J=7.5Hz,1H),6.80(d,J=8.6Hz,2H),3.10(d,J=12.9Hz,2H),3.02(s,6H),2.90(s,1H).

[0686] 13 C NMR (101MHz, DMSO): δ151.57,142.56,141.72,134.48,131.71,131.03,129.08, 128.61,125.82,122.53,120.98,119.43,111.56,102.86,57.55,41.76,39.59.

[0687] Example 3: Synthesis of unnatural amino acids PAPd and DMPd

[0688] Referring to the synthesis method of Example 2, the initial raw material 2,6-di(bromomethyl)benzene is replaced with 2,6-di(bromomethyl)pyridine, and the unnatural amino acid PAPd, (S,Z)-2-amino-3-(6-(2-(4-aminophenyl)-1-cyanovinyl)pyridin-2-yl)propanoic acid, can be finally synthesized. Based on the synthetic route of PAPd, 4-(boc-amino)benzaldehyde is replaced with p-dimethylaminobenzaldehyde to finally synthesize the unnatural amino acid DMPd, (S,Z)-2-amino-3-(6-(1-cyano-2-(4-(dimethylamino)phenyl)vinyl)pyridine-2-yl)propanoic acid. The synthetic route is shown below.

[0689] The 2,6-bis(bromomethyl)pyridine in the synthetic route can be prepared from the raw material 2,6-pyridine dimethanol (as described below), and the remaining synthetic steps can refer to the synthesis in Example 2 and will not be repeated here.

[0690]

[0691] (1) Synthesis of 2,6-bis(bromomethyl)pyridine

[0692] Take 4.17g (30mmol) of raw material 2,6-pyridine dimethanol into a 250mL round-bottom flask, add 100mL of anhydrous dichloromethane and 2.37g (30mmol) of ultra-dry pyridine, then evacuate and replace with nitrogen. Put the reaction bottle in an ice bath, and slowly add phosphorus tribromide (75mmol) to it with a syringe. Stir continuously and react overnight. After the reaction is nearly completed by thin layer chromatography (TLC) the next day, methanol is slowly added to the reaction system under an ice bath to quench the remaining phosphorus tribromide, and then potassium hydroxide is used to adjust the pH of the system to 5-6. The reaction solution is extracted and separated with dichloromethane, and the organic phase is washed twice with saturated brine and then dried with anhydrous sodium sulfate. Finally, the solvent is spin-dried to obtain a white solid. At this time, after TLC detection, the product is relatively pure with a yield of 77.3%, and can be directly used for the next step reaction.

[0693] NMR characterization:

[0694] 1H NMR (400MHz, DMSO): δ7.83 (t, J = 7.7Hz, 1H), 7.48 (d, J = 7.7Hz, 2H), 4.67 (s, 4H).

[0695] 13 C NMR (101MHz, DMSO): δ156.56,138.45,123.13,34.40.

[0696] Mass spectrometry characterization:

[0697] LC-MS(ESI)calcd for C7H7Br2N(M+1):265.9;obsd:265.

[0698] (2) PAPd amino acid. PAPd amino acid was prepared as a reddish brown solid with a comprehensive yield of 28.8%.

[0699] (3) DMPd amino acid. DMPd amino acid was prepared as a dark red solid with a comprehensive yield of 31.4%.

[0700] Mass spectrometry characterization of PAPd:

[0701] LC-MS(ESI)calcd for C 17 H 16 N4O2(M+1):309.14;obsd:309.14

[0702] NMR characterization of PAPd:

[0703] 1 H NMR (400MHz, MeOD): δ8.18(s,1H),7.87(d,J=8.6Hz,2H),7.80(t,J=7.8Hz,1H),7.66(d,J=8.0Hz,1H),7.24(d, J=7.6Hz,1H),6.72(d,J=8.8Hz,2H),4.07(dd,J=8.6,3.3Hz,1H),3.47(d,J=3.3Hz,1H),3.28(d,J=4.4Hz,1H).

[0704] 13 C NMR (101MHz, DMSO): δ177.64,161.15,152.54,151.12,144.78,137.30,132.21,122.52,120.26,119.30,116.71,113.50,101.79,56.32,44.13.

[0705] Mass spectrometry characterization of DMPd: LC-MS (ESI) calcd for (M+1) + :337.17; obsd:337.17

[0706] NMR Characterization of DMPd:

[0707] 1 H NMR (400MHz, DMSO): δ8.31(s,1H),7.94(d,J=8.9Hz,2H),7.76(t,J=7.8Hz,1H),7.50(d,J=7. 8Hz,1H),7.21(d,J=7.6Hz,1H),6.83(d,J=8.8Hz,2H),3.48(s,2H),3.17(s,1H),3.05(s,6H).

[0708] 13 C NMR (101MHz, DMSO) δ170.64,155.45,151.76,151.29,145.92,138.66,132.09,122.76,119.12,118.12,112.48,101.80,50.61,40.11,36.10.

[0709] Example 4 Synthesis of unnatural amino acid PAFCN

[0710] Based on the synthesis route of mPAPF in Example 2, the raw material 2,6-di(bromomethyl)benzene is replaced with 2,6-di(bromomethyl)benzonitrile, and the amino acid PAFCN, (S,Z)-2-amino-3-(3-(2-(4-aminophenyl)-1-cyanovinyl)-2-cyanophenyl)propanoic acid ((S,Z)-2-amino-3-(3-(2-(4-aminophenyl)-1-cyanovinyl)-2cyanophenyl)propanoic acid) can be finally synthesized.

[0711] The first step of the synthetic route requires the preparation of 2,6-bis(bromomethyl)benzonitrile (as shown below) via 2,6-dimethylbenzonitrile. The overall synthetic route is shown below.

[0712]

[0713] Synthesis of 2,6-Bis(bromomethyl)benzonitrile

[0714] 30mM (3.93g) of 2,6-dimethylbenzonitrile raw material was added, 0.1eq of AIBN (0.492g) was added, and 100mL of anhydrous acetonitrile was used as solvent. The air was evacuated, nitrogen was added for protection, and after heating at 90 degrees for 15min, 3eq of NBS (15.9g) was added. After about 12 hours, the reaction was completed, and the ratio of monobrominated and dibrominated products was about 6:4. After treatment, the reaction solution was first treated with a saturated sodium sulfite solution to consume the remaining NBS, and then extracted with ethyl acetate. The obtained organic phase was washed twice with saturated brine and then dried over anhydrous sodium sulfate. Finally, it was separated by silica gel column chromatography, and the eluent was ethyl acetate / petroleum ether = 1 / 25. After the solvent was dried, 1.98g (6.9mmol) of white solid was obtained with a yield of 23.0%. The remaining synthesis steps refer to the method of mPAPF. Finally, the comprehensive yield of PAFCN amino acid was 9.5%

[0715] Mass spectrometry characterization of PAFCN: LC-MS (ESI) calcd for (M+1) + :333.14; obsd:333.14

[0716] NMR characterization of PAFCN:

[0717] 1 H NMR (400MHz, DMSO): δ7.72(d,J=8.8Hz,2H),7.66(d,J=7.8Hz,1H),7.54(t,J=8.7Hz,2H),7.45(s,1H),6.6 6(d,J=8.7Hz,2H), 3.61(dd,J=8.9,5.7Hz,1H), 3.45(dd,J=14.7,5.7Hz,1H), 3.07(dd,J=14.6,8.9Hz,1H).

[0718] 13 C NMR (101MHz, DMSO): δ168.93,152.78,149.57,143.23,140.17,133.01,131.83, 129.98,127.52,120.07,118.83,116.30,113.40,110.96,98.05,54.03,36.13.

[0719] Example 5: Performance Test

[0720] 1. Fluorescence spectroscopy measurement

[0721] (1) Absorption spectrum. The non-natural amino acid prepared above was first dissolved in DMSO to form a 100 mM stock solution, and then diluted into various solvents (methanol, glycerol, acetonitrile, DMSO, PBS (phosphate buffer)) to a final concentration of 100 uM. The absorbance between 300-500 nm was measured using a Hitachi UH5300 spectrophotometer with an interval of 2 nm.

[0722] (2) Fluorescence emission spectrum: The 100 uM solution prepared by the above method was used to measure the fluorescence emission spectrum using a Hitachi F-7000 fluorescence spectrometer, with an excitation wavelength of 365 nm, a receiving wavelength range of 380-600 nm, and PMT=600V.

[0723] The inventors dissolved pAPCF in different solvents and then measured the corresponding absorption and fluorescence emission spectra ( Figure 1 and Table 1).

[0724] Table 1 Maximum absorption wavelength and emission wavelength of pAPCF in various solvents

[0725]

[0726] From the results, we can see that the fluorescence wavelength of pAPCF is in the range of 435-470nm, which is approximately blue-green. In different solvents, the spectrum of pAPCF is also different. The maximum fluorescence emission wavelengths shown in several tested solvents can differ by nearly 40nm (Em=435nm in MeCN; Em=470nm in DMSO). A basic rule is that the more polar the solvent, the more red-shifted the fluorescence emission wavelength. In addition, the inventors also noticed during the test that the fluorescence brightness of pAPCF in glycerol was significantly stronger, so the viscosity dependence of its fluorescence will be characterized in detail next.

[0727] For other unnatural amino acids, the inventors expected that their fluorescence wavelengths would also shift, considering that their conjugated structures contained dimethylamino groups that were more electron-rich or pyridine rings or cyano groups that were more electron-deficient than benzene rings. The inventors dissolved these unnatural amino acids in glycerol and then measured their spectra. The results were as follows: Figure 2 As shown in Table 2, compared with the original pAPCF, the maximum fluorescence emission wavelengths of the derived non-natural amino acids have all been red-shifted, and DMPd has even reached the green light band (Em max = 514 nm). This suggests that the derived non-natural amino acids may provide different wavelength options from pAPCF in subsequent studies, and also proves that chemical derivatization based on the pAPCF skeleton (especially changing the electron-donating group and the electron-withdrawing group) can effectively change the fluorescence wavelength of the non-natural amino acids of the present invention.

[0728] Table 2 Maximum fluorescence excitation, emission wavelength and fluorescence activation multiples of various unnatural amino acids in glycerol solvent

[0729]

[0730]

[0731] 2. Characterization of Viscosity Dependence of Fluorescence

[0732] A series of glycerol / water mixed solvents with different glycerol volume ratios were prepared: 0, 20%, 40%, 60%, 80%, 90%, 100%. The non-natural amino acid mother solutions prepared above were added to different solvent systems to make the final concentration 20uM, and three parallel replicates were set. Finally, the microplate reader was used to read the results, Ex = 380nm, Em = 465nm.

[0733] The inventors used a glycerol / water mixture to prepare a series of solvents with different viscosities, and then investigated the changes in the fluorescence intensity of pAPCF and PAPd as the viscosity increased. Figure 3 As shown in the figure, in pure methanol solvent, pAPCF has almost no fluorescence, but as the viscosity of the system increases, the fluorescence of pAPCF increases accordingly, and the fluorescence intensity in 100% glycerol is 112 times higher than that in methanol. This result fully demonstrates that pAPCF has the typical characteristics of a fluorescent molecular rotor, that is, there is almost no fluorescence background in a low-viscosity solution, and the fluorescence intensity has a good positive correlation with the environmental viscosity. This experimental result also laid the foundation for the subsequent use of pAPCF as a GFP chromophore analog and the construction of a fluorescent probe.

[0734] Similarly, the inventors also determined the dependence of various other unnatural amino acids on viscosity. Figure 4 As shown in the figure, these unnatural amino acids all have typical fluorescence viscosity dependence characteristics of fluorescent molecular rotors. They have almost no fluorescence in low-viscosity methanol solvents, but the fluorescence intensity gradually increases with the increase of glycerol concentration in the system. The fluorescence intensity in 100% glycerol is 217 times (PAPd), 151 times (DMPd), 60 times (mPAPF), 97 times (PAFCN), and 264 times (mDMPF) higher than that in methanol. In addition, the fluorescence colors displayed by them when excited in glycerol also cover a wider range from blue to green (such as Figure 1 ).

[0735] Example 6 Screening for mutants of the MjTyrRS system that can be used in prokaryotic cells

[0736] In this embodiment, a library of aminoacyl tRNA synthetase / tRNA pairs (aaRS / tRNA) mutants was constructed, and fluorescent rotor-type non-natural amino acids were screened on a small scale to obtain aaRS / tRNA pairs that can efficiently identify and catalyze fluorescent rotor-type non-natural amino acids, thereby realizing the genetic coding of fluorescent rotor-type non-natural amino acids. Certain sites of the fluorescent protein sfGFP (amino acid sequence as shown in SEQ ID NO: 19) can highly tolerate point mutations of amino acids, so the coding nucleotides of a certain permissive site (such as site 151) in the middle of sfGFP are mutated into stop codons-TAG to prepare for the introduction of fluorescent rotor-type non-natural amino acids into the site (i.e., replacing the original amino acid residues at the site with non-natural amino acids). The sfGFP (151TAG) reporter gene (its nucleotide sequence is shown in SEQ ID NO: 22) is used to evaluate the introduction efficiency of fluorescent rotor-type non-natural amino acids. At the same time, a cell flow sorting technique based on fluorescence is used for screening. To this end, the inventors first used a library of aaRS mutants to perform a simple screening of pAPCF in Escherichia coli.

[0737] Screening method: Screening and identification of specific aaRS / tRNA pairs in E. coli

[0738] The BL21 (DE3) glycerol bacteria carrying the aaRS / tRNA library and the reporter gene sfGFP (151TAG) were revived and cultured, and then IPTG was added to 0.5mM. No unnatural amino acids were added to the negative group, and the corresponding unnatural amino acids were added to 1mM to the positive group, and the culture was continued to induce the expression of GFP. The bacteria were collected and washed once with PBS buffer, and then the absorbance value of the bacterial solution at 600nm and the GFP fluorescence intensity were read using an ELISA reader, and the GFP / OD600 value was calculated as an indicator of TAG read-through efficiency. The candidate aaRS / tRNA group can show a very low GFP / OD600 value under negative conditions (meaning that there is no misrecognition of natural amino acids and no read-through of TAG stop codons), and a very high GFP / OD600 value under positive conditions (meaning that unnatural amino acids can be efficiently recognized and TAG stop codons can be read through, resulting in the expression of complete sfGFP protein). The selected bacterial solution containing candidate aaRS / tRNA was stored and awaited for subsequent experiments.

[0739] This example compares the fluorescence intensity of complete sfGFP expression induced by adding pAPCF in the presence of each group of aaRS mutants. The final experimental results found an aaRS mutant from the archaeon Methanococcus.Jannaschii (Mj), and named this MjTyrRS mutant that selectively and efficiently recognizes pAPCF as MjFmrRS (amino acid sequence as shown in SEQ ID NO: 15), which contains point mutations Y32G, L65Y, F108H, Q109G, Q158G, and D286R compared to the wild-type MjTyrRS. This conclusion is supported by the sfGFP fluorescence numerical measurement and protein spectrum identification ( Figure 5 ). Due to the MjTyrRS / tRNA tyr (tRNA tyr The nucleotide sequence is shown in SEQ ID NO: 16) can be orthogonal to prokaryotes, so the MjTyrRS system can be used in prokaryotes. Subsequent studies will use MjFmrRS / tRNA tyr The fluorescent protein synthesis of pAPCF was studied in the prokaryotic system.

[0740] Example 7 Screening for pyrrolysyl tRNA synthetase mutants

[0741] The above Example 6 has already used MjFmrRS / tRNA tyr The efficient introduction of pAPCF amino acids into the expressed protein / peptide in the E. coli expression system. In order to expand the application of fluorescent rotor-type unnatural amino acids in eukaryotic cells, the inventors used the pyrrolysyl-tRNA synthetase (PylRS) family that can be used in both prokaryotic and eukaryotic systems to conduct library screening to obtain mutants that can efficiently recognize fluorescent rotor-type unnatural amino acids.

[0742] Step 1: Construction of PylRS mutant library

[0743] The PylRS system was used, and the Methanogenic archaeon ISO4-G1PylRS (G1pylRS, whose amino acid sequence is shown in SEQ ID NO: 23) family with a large binding pocket was used as the basis for constructing the aaRS mutation library. The following key amino acid sites were selected in the substrate binding pocket of G1pylRS for NNK saturation mutation (saturation mutation of 20 natural amino acids was performed on the target site, usually using the degenerate codon NNK (N = A / T / G / C, K = G / T)): L124, Y125, M128, N165, V167, Y204. Finally, the pBK-G1PylRS-Lib plasmid library was obtained.

[0744] Step 2: Fluorescence flow cytometry screening of the PylRS mutant library

[0745] The constructed pBK-G1PylRS-Lib plasmid and the screening plasmid pRep-RFP-GFP (the nucleotide sequence encoding GFP is shown in SEQ ID NO: 20; the nucleotide sequence encoding RFP is shown in SEQ ID NO: 21) were transferred into DH10B Escherichia coli for culture. Then, induction was performed. For the positive screening process, L-arabinose was added to a final concentration of 0.2%, and non-natural amino acids were added to 1 mM; for the negative screening process, only L-arabinose was added for induction. After about 16 hours of culture, the bacteria were collected. During fluorescence flow sorting, the fluorescence signal of GFP was used as an internal reference, and the fluorescence signal of RFP was used as a standard to reflect the TAG read-through efficiency. In this experiment, the TAG stop codon was designed to be located on the T7 RNA polymerase gene (whose nucleotide sequence is shown in SEQ ID NO: 1) that controls the transcription of RFP. Therefore, only when the TAG stop codon can be read through can the efficient expression of RFP be caused. For positive screening, both GFP and RFP of positive bacteria are highly expressed, so the P5 gate in the upper right area is collected; for negative screening, in order to exclude false positives (false positive mutants will recognize natural amino acids and cause TAG to be read through in the absence of non-natural amino acids, thereby leading to high expression of RFP), bacteria that only highly express GFP are collected, so the P4 gate in the lower right area is collected ( Figure 6 The enriched bacteria were revived and cultured, and a portion was taken for the next round of screening according to the above method. After 3-5 rounds of alternating positive and negative screening, a PylRS mutant that specifically and efficiently recognized the target non-natural amino acid was obtained.

[0746] Step 3: Verify the efficiency of PylRS mutants

[0747] Based on fluorescence flow sorting, the following PylRS mutants were obtained: C-C9 PylRS (amino acid sequence shown in SEQ ID NO: 12) and AC2 PylRS (amino acid sequence shown in SEQ ID NO: 13). pyl and pUltra-AC2 PylRS-tRNA pyl plasmids, which contain the encoding C-C9PylRS and tRNA pyl The nucleotide sequence of AC2 PylRS and tRNA pyl The nucleotide sequence of pyl The nucleotide sequence of pUltra-CC9 PylRS-tRNA is shown in SEQ ID NO: 14.pyl and pUltra-AC2 PylRS-tRNA pyl They were respectively transferred into BL21 (DE3) together with the pET-sfGFP (151TAG) reporter plasmid, and the GFP protein expression was induced according to the method steps of Example 6. The GFP / OD600 value was calculated to obtain quantitative data on the efficiency and selectivity of the PylRS mutant for introducing fluorescent rotor-type non-natural amino acids.

[0748] The PylRS family can be used in both prokaryotic and eukaryotic systems. Experiments have shown that C-C9 PyRS can simultaneously and efficiently recognize amino acids such as pAPCF, PAPd, PAFCN, and DMPd, while AC2 PylRS can simultaneously and efficiently recognize amino acids such as mPAPF, mDMPF, and DMPd ( Figure 7 ). In brief, in order to verify the efficiency of the introduction of unnatural amino acids in eukaryotic cells, a gene encoding C-C9 PylRS or AC2 PylRS and tRNA pyl A plasmid containing the nucleotide sequence of pyl Plasmid; see Example 9 for detailed description) and pCDNA3.1-mCherry-EGFP (39TAG) plasmid (see Example 9 for detailed description) were simultaneously transfected into HEK293T cells, and the cell culture medium was then replaced with fresh culture medium containing 50-500uM of a specified non-natural amino acid (e.g., pAPCF or PAPd), and culture was continued for 36-48 hours. The cells were lysed, the mCherry red fluorescence and EGFP enhanced green fluorescence were measured, and the EGFP / mCherry ratio corresponding to each well was calculated as an indicator of the efficiency of introduction of non-natural amino acids (e.g., pAPCF or PAPd). The results showed that C-C9 PyRS can simultaneously and efficiently recognize amino acids such as pAPCF, PAPd, PAFCN, and DMPd, and introduce them into polypeptides expressed using a eukaryotic cell expression system; while AC2PylRS can simultaneously and efficiently recognize amino acids such as mPAPF, mDMPF, and DMPd, and introduce them into polypeptides expressed using a eukaryotic cell expression system ( Figure 7 ).

[0749] Example 8: Using the protein Z and Affibody interaction model to verify the fluorescent probe function of pAPCF

[0750] In this embodiment, the inventors used a classic model based on the interaction between protein Z and Affibody to verify the fluorescent probe function of the fluorescent rotor-type non-natural amino acid pAPCF. Protein Z is a three-helix bundle domain containing 58 amino acids derived from Staphylococcus protein A, which can bind to the Fc part of IgG of different species, and there are 13 amino acids in the binding region that can be highly variable. High-affinity protein-Affibody can be obtained by mutation screening of protein Z through phage display technology. At the same time, the mutant Affibody can also bind to the original protein Z, and the interaction between the two has a moderate affinity and a simple structure, which is a classic model for studying protein interactions.

[0751] In this example, pAPCF is introduced into the interface or its vicinity where protein Z interacts with Affibody to verify the perception of the fluorescent rotor-type non-natural amino acid on protein interaction of the present application. Figure 8 As shown in a, in the absence of Affibody, the pAPCF on the interface of protein Z (e.g., site 17) can rotate freely, thereby not emitting fluorescence. In the presence of Affibody, protein Z interacts with Affibody, resulting in the restriction of the intramolecular rotation of pAPCF located on the interaction interface (e.g., site 17), thereby generating fluorescence. Therefore, pAPCF can be used as a fluorescent probe to detect the interaction between protein Z and Affibody. The amino acid residues located at the interface interacting with Affibody or its vicinity in protein Z mainly include amino acid residues (Q10, F13, Y14, L17, N28, I31, K35) on sites 10, 13, 14, 17, 28, 31, and 35. In the present embodiment, taking sites 13 and 17 of protein Z as an example, the amino acid residues at sites 13 and 17 of protein Z are replaced with pAPCF respectively to verify the fluorescent probe function of pAPCF. The present embodiment mainly includes the following experimental steps.

[0752] Step 1: Construction of protein Z mutant introduced into pAPCF

[0753] The amino acid residue L at position 17 of the wild-type protein Z (abbreviated as Z-wt, whose amino acid sequence is shown in SEQ ID NO: 2) (which is located at the interface where protein Z interacts with Affibody) is replaced with pAPCF, and the F13A point mutation is introduced to obtain the Z-13A-17pAPCF mutant (abbreviated as Z-17, whose amino acid sequence is shown in SEQ ID NO: 3). In addition, the amino acid residue F at position 13 of the wild-type protein Z (which is located near the interface where protein Z interacts with Affibody) is replaced with pAPCF to obtain the Z-13pAPCF mutant (abbreviated as Z-13, whose amino acid sequence is shown in SEQ ID NO: 4, sequence provided). In addition, a 6*His tag (whose amino acid sequence is shown in SEQ ID NO: 5) is fused to the C-terminus of proteins Z-wt, Z-17 and Z-13 to facilitate protein purification.

[0754] Step 2: Expression and purification of MBP-Affibody, protein Z and its mutants

[0755] MBP-Affibody (abbreviated as Afb, whose amino acid sequence is shown in SEQ ID NO: 6) and Z-wt, Z-13, and Z-17 proteins carrying a soluble expression tag MBP at the N-terminus and fused to a 6*His tag at the C-terminus were expressed and purified in Escherichia coli by conventional methods. In particular, in order to achieve the expression of non-natural amino acids pAPCF in proteins, plasmids encoding Z-13 or Z-17 proteins (wherein pAPCF is encoded by the selector codon TAG, which is a nonsense codon commonly used in gene codon expansion technology for encoding non-natural amino acids) and pUltra-MjFmrRS plasmids constructed as described above (which encode MjFmrRS and corresponding tRNA capable of recognizing pAPCF) were simultaneously transferred into BL21 (DE3) (purchased from Thermo Fisher), and protein expression was induced in a culture medium containing 0.1 mM IPTG (purchased from INALCO) and 0.8 mM pAPCF. Then, the expressed protein carrying the 6*His tag was purified using Ni-NTAAgarose (Qiagen, Germany).

[0756] Step 3. Measurement of the interaction between MBP-Affibody and protein Z mutant

[0757] The molar concentrations of the obtained Afb, Z-13 and Z-17 proteins were determined. The concentration of Z-13 or Z-17 protein was then adjusted to 25uM, and then Afb with gradient concentrations of 0, 25, 50, 100, 200, and 250uM was added thereto, respectively, so that the total volume of each sample was 100ul, and after mixing, the mixture was placed on ice for 20min, and then the fluorescence intensity was measured with an ELISA reader.

[0758] Step 4: Wild-type protein Z competitively binds to MBP-Affibody

[0759] The concentration of Z-13 or Z-17 was adjusted to 20uM, and 20, 40, and 100uM of Z-wt were added thereto, respectively, and finally 40uM of Afb was added to each sample to make a total volume of 100ul. After mixing, the mixture was placed on ice for 20min, and the fluorescence intensity was measured with an enzyme-labeled instrument.

[0760] The results showed that the single Z-17 protein had almost no fluorescence. After gradually adding Affibody to the system, the fluorescence of the system also increased significantly with the increase of Affibody concentration. The fluorescence intensity was dependent on Affibody concentration ( Figure 8 b and Figure 8 c). In the competitive inhibition experiment of step 4, Z-wt effectively weakened the binding of Z-17 and Affibody, and the fluorescence intensity of the system gradually decreased with the increase of Z-wt concentration ( Fig. 9 ). In parallel experiments, similar detection results were obtained using the Z-13 mutant. The fluorescence intensity of the Z-13-Affibody interaction system also increased with the increase of Affibody concentration, and was reduced by competitive inhibition of Z-wt ( Fig.10 ). The above results indicate that the fluorescent rotor-type unnatural amino acid pAPCF can be used as a fluorescent probe for detecting protein interactions and is not site-dependent.

[0761] Example 9: Use of pAPCF as a precise fluorescent probe for detecting protein aggregates such as abnormal aggregation of SOD1

[0762] This example and Example 10 both use the novel aminoacyl-tRNA synthetase (aaRS) mutant designed by the present application to achieve codon expansion of pAPCF and PAPd in ​​prokaryotic / eukaryotic cells, and verify the availability of the fluorescent rotor-type non-natural amino acids of the present application in prokaryotic / eukaryotic cells.

[0763] In this example, pAPCF is directly introduced into the flexible exposed region (mainly including amino acid residues at positions 25, 40, 47 and 67 (S25, E40, V47, L67)) in the superoxide dismutase 1 (SOD1) mutant. pAPCF can rotate freely in the normally folded free SOD1 and therefore does not produce fluorescence. Further, after giving the cell drug stimulation-proteasome inhibitor MG132 (which can interfere with the protein degradation homeostasis of the cell), the abnormal SOD1 will accumulate and aggregate in the cell, thereby encapsulating pAPCF in the SOD1 aggregate, causing pAPCF to be unable to rotate freely, thereby activating the fluorescence of pAPCF. This example proves that the fluorescent rotor-type non-natural amino acids of the present application can not only be normally expressed in prokaryotic / eukaryotic cells and incorporated into the target polypeptide, but also can be used as fluorescent probes for detecting abnormal aggregation states of proteins (i.e., identifying the degree of aggregation of proteins by detecting the intensity and size of the fluorescence signal of the aggregates).

[0764] The following plasmids were used in this example:

[0765] (1) pCMV-SOD1(A4V)-40TAG-mCherry plasmid, encoding a fusion protein SOD1(A4V)-40pAPCF-mCherry. The fusion protein comprises a SOD1 mutant SOD1(A4V)-40pAPCF (whose amino acid sequence is shown in SEQ ID NO:7), which, compared with the wild-type SOD1 (Genbank ID:AH003628.2), has a point mutation A4V (this point mutation makes SOD1 unstable and prone to aggregation), replaces the amino acid at position 40 (which is located in the flexible exposed region of SOD1) with pAPCF, and is connected to the red fluorescent protein mCherry (which can emit red fluorescence for co-localization with the fluorescent signal of pAPCF, whose amino acid sequence is shown in SEQ ID NO:9) at the C-terminus of the SOD1 mutant via a linker (whose amino acid sequence is shown in SEQ ID NO:8). The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 10, wherein pAPCF is encoded by a nonsense codon TAG (TAG is a nonsense codon commonly used in gene codon expansion technology for encoding unnatural amino acids). The nucleotide sequence encoding the fusion protein is introduced into the pCMV eukaryotic expression vector by Gibson ligation to obtain the target plasmid.

[0766] (2) pCMV-SOD1(V31A)-25TAG-mCherry plasmid, encoding a fusion protein SOD1(V31A)-25pAPCF-mCherry. The fusion protein comprises a SOD1 mutant SOD1(V31A)-25pAPCF (whose amino acid sequence is shown in SEQ ID NO: 11), which, compared with the wild-type SOD1 (Genbank ID: AH003628.2), has a point mutation V31A (this point mutation makes SOD1 unstable and prone to aggregation, but the aggregation tendency is weaker than that of the A4V mutant), the amino acid at position 25 (which is located in the flexible exposed region of SOD1) is replaced with pAPCF, and is connected to the C-terminal red fluorescent protein mCherry of the SOD1 mutant (which can emit red fluorescence for co-localization with the fluorescent signal of pAPCF, and its amino acid sequence is shown in SEQ ID NO: 9) through a linker (whose amino acid sequence is shown in SEQ ID NO: 8). The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 24, wherein pAPCF is encoded by the nonsense codon TAG (TAG is a nonsense codon commonly used in gene codon expansion technology for encoding unnatural amino acids). The nucleotide sequence encoding the fusion protein is introduced into the pCMV eukaryotic expression vector by Gibson ligation to obtain the target plasmid.

[0767] (3)pCMV-C-C9 pylRS-tRNA pyl The plasmid, whose backbone is a pCMV eukaryotic expression vector, contains a C-C9PylRS mutant (whose amino acid sequence is shown in SEQ ID NO: 12) and a tRNA pyl (The nucleotide sequence of which is shown in SEQ ID NO: 14).

[0768] (4) pUltra-MjFmrRS plasmid, whose backbone is the pUltra expression vector, containing the encoding MjFmrRS (whose amino acid sequence is shown in SEQ ID NO: 15) and tRNA tyr (The nucleotide sequence is shown in SEQ ID NO: 16). MjFmrRS can selectively and efficiently recognize pAPCF and accurately introduce pAPCF into the site corresponding to the nonsense codon TAG.

[0769] (5) pET22b-SOD1 (wt) plasmid: The target plasmid was obtained by synthesizing the human wild-type SOD1 gene (Genbank ID: AH003628.2) and cloning it into the pET22b protein expression vector.

[0770] (6) pET22b-SOD1(V31A)-25TAG plasmid, whose backbone is the pET22b protein expression vector, encoding the SOD1 mutant SOD1(V31A)-25pAPCF (whose amino acid sequence is shown in SEQ ID NO: 11).

[0771] (7) pCDNA3.1-mCherry-EGFP (39TAG) plasmid, which encodes mCherry-EGFP fusion protein (its amino acid sequence is shown in SEQ ID NO: 17), wherein mCherry fluorescent protein is fused to the N-terminus of EGFP, and the amino acid at position 39 of EGFP is replaced with unnatural amino acid pAPCF or PAPd (which is encoded by the selector codon TAG). The nucleotide sequence encoding the fusion protein is introduced into the pCDNA3.1 expression vector to obtain the target plasmid.

[0772] This embodiment mainly includes the following experimental steps.

[0773] Step 1: Expression and purification of SOD1(V31A)-25pAPCF protein and temperature-induced abnormal aggregation of SOD1

[0774] The above plasmid was used to express SOD1(V31A)-25pAPCF protein in Escherichia coli. The protein expression and purification method is described in Example 8. In brief, pET22b-SOD1(V31A)-25TAG and pUltra-MjFmrRS plasmids were simultaneously transferred into BL21(DE3) super competent cells, and protein expression was induced at 18°C ​​for 16h in a medium containing 0.2mM IPTG and 1mM pAPCF. The purified protein samples were then incubated at 37°C, 45°C, 52°C, and 60°C for 0.5 hours, after which the turbidity and fluorescence intensity were measured, and the fluorescence of the protein precipitate was observed.

[0775] The results showed that SOD1(V31A)-25pAPCF obtained by expression and purification in E. coli gradually aggregated to form insoluble small particles after heating (as shown by the increase of absorbance at 395nm), and the fluorescence intensity of pAPCF also increased accordingly ( Fig.11 ). The experimental results show that the fluorescent rotor-type unnatural amino acid pAPCF of the present application can not only be normally expressed in prokaryotic cells and incorporated into the target polypeptide, but also, at the in vitro protein level, can be used as a fluorescent probe to detect abnormal protein aggregation.

[0776] Step 2: Subculture and plasmid transfection of HEK293T cells

[0777] This experiment used eukaryotic cells HEK293T. In order to verify the efficiency of the introduction of unnatural amino acids in eukaryotic cells, pCMV-C-C9 PylRS-tRNA pyl Plasmid and pCDNA3.1-mCherry-EGFP (39TAG) plasmid were transfected into HEK293T cells at the same time, and then the cell culture medium was replaced with fresh culture medium containing 50-500uM pAPCF or 50-500uM PAPd, and culture was continued for 36-48 hours. The cells were lysed, the mCherry red fluorescence and EGFP enhanced green fluorescence were measured, and the EGFP / mCherry ratio corresponding to each well was calculated as an indicator of the efficiency of pAPCF and PAPd introduction. The results showed that both unnatural amino acids pAPCF and PAPd can be effectively introduced into polypeptides expressed using eukaryotic cell expression systems.

[0778] The above-mentioned pCMV-SOD1(A4V)-40TAG-mCherry plasmid or pCMV-SOD1(V31A)-25TAG-mCherry plasmid was combined with pCMV-C-C9 PylRS-tRNA pyl The plasmids were co-transfected into HEK293T cells at a growth density of about 80%, and the cells were cultured for 4 hours. Then, the culture medium was replaced with a fresh culture medium containing 200 uM pAPCF, and the cells were cultured for 24 hours.

[0779] Step 3: Confocal imaging experiment to explore the function of pAPCF probe

[0780] The cells obtained in step 2 were inoculated into confocal dishes, and 1 ml of fresh DMEM medium (containing 200 uM pAPCF) was added to each dish, and the culture was continued for about 20 hours. Six hours before shooting with a laser confocal microscope, the proteasome inhibitor MG132 (1 mM mother solution, dissolved in DMSO) was added to the experimental group cells to 0.5 or 5 uM, and the culture was continued. 0.5 h before shooting, 5 uM of the nuclear dye Draq 5 was added and the culture was continued. Finally, a confocal imaging experiment was performed. The pAPCF fluorescence signal was used to detect the aggregation of SOD1, with 405 nm excitation set and 450-540 nm fluorescence received. mCherry was used for co-localization observation, with 561 nm excitation set and 590-650 nm fluorescence received.

[0781] The results showed that at the HEK293T cell level, after induction with the proteasome inhibitor MG132, SOD1(A4V)-40pAPCF and SOD1(V31A)-25pAPCF mutant proteins accumulated and aggregated in the cells, and pAPCF fluorescence was detected. Obvious fluorescent spots were observed using laser confocal microscopy, in which the mCherry signal traced the aggregation area of ​​SOD1, and the significantly activated pAPCF fluorescence co-localized well with mCherry. In addition, the aggregation degree of SOD1 increased with the increase of MG132 concentration, and the fluorescence signal of pAPCF also increased accordingly ( Fig.12 ). The experimental results show that the fluorescent rotor-type unnatural amino acid pAPCF of the present application can not only be normally expressed in eukaryotic cells and incorporated into the target polypeptide, but also, at the cellular level, can be used as a fluorescent probe to detect abnormal protein aggregation.

[0782] The above experimental results verified the ability of pAPCF to detect protein aggregation in living cells, which shows that it is a feasible strategy to directly introduce pAPCF into the specific site of the target protein as a fluorescent probe, and compared with other methods, pAPCF can directly and accurately sense the state of the target protein. In addition, in the live cell experiment using pAPCF, there is no need for complex processing such as washing, and the signal to be detected can be directly observed, which is simpler and more efficient.

[0783] Example 10: Using the rapamycin-induced FKBP and FRB protein interaction model to validate the PAPd fluorescence probe Needle function

[0784] This embodiment uses HEK293T cells to detect the fluorescent probe function of fluorescent rotor-type non-natural amino acids at the level of mammalian living cells. The inventors selected a model of FKBP and FRB protein interaction induced by rapamycin. FK506 binding protein (FKBP, FK506 binding protein) is a heat shock protein that is widely expressed in mammals and can specifically bind to the immunosuppressant FK506 (Tacrolimus) and rapamycin (Rapamycin). And FRB (FKBP-rapamycin binding domain) is a domain of mammalian rapamycin target protein (mTOR). Under the action of rapamycin, FKBP combined with rapamycin can further combine with FRB to form a stable ternary complex.

[0785] The interaction interface between FKBP and FRB protein mainly involves amino acid residues (V76, T80, D84, Y87, R91) on sites 76, 80, 84, 87 and 91 of FRB protein and amino acid residues (K45, H88) on sites 45 and 88 of FKBP protein. In this embodiment, PAPd is introduced into the site of the interaction interface between FKBP and FRB protein or its adjacent position (taking sites 87 and 84 of FRB protein as an example), and then the feasibility of PAPd as a fluorescent probe for detecting protein interactions is explored. In short, the amino acid residues on site 87 on the FRB protein that directly contacts FKBP and the amino acid residues on site 84 located in the edge region of the interaction interface are replaced with PAPd, respectively, to verify the fluorescent probe function of PAPd.

[0786] This embodiment mainly includes the following experimental steps.

[0787] Steps include constructing mutants that introduce PAPd into different sites of the FKBP-FRB protein complex, and conducting confocal imaging experiments to explore the function of the PAPd probe.

[0788] Step 1. Plasmid transfection and fluorescence measurement

[0789] The cells, cell culture method and plasmid transfection method used in this step are the same as those in Example 9. The following plasmids were used during the experiment:

[0790] (1) pCMV-C-C9 PylRS-tRNA mentioned in the previous examples pyl Plasmids;

[0791] (2) The pCDNA3.1-mCherry-EGFP (39TAG) plasmid mentioned in the above embodiment encodes the mCherry-EGFP fusion protein (its amino acid sequence is shown in SEQ ID NO: 17), wherein the mCherry fluorescent protein is fused to the N-terminus of EGFP, and the amino acid at position 39 of EGFP is replaced with the unnatural amino acid pAPCF or PAPd (which is encoded by the selector codon TAG). The nucleotide sequence encoding the fusion protein is introduced into the pCDNA3.1 expression vector to obtain the target plasmid.

[0792] Briefly, pCMV-C-C9 PylRS-tRNA pylPlasmid and pCDNA3.1-mCherry-EGFP (39TAG) plasmid were transfected into HEK293T cells at the same time, and then the cell culture medium was replaced with fresh culture medium containing 50-500uM pAPCF or 50-500uMPAPd, and culture was continued for 36-48 hours. The cells were lysed, the mCherry red fluorescence and EGFP enhanced green fluorescence were measured, and the EGFP / mCherry ratio corresponding to each well was calculated as an indicator of the efficiency of pAPCF and PAPd introduction. The results showed that both unnatural amino acids pAPCF and PAPd can be effectively introduced into polypeptides expressed using eukaryotic cell expression systems.

[0793] Step 2: Expression of FRB mutants containing PAPd in ​​HEK293T cells

[0794] The codon nucleotides encoding positions 84 and 87 of the FRB protein were mutated to TAG for the introduction of PAPd. In brief, the following two plasmids were constructed:

[0795] (1) pCDNA3.1-FRB-84TAG-mCherry plasmid, encoding FRB-84PAPd-mCherry fusion protein, which includes a FRB-84PAPd mutant in which the amino acid residue at position 84 is replaced with a non-natural amino acid PAPd (whose amino acid sequence is shown in SEQ ID NO: 18), and a red fluorescent protein mCherry (which can emit red fluorescence for co-localization with the fluorescent signal of PAPd, whose amino acid sequence is shown in SEQ ID NO: 9) connected to the C-terminus of the FRB-84PAPd mutant via a linker (whose amino acid sequence is shown in SEQ ID NO: 25). The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 26, wherein the non-natural amino acid is encoded by a nonsense codon TAG. The nucleotide sequence encoding the fusion protein is introduced into the pCDNA3.1 expression vector to obtain the target plasmid.

[0796] (2) pCDNA3.1-FRB-87TAG-mCherry plasmid, which encodes FRB-87PAPd-mCherry fusion protein, which includes a FRB-87PAPd mutant in which the amino acid residue at position 87 is replaced with a non-natural amino acid PAPd (whose amino acid sequence is shown in SEQ ID NO: 27), and a red fluorescent protein mCherry (which can emit red fluorescence for co-localization with the fluorescent signal of PAPd, and whose amino acid sequence is shown in SEQ ID NO: 9) connected to the C-terminus of the FRB-87PAPd mutant via a linker (whose amino acid sequence is shown in SEQ ID NO: 25). The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO: 28, wherein the non-natural amino acid is encoded by a nonsense codon TAG. The nucleotide sequence encoding the fusion protein is introduced into the pCDNA3.1 expression vector to obtain the target plasmid.

[0797] In addition, the pCDNA3.1-FKBP plasmid was constructed, which can express wild-type FKBP protein. The FRB-related plasmid and FKBP plasmid obtained above were combined with pCMV-C-C9 pylRS-tRNA pyl Co-transfected into HEK293T cells.

[0798] Step 3: Confocal imaging experiment to explore the function of PAPd probe

[0799] The HEK293T cells obtained in the previous step were expressed in DMEM medium containing 50uM PAPd for about 48h and photographed using a Zeiss LSM880 laser confocal microscope. The image data was recorded and the shooting parameters were kept unchanged. Subsequently, the negative control cells were not treated with drugs, while rapamycin was added to the experimental group cells to a final concentration of 100nM, and the recording was continued for 15min, every 30s. Finally, the software was used for quantitative analysis of fluorescence intensity.

[0800] The results showed that without the addition of rapamycin, HEK293T cells were able to express FRB-87PAPd-mCherry and FKBP, but PAPd did not produce a fluorescent signal. After the addition of 100nM rapamycin, the cells produced a clear PAPd fluorescent signal within 10 minutes ( Fig.13 The experimental results also showed that similar fluorescence enhancement could be detected when site 84 at the edge of the interaction region was used as a PAPd introduction site, but the fluorescence enhancement amplitude was 30%.

[0801] In addition, similar methods and steps as above were used to express mCherry-FKBP fusion protein (whose amino acid sequence is shown in SEQ ID NO: 29, and after expression, it was localized to the cell membrane) and FRB-87PAPd mutant (whose amino acid sequence is shown in SEQ ID NO: 27, and after expression, it was distributed in the cytoplasm) carrying a membrane localization signal peptide and a transmembrane domain (related elements from PDGFR protein, GenBank: AAA60049.1) in HEK293T cells, and treated with rapamycin.

[0802] The results showed that after adding rapamycin, activated PAPd fluorescence could be detected in HEK293T cells, and it produced a good fluorescence co-localization effect with the mCherry fluorescence localized on the cell membrane ( Fig.14 ). This indicates that after the addition of rapamycin, the mCherry-FKBP fusion protein fixed to the cell membrane recruits FRB-87PAPd to transfer to the cell membrane, and protein interaction occurs in the cell, thereby generating fluorescence.

[0803] The above experimental results indicate that fluorescent rotor-type unnatural amino acids represented by PAPd can be used as direct and sensitive biological probes for detecting protein interactions in the environment of living cells, and their fluorescence response amplitude can reflect the interaction strength of different regions of the protein.

[0804] Example 11: Application of pAPCF in the preparation of glucose sensor

[0805] Glucose binding protein (D-glucose / D-galactose-binding protein, GBP) is part of the glucose transport system in the periplasmic space of Escherichia coli, responsible for transferring the bound glucose molecules to the Trg receptors on the membrane. The structure of the GBP protein is two globular domains connected by a hinge region, and the binding site of the glucose molecule is located in the gap between the two domains. After binding to the glucose molecule, GBP undergoes significant conformational changes, from the original open conformation to the closed conformation of the complex. In this embodiment, the inventor uses pAPCF as a probe sensitive to the protein microenvironment, and introduces it into the site of conformational changes in GBP (mainly involving amino acid residues (V19, E149, M182, Q187, A213 and L238) on sites 19, 149, 182, 187, 213 and 238), and develops a new type of glucose sensor. Surface plasmon resonance (SPR) technology is used to detect the interaction between protein and glucose. In short, a layer of a specific biomolecule film is fixed on the surface of the sensor metal chip, and then the sample to be tested flows over the chip surface. When the analyte molecules contained in the sample to be tested can interact with the biomolecule film and adsorb onto the chip, it will cause the refractive index of the sensor metal chip surface to change, resulting in a change in the minimum energy incident angle (SPR angle). By analyzing the change in the SPR angle, information such as the concentration, affinity, and binding kinetic constant of the analyte in the sample to be tested can be obtained. This embodiment mainly includes the following experimental steps.

[0806] Step 1: GBP protein expression optimization

[0807] GBP protein is an intercellular protein and needs to rely on a signal peptide for transport. In order to obtain the maximum intercellular protein yield, the original wild-type signal peptide of GBP was replaced with a StII signal peptide (whose amino acid sequence is shown in SEQ ID NO:30). In addition, a 9x his tag (whose amino acid sequence is shown in SEQ ID NO:32) was fused to the C-terminus of the GBP protein (whose amino acid sequence is shown in SEQ ID NO:31) to facilitate the use of Ni 2+ The resin captures and purifies the expressed GBP protein. Therefore, a pET22b-StII-GBP-9his expression plasmid was constructed, which encodes a StII-GBP-9his fusion protein, wherein a 9x his tag is fused and expressed at the C-terminus of GBP, and a StII signal peptide is fused and expressed at the N-terminus. The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO:33. The nucleotide sequence encoding the fusion protein is introduced into the pET22b expression vector to obtain the target plasmid.

[0808] Step 2: Construction, expression and purification of GBP protein mutants

[0809] Referring to the method described in the previous example, the following GBP mutants were prepared as sensors responsive to glucose:

[0810] GBP-149pAPCF protein (GBP-149 for short): The amino acid residue at site 149 of GBP protein was replaced with pAPCF (whose amino acid sequence is shown in SEQ ID NO: 34), and then a 9xhis tag was fused and expressed at its C-terminus. The nucleotide sequence encoding the fusion protein was introduced into the pET22b expression vector to obtain the target plasmid pET22b-StII-GBP(149TAG)-9his.

[0811] GBP-238pAPCF protein (GBP-238 for short): The amino acid residue at site 238 of GBP protein was replaced with pAPCF (whose amino acid sequence is shown in SEQ ID NO: 35), and then a 9xhis tag was fused and expressed at its C-terminus. The nucleotide sequence encoding the fusion protein was introduced into the pET22b expression vector to obtain the target plasmid pET22b-StII-GBP(238TAG)-9his.

[0812] The expression and purification method of the mutant protein was as described in Example 8. The plasmids pET22b-StII-GBP(149 / 238TAG)-9his and pUltra-MjFmrRS were simultaneously transferred into BL21(DE3), and then the mutant protein expression was induced and purified with Ni. 2+ The specific experimental steps are as follows.

[0813] (1) The plasmids pET22b-StII-GBP(TAG)-9his (which encodes the target GBP mutant) and pUltra-MjFmrRS were transformed into BL21(DE3), and single clones were selected for culture.

[0814] (2) The next day, the bacterial solution was inoculated into 200 ml LB at a ratio of 1:50. When the culture reached OD600 = 0.6, 0.1 mM IPTG and 0.8 mM pAPCF were added to induce expression at 20°C for 18 h.

[0815] (3) After harvesting the bacteria, discard the supernatant, wash once with PBS, and then resuspend in 10 ml of lysis buffer (25 mM tris-HCl, 500 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF, pH 8.0), snap-freeze in liquid nitrogen, and then thaw in a 37°C water bath.

[0816] (4) Add 1 mg / ml lysozyme, incubate on ice for 1 h, and then sonicate for 3 min.

[0817] (5) The lysate was centrifuged at high speed for 20 min at 4°C, the supernatant was aspirated, and 200 μl of Ni 2+ -NTA binding, incubate on ice for 1.5 hours.

[0818] (6) The column was then washed for several column volumes with 20 mM and 40 mM imidazole wash buffer (25 mM tris-HCl, 500 mM NaCl, 20 or 40 mM imidazole, 10% glycerol, pH 8.0).

[0819] (7) Finally, the target mutant protein was washed off with elution buffer (25 mM tris-HCl, 500 mM NaCl, 10% glycerol, pH 7.4).

[0820] (8) The protein eluate was concentrated using a 10K ultrafiltration tube.

[0821] (9) Dialysis treatment: Dilute the GBP protein into denaturation buffer (2M guanidine hydrochloride, 1mM EDTA, 25mM tris-HCl, pH=8) to a concentration of about 0.5mg / ml, and then seal it in a dialysis bag. Then add it to 1L of renaturation buffer (10mMtris-HCl, 150mM NaCl, 1mM CaCl2, pH=7.4). Stir slowly at 4℃ for 4h. Then replace with fresh renaturation buffer, and do this 3 times. Finally, take out the liquid in the dialysis bag, concentrate it with a 10K ultrafiltration tube, and replace the liquid with PBS, and replace it 3 times.

[0822] Step 3: Fluorescence quantification of glucose by GBP-238

[0823] GBP-238 was used to conduct a fluorescence quantitative experiment of glucose binding. The concentration of GBP-238 was set to 5uM, and the concentration of glucose was set to 0, 1, 3, 10, 30, 50, 100, 300uM, and a 100ul solution system was prepared. The fluorescence intensity was measured with an ELISA instrument, and a fitting curve was drawn between the fluorescence intensity and the glucose concentration.

[0824] Step 4: Use SPR technology to measure the affinity of GBP-238 for glucose

[0825] Using the Biacore 8K molecular interaction detector, the purified GBP-238 protein was fixed on a dextran CM5 chip, and then a series of glucose concentration gradients were prepared with PBS-P buffer: 0, 0.1, 0.5, 1, 2.5, 5, 12.5, 25, 50uM. The experimental conditions were a flow rate of 30uL / min, a binding time of 60sec, and a dissociation time of 60sec. Finally, the instrument's own software was used for data analysis and curve fitting to analyze whether the binding affinity of the GBP-238 mutant to glucose changed significantly.

[0826] The experimental results are as follows Fig.15 As shown. The experimental results show that the fluorescence intensity of GBP-238 is strictly dependent on the glucose concentration. Therefore, the glucose concentration in unknown samples can be measured according to the standard working curve. In addition, compared with other monosaccharides or disaccharides, GBP-238 has specific response to glucose. Except for galactose, one of the known natural substrates of GBP, other sugar molecules cannot affect the fluorescence signal of GBP-238 ( Fig.15 a). In addition, the dynamic range of GBP-238 fluorescence signal in the presence or absence of glucose is up to 15 times, which is better than the traditional glucose sensor developed based on FRET or CP-GFP principle. SPR measurement results also show that the dissociation constant of GBP-238 and glucose has reached 1.39uM, which is comparable to the dissociation coefficient of wild-type GBP to glucose of 0.2uM ( Fig.15 b).

[0827] The above results illustrate that the GBP mutant (e.g., GBP-238) obtained by introducing pAPCF at the conformational change site of GBP not only retains the binding specificity and affinity for glucose well, but also has a fluorescence response and a wide dynamic range that is strictly dependent on the glucose concentration. The fluorescent rotor type non-natural amino acid of the present application can be used to develop a new sensor for detecting glucose. In particular, in the absence of glucose, the mutant protein (e.g., GBP-149 and GBP-238) is in the first conformation, wherein the intramolecular rotation of pAPCF is restricted, and thus a fluorescent signal is generated. When glucose is present in the protein solution, the mutant protein (e.g., GBP-149 and GBP-238) will undergo a conformational change, transforming into a second conformation, wherein pAPCF can rotate freely, and the fluorescent signal it produces decreases. Therefore, the GBP mutant containing the fluorescent rotor type non-natural amino acid of the present application at the conformational change site can be used as a sensor for responding to glucose.

[0828] Example 12: Application of pAPCF in the preparation of glutamine sensor

[0829] GlnH is a high-affinity glutamine-binding protein from Escherichia coli. After binding to glutamine (gln), the protein conformation undergoes significant changes, mainly involving amino acid residues at sites 51, 74, 116, 117, 138 and 159 (S51, E74, S116, G117, N138, P159). In this embodiment, referring to the scheme described in the previous embodiment, the inventors replaced the amino acids at sites (sites 51, 116, 117) where the conformational changes of glnH before and after binding to glutamine were large with pAPCF, and additionally introduced point mutations R75M and Y86A (this is to adjust the appropriate affinity for gln to adapt to the concentration range of gln in the actual detection scenario). Finally, three glnH mutants (gH-51, gH-116 and gH-117, whose amino acid sequences are shown in SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively) were obtained, and the fluorescence response of the glnH mutants to gln was analyzed.

[0830] The experimental results are as follows Fig.16 The results showed that the three glnH mutants all had fluorescence responses to gln, among which the mutant with the amino acid at position 51 replaced by pAPCF (referred to as gH-51) had the largest fluorescence response amplitude to gln, with an apparent Kd constant of 1.40 mM and a dynamic range of up to 900%. In addition, gH-51 had good substrate selectivity for glutamine and did not produce fluorescence responses to several other glutamine-like amino acids.

[0831] The above results show that the glnH mutant obtained by introducing pAPCF at the conformational change site of glnH not only retains the binding specificity and affinity for gln well, but also has a fluorescence response that is strictly dependent on the concentration of gln and a wide dynamic range. The fluorescent rotor-type non-natural amino acid of the present application can be used to develop a new sensor for detecting gln. In particular, in the absence of glutamine, the glnH mutant protein is in a first conformation, in which the intramolecular rotation of pAPCF is restricted, and thus a fluorescent signal is generated. When glutamine is present in the protein solution, the glnH mutant protein will undergo a conformational change and transform into a second conformation, in which pAPCF can rotate freely and the fluorescent signal it generates decreases. Therefore, the glnH mutant containing the fluorescent rotor-type non-natural amino acid of the present application at the conformational change site can be used as a sensor that responds to glutamine.

[0832] Example 13: Application of PAPd in ​​the preparation of GTP / GDP sensor

[0833] Fe from Methanococcus jannaschii 2+The G protein domain (MjFeoB) of the transporter FeoB can bind GTP or GDP molecules, and after binding, some regions in the MjFeoB structure can undergo significant conformational changes. The conformational changes of MjFeoB before and after binding GTP or GDP molecules mainly involve amino acid residues (W33, V36, S145, A146, A147, K148) on sites 33, 36, 145, 146, 147 and 148. In the present embodiment, the inventors selected the amino acid at site 33 of MjFeoB to be replaced with the fluorescent rotor type non-natural amino acid of the present application, and analyzed the fluorescence response of the MjFeoB mutant combined with GTP or GDP.

[0834] Referring to the scheme described in the above embodiment, the inventors replaced the amino acid at the site (site 33) where MjFeoB has a large conformational change before and after binding to GTP or GDP molecules with PAPd, and obtained the MjFeoB-33PAPd mutant (whose amino acid sequence is shown in SEQ ID NO: 39).

[0835] Referring to the scheme described in the previous example, the expression of MjFeoB mutant was induced in Escherichia coli and the protein was purified. The purified MjFeoB-33PAPd protein was diluted in PIPES buffer (50mM PIPES, 100mM KCl, 5mMMgCl2, 10% glycerol, pH = 7.4) and divided into three groups, one of which was not treated as a control group, and the other two groups were added with 1mM GTP or 10mM EDTA, respectively, and 3 parallel experiments were set up for each group. After incubation at room temperature for 5min, fluorescence was measured using an enzyme reader.

[0836] The experimental results are as follows Fig.17 As shown. The results showed that the MjFeoB-33PAPd mutant had a strong fluorescence response to the concentration change of GTP. As the concentration of GTP increased, the MjFeoB-33PAPd mutant showed a corresponding decrease in fluorescence intensity. The titration experiment showed that the apparent affinity of MjFeoB-33PAPd for GTP was Kd = 3.52uM, and the dynamic range was about 400%. In addition, MjFeoB-33PAPd also had a similar fluorescence response to GDP.

[0837] The above results show that the MjFeoB mutant obtained by introducing PAPd at the conformational change site of MjFeoB not only retains the binding specificity and affinity for GTP / GDP well, but also has a fluorescence response that is strictly dependent on the GTP / GDP concentration and a wide dynamic range. The fluorescent rotor-type non-natural amino acid of the present application can be used to develop a new sensor for detecting GTP / GDP. In particular, in the absence of GTP / GDP, the MjFeoB mutant protein is in a first conformation, in which the intramolecular rotation of PAPd is restricted, and thus a fluorescent signal is generated. When GTP / GDP is present in the protein solution, the MjFeoB mutant protein will undergo a conformational change and transform into a second conformation, in which PAPd can rotate freely and the fluorescent signal it generates decreases. Therefore, the MjFeoB mutant containing the fluorescent rotor-type non-natural amino acid of the present application at the conformational change site can be used as a sensor that responds to GTP / GDP.

[0838] Example 14: Application of calcium ion probe

[0839] Calmodulin (CaM) can acquire the ability to bind to various target proteins through conformational changes after binding to calcium ions. The monomer form of CaM is a loose dumbbell-shaped structure. 2+ When binding to CaM, CaM changes to a near-globular structure and binds to downstream proteins at the same time. The M13 peptide is an amino acid sequence from smooth muscle myosin light chain kinase, one of the downstream proteins of CaM, which can quickly bind to CaM bound to calcium ions. Therefore, the CaM-M13 combination is often used to develop CaM based on CP-GFP or FRET principles. 2+ Sensor. After binding to M13, the microenvironment of some sites in CaM will change with conformational changes (for example, from an exposed open area to an internal restricted area, or from an internal restricted area to an exposed open area), including amino acid residues (F19, F68, M72, A88, V91, F92, V108, F141) on sites 19, 68, 72, 88, 91, 92, 108, 141). In this embodiment, the open area originally located in the monomer CaM is selected, but the site (taking sites 19, 68, 92, 141 as an example) that is changed to the internal restricted area after binding to M13 is selected, and the amino acids at these sites are replaced with the fluorescent rotor type non-natural amino acids of the present application, respectively. Then, the function of the CaM mutant as a calcium ion probe is verified in Escherichia coli and eukaryotic cells, respectively. This embodiment mainly includes the following experimental steps.

[0840] Step 1: Construction of CaM-M13 mutant with PAPd introduced

[0841] A polynucleotide fragment expressing a CaM-M13 fusion protein was constructed, wherein the M13 short peptide was located at the C-terminus of CaM, and CaM and M13 were connected by a linker sequence GSGS (SEQ ID NO: 41). The polynucleotide fragment was cloned into an expression vector pET22b. Then, referring to the scheme described in the previous embodiment, a TAG mutation was introduced into the polynucleotide fragment at the position of site 19, 68, 92 or 141 encoding CaM by point mutation, thereby constructing the corresponding pET22b-CaM (19 / 68 / 92 / 141TAG)-M13 plasmid, which encodes the following CaM-M13 mutants introduced with a non-natural amino acid (PAPd) at site 19, 68, 92 or 141 of CaM, respectively:

[0842] CaM-19PAPd-M13, which comprises CaM-19PAPd (whose amino acid sequence is shown in SEQ ID NO:40) and M13 short peptide (whose amino acid sequence is shown in SEQ ID NO:42), which are connected by a linker sequence GSGS;

[0843] CaM-68PAPd-M13, which comprises CaM-68PAPd (whose amino acid sequence is shown in SEQ ID NO:43) and M13 short peptide (whose amino acid sequence is shown in SEQ ID NO:42), which are connected by a linker sequence GSGS;

[0844] CaM-92PAPd-M13, which comprises CaM-92PAPd (whose amino acid sequence is shown in SEQ ID NO:44) and M13 short peptide (whose amino acid sequence is shown in SEQ ID NO:42), which are connected by a linker sequence GSGS;

[0845] CaM-141PAPd-M13 comprises CaM-141PAPd (whose amino acid sequence is shown in SEQ ID NO:45) and M13 short peptide (whose amino acid sequence is shown in SEQ ID NO:42), which are connected by a linker sequence GSGS.

[0846] Then, the plasmids encoding the CaM-M13 mutants were co-transfected with pUltra-CC9 pylRS into BL21(DE3). After that, 0.2mM IPTG and 0.8mM PAPd were added to the culture medium, and the bacterial culture was placed in a 25°C shaker for about 16 hours to obtain bacteria expressing the CaM-M13 mutants. The bacterial lysate was used directly for preliminary verification. In brief, 1mM Ca was added to the bacterial lysate. 2+ or 1 mM EGTA (which can chelate Ca 2+) and then analyze the changes in fluorescence.

[0847] Step 2: Calcium ion fluorescence response experiment of CaM-92PAPd-M13

[0848] CaM-92PAPd-M13 was selected for the next step of the experiment, and the mutant was expressed and purified. Then, the fluorescence response and affinity of CaM-92PAPd-M13 to calcium ions were quantitatively determined.

[0849] Step 3: Confocal imaging experiment to explore the function of CaM-92PAPd-M13 calcium ion sensor

[0850] HEK293T cells were used in this experiment. The polynucleotide fragment encoding the CaM-M13 mutant (CaM-92PAPd-M13) was cloned into the pCDNA3.1 eukaryotic expression vector to obtain the pCDNA3.1-hCaM-92TAG-M13 plasmid. The plasmid was then combined with pCMV-CC9 pylRS-tRNA pyl The cells were co-transfected into HEK293T cells. The cells were then cultured in DMEM medium containing 50uM PAPd for about 48h before confocal imaging experiments.

[0851] Before drug treatment, the image data was recorded and the shooting parameters were kept unchanged. Subsequently, the negative control cells were not treated with drugs; for the experimental group cells, carbachol was carefully added to the confocal dish to 200uM and recorded; then, drug treatment was performed again, ionomycin was carefully added to the confocal dish to 10uM and CaCl2 was added to 20mM, and recorded again. Finally, the software was used to quantitatively measure the average fluorescence intensity of the cells.

[0852] The experimental results are as follows Figure 18-21 The results showed that the four CaM-M13 mutants introduced with PAPd had a significant effect on Ca 2+ The responses are obvious ( Fig.18 ). Among them, CaM-19PAPd-M13 and CaM-92PAPd-M13 are 2+ The concentration showed a positive response trend, with 11-fold and 5-fold signal amplitude changes, respectively, while CaM-68PAPd-M13 and CaM-141PAPd-M13 had a positive response to Ca 2+ The concentration showed a negative response trend.

[0853] The results also showed that the CaM-92PAPd-M13 protein can sensitively respond to Ca 2+The fluorescence intensity increases with the increase of CaM-92PAPd-M13 concentration, with a dynamic range of nearly 1400%. 2+ The EC50 of the fluorescence response was 39 nM ( Fig.19 In addition, the experimental results also showed that other mutants introduced into pAPCF had no significant effect on Ca 2+ Also has similar responsiveness, but the response is weaker.

[0854] The results also showed that in HEK293T cells expressing CaM-92PAPd-M13, under normal conditions, there was almost no fluorescence signal in the cells; however, when carbachol was given to the culture medium to stimulate the intracellular Ca 2+ When released from calcium stores, a rapidly activated PAPd fluorescence signal was observed within 1 minute; further, when ionomycin was added to induce calcium ionophores to transfer exogenous Ca 2+ When transported into the cell, the greater Ca 2+ concentration caused a further enhancement of PAPd fluorescence ( Fig. 20 ).

[0855] In addition, in order to further improve the signal-to-noise ratio of the calcium sensor and maintain a relatively low fluorescence signal at the background calcium ion level, the inventors also introduced the F141L mutation into the CaM mutant to obtain an improved CaM-92PAPd-F141L-M13 calcium ion sensor, which comprises CaM-92PAPd-F141L (its amino acid sequence is shown in SEQ ID NO: 46) and M13 short peptide (its amino acid sequence is shown in SEQ ID NO: 42), which are connected by a linker sequence GSGS; and its performance was tested as above. The results show that CaM-92PAPd-F141L-M13 has a good performance in terms of Ca 2+ The EC50 of the fluorescence response was increased to 124nM, and the dynamic range of the signal response was significantly improved to nearly 3500% ( Fig.21 ).

[0856] The above results show that the CaM-M13 mutants obtained by replacing the amino acid residues at the conformational change sites of CaM-M13 (such as sites 19, 68, 92, and 141) with PAPd not only retain the Ca 2+ The binding specificity and affinity of 2+ The fluorescent rotor-type unnatural amino acid of the present application can be used to develop a method for detecting Ca 2+ New sensor.

Claims

1. A non-natural polypeptide, wherein The non-natural polypeptide comprises a non-natural amino acid of formula I: Among them, X 1a , X 2a , X 3a and X 4a Independently selected from CH and N; preferably X 1a , X 2a , X 3a and X 4a 3 of them are CH; more preferably all 4 of them are CH; R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl, preferably C1-C3 alkyl; preferably R1 is selected from: -NH2, -NHR a and-NR b R c , where R a , R b , R c Independently selected from C1-C6 alkyl, preferably C1-C3 alkyl; R2 is selected from: cyano, sulfonic acid, nitro and trihalomethyl (e.g. trifluoromethyl, trichloromethyl); preferably R2 is selected from: cyano, sulfonic acid and trichloromethyl; more preferably R2 is selected from: cyano; X 1b Selected from: N and CR3; R3 is selected from: H, cyano, sulfonic acid, nitro and trihalomethyl (e.g. trifluoromethyl, trichloromethyl); preferably R3 is selected from: H, cyano, sulfonic acid and trichloromethyl; more preferably R3 is selected from: H and cyano; X 2b Selected from CR 42 and N, preferably CR 42 , X 3b Selected from CR 43 and N, preferably CR 43 , X 4b Selected from CR 44 and N, preferably CR 44 ,and X 5b Selected from CR 45 and N, preferably CR 45 ; R 42 , R 43 , R 44 , R 45 Each independently selected from: H and -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3, and R 42 , R 43 , R 44 , R 45 At least one of is not H; preferably R 42 , R 43 , R 44 , R 45 1 of the following (preferably R 42 or R 43 ) is selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1-3, and the rest are H.

2. The non-natural polypeptide according to claim 1, wherein The unnatural amino acid has the structure of Formula IA or Formula IB: Wherein, R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl; Preferably, R1 is selected from: -NH2, -NHR a and-NR b R, where R a , R b , R c Independently selected from C1-C3 alkyl, preferably methyl, ethyl, propyl; More preferably, R1 is selected from: -NH2 and -NR a R b , where R a , R b independently selected from methyl; R3 is independently selected from: H, cyano, sulfonic acid, nitro and trihalomethyl (eg, trifluoromethyl and trichloromethyl); Preferably, R3 is selected from: cyano, sulfonic acid and trichloromethyl; More preferably, R3 is selected from cyano; R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3; Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH); Preferably, Formula IA is selected from: Wherein, R1, R3, and R4 in Formula I-A1 and Formula I-A2 are defined as the corresponding variables in Formula IA; or, Wherein, R1 is selected from: -NH2, -NHR a 、-NR b R c , -OH and -OR d , where R a , R b , R c , R d Independently selected from C1-C6 alkyl; Preferably, R1 is selected from: -NH2, -NHR a and-NR b R, where R a , R b , R c Independently selected from C1-C3 alkyl, preferably methyl, ethyl and propyl; More preferably, R1 is selected from: -NH2, -NR a R b , where R a , R b independently selected from methyl; R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3; Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH); Preferably, Formula IB is selected from: Wherein, R1 and R4 in Formula I-B1 and Formula I-B2 are defined as the corresponding variables in Formula IB.

3. The non-natural polypeptide according to claim 1 or 2, wherein The unnatural amino acid is selected from the following:

4. The non-natural polypeptide according to any one of claims 1 to 3, wherein The non-natural amino acid is restricted from rotating intramolecularly in the spatial structure of the non-natural polypeptide, or the non-natural amino acid is able to rotate freely intramolecularly in the spatial structure of the non-natural polypeptide.

5. The non-natural polypeptide of claim 4, wherein The intramolecular rotation state of the non-natural amino acid in the spatial structure of the non-natural polypeptide can change, for example, from free intramolecular rotation to restricted intramolecular rotation, or from restricted intramolecular rotation to free intramolecular rotation; Preferably, the change in the intramolecular rotation state is caused by a change in the spatial conformation of the non-natural polypeptide, or by the binding of the non-natural polypeptide to a target molecular entity.

6. The non-natural polypeptide of any one of claims 1 to 5, wherein The non-natural polypeptide can specifically bind to the target molecular entity to form a complex; Preferably, the non-natural amino acid has a first intramolecular rotation state in the spatial structure of the non-natural polypeptide not bound to the target molecular entity, and has a second intramolecular rotation state in the spatial structure of the complex, and the first intramolecular rotation state is different from the second intramolecular rotation state; Preferably, the first intramolecular rotation state is free intramolecular rotation, and the second intramolecular rotation state is restricted intramolecular rotation; or, the first intramolecular rotation state is restricted intramolecular rotation, and the second intramolecular rotation state is free intramolecular rotation; Preferably, the target molecular entity is another polypeptide, or a substrate of the non-natural polypeptide, or the non-natural polypeptide itself.

7. The non-natural polypeptide of claim 6, wherein The target molecular entity is another polypeptide, and the contact or binding between the non-natural polypeptide and the other polypeptide causes the intramolecular rotation state of the non-natural amino acid to change (for example, from free intramolecular rotation to restricted intramolecular rotation); Preferably, the non-natural amino acid is located at or near the interface where the non-natural polypeptide and the other polypeptide contact or bind to each other; Preferably, the non-natural polypeptide and the other polypeptide form a molecular pair that specifically binds to each other or interacts with each other; Preferably, the non-natural polypeptide and the additional polypeptide form an antigen-antibody molecule pair that specifically binds to each other; for example, the non-natural polypeptide comprises or is an antibody, and the additional polypeptide comprises or is an antigen that the antibody specifically binds to; or, the non-natural polypeptide comprises or is an antigen, and the additional polypeptide comprises or is an antibody that specifically binds to the antigen; Preferably, the non-natural polypeptide and the other polypeptide constitute a protein Z-Affibody molecule pair that specifically binds to each other; for example, the non-natural polypeptide comprises or is protein Z containing the non-natural amino acid, and the other polypeptide comprises or is Affibody; or, the non-natural polypeptide comprises or is Affibody containing the non-natural amino acid, and the other polypeptide comprises or is protein Z; Preferably, the non-natural polypeptide and the additional polypeptide form a FKBP-FRB molecular pair that specifically binds to each other; for example, the non-natural polypeptide comprises or is a FRB containing the non-natural amino acid, and the additional polypeptide comprises or is FKBP; or, the non-natural polypeptide comprises or is a FKBP containing the non-natural amino acid, and the additional polypeptide comprises or is FRB; Preferably, the non-natural polypeptide and the other polypeptide constitute a spike protein and an ACE2 receptor molecule pair that specifically bind to each other; for example, the non-natural polypeptide comprises or is a spike protein containing the non-natural amino acid, and the other polypeptide comprises or is an ACE2 receptor; or, the non-natural polypeptide comprises or is an ACE2 receptor containing the non-natural amino acid, and the other polypeptide comprises or is a spike protein; Preferably, the non-natural polypeptide and the additional polypeptide form a pair of α-tubulin and β-tubulin molecules that specifically bind to each other; for example, the non-natural polypeptide comprises or is α-tubulin containing the non-natural amino acid, and the additional polypeptide comprises or is β-tubulin; or, the non-natural polypeptide comprises or is β-tubulin containing the non-natural amino acid, and the additional polypeptide comprises or is α-tubulin.

8. The non-natural polypeptide of claim 7, wherein The non-natural polypeptide comprises or is a protein Z containing the non-natural amino acid; Preferably, the protein Z containing the non-natural amino acid can interact with Affibody and change the intramolecular rotation state of the non-natural amino acid (for example, from free intramolecular rotation to restricted intramolecular rotation); preferably, the non-natural amino acid is located at the interface where protein Z interacts with Affibody or at a position adjacent thereto, for example, at the following sites of protein Z: sites corresponding to sites 10, 13, 14, 17, 28, 31 or 35 of SEQ ID NO:2; Preferably, the unnatural amino acid is located at the following position of protein Z: the position corresponding to position 13 or 17 of SEQ ID NO: 2; preferably, the unnatural amino acid is pAPCF; Preferably, the protein Z containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:3 or SEQ ID NO:4 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:4; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 13 or 17 of SEQ ID NO:2; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

9. The non-natural polypeptide of claim 7, wherein The non-natural polypeptide comprises or is a FRB protein containing the non-natural amino acid; Preferably, the FRB protein containing the non-natural amino acid can interact with FKBP in the presence of rapamycin, and changes the intramolecular rotation state of the non-natural amino acid (for example, from free intramolecular rotation to restricted intramolecular rotation); preferably, the non-natural amino acid is located at the interface where the FRB protein interacts with FKBP or at a position adjacent thereto, for example, at the following sites of the FRB protein: sites corresponding to sites 76, 80, 84, 87 or 91 of SEQ ID NO: 18; Preferably, the unnatural amino acid is located at the following site of the FRB protein: a site corresponding to site 84 or 87 of SEQ ID NO: 18; preferably, the unnatural amino acid is pAPCF or PAPd; Preferably, the FRB protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO: 18 or SEQ ID NO: 27 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 27, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO: 18 or SEQ ID NO: 27; preferably, the non-natural amino acid (e.g., pAPCF or PAPd) is located at a position corresponding to position 84 or 87 of SEQ ID NO: 18; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

10. The non-natural polypeptide of claim 6, wherein The target molecular entity is the non-natural polypeptide itself, and the binding or aggregation of the non-natural polypeptides causes the intramolecular rotation state of the non-natural amino acid to change (for example, from free intramolecular rotation to restricted intramolecular rotation); Preferably, the non-natural amino acid is located in an exposed region of the non-natural polypeptide or in a proximal position thereof; Preferably, the non-natural polypeptide comprises or is: superoxide dismutase 1 (SOD1) containing the non-natural amino acid, Huntingtin protein (see, e.g., GenBank: AAB38240.1) containing the non-natural amino acid, Nucleophosmin (see, e.g., GenBank: AAA36380.1) containing the non-natural amino acid, or Fibrillarin (see, e.g., GenBank: AAA52453.1) containing the non-natural amino acid.

11. The non-natural polypeptide of claim 10, wherein The non-natural polypeptide comprises or is superoxide dismutase 1 (SOD1) containing the non-natural amino acid; Preferably, the SOD1 of the non-natural amino acid can change the intramolecular rotation state of the non-natural amino acid when it aggregates (for example, from free intramolecular rotation to restricted intramolecular rotation); Preferably, the unnatural amino acid is located in an exposed region of SOD1, for example, at the following sites of SOD1: a site corresponding to site 25, 40, 47 or 67 of SEQ ID NO:7; Preferably, the non-natural amino acid is located at the following position of SOD1: the position corresponding to position 25 or 40 of SEQ ID NO:7; preferably, the non-natural amino acid is pAPCF; preferably, the SOD1 containing the non-natural amino acid further contains a mutation selected from the following: the amino acid residue at the position corresponding to position 4 of SEQ ID NO:7 is replaced by V, the amino acid residue at the position corresponding to position 31 of SEQ ID NO:7 is replaced by A, or a combination thereof; Preferably, the SOD1 protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:11 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:11, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:7 or SEQ ID NO:11; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 25 or 40 of SEQ ID NO:7; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

12. The non-natural polypeptide of claim 6, wherein The target molecular entity is a substrate of the non-natural polypeptide, and the contact or binding between the non-natural polypeptide and the substrate causes a change in the intramolecular rotation state of the non-natural amino acid (e.g., from free intramolecular rotation to restricted intramolecular rotation; or from restricted intramolecular rotation to free intramolecular rotation); Preferably, the non-natural amino acid is located in the following region of the non-natural polypeptide or in a vicinity thereof, wherein the region has a different spatial conformation in the non-natural polypeptide when bound or not bound to the substrate; Preferably, the non-natural polypeptide and the substrate form a molecular pair that specifically binds to each other or a molecular pair that interacts with each other; Preferably, the non-natural polypeptide and the substrate form a glucose-binding protein-glucose molecule pair that specifically interacts with each other; for example, the non-natural polypeptide comprises or is a glucose-binding protein, and the substrate comprises or is glucose; Preferably, the non-natural polypeptide and the substrate form a glutamine binding protein-glutamine molecule pair that interacts specifically with each other; for example, the non-natural polypeptide comprises or is a glutamine binding protein, and the substrate comprises or is glutamine; Preferably, the non-natural polypeptide and the substrate constitute a G protein domain-GTP / GDP molecule pair that interacts specifically with each other; for example, the non-natural polypeptide comprises or is a G protein domain, and the substrate comprises or is GTP or GDP; Preferably, the non-natural polypeptide and the substrate form a calmodulin-Ca 2+ Molecular pair; for example, the non-natural polypeptide comprises or is calmodulin, and the substrate comprises or is Ca 2+ ; Preferably, the non-natural polypeptide and the substrate form a mineralocorticoid receptor-aldosterone molecular pair that interacts specifically with each other; for example, the non-natural polypeptide comprises or is a mineralocorticoid receptor, and the substrate comprises or is aldosterone.

13. The non-natural polypeptide of claim 12, wherein The non-natural polypeptide comprises or is a glucose binding protein containing the non-natural amino acid; Preferably, the glucose-binding protein containing the non-natural amino acid is capable of undergoing a conformational change after binding to a glucose molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation); preferably, the non-natural amino acid is located in the following region of the glucose-binding protein or its adjacent position, and the region has different spatial conformations in the glucose-binding protein when it is bound or not bound to glucose; for example, the non-natural amino acid is located at the following sites of the glucose-binding protein: sites corresponding to sites 19, 149, 182, 187, 213 or 238 of SEQ ID NO:31; Preferably, the non-natural amino acid is located at the following position of the glucose binding protein: the position corresponding to position 149 or 238 of SEQ ID NO: 31; preferably, the non-natural amino acid is pAPCF; Preferably, the glucose-binding protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:34 or SEQ ID NO:35 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:34 or SEQ ID NO:35, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:34 or SEQ ID NO:35; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 149 or 238 of SEQ ID NO:31; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

14. The non-natural polypeptide of claim 12, wherein The non-natural polypeptide comprises or is a glutamine binding protein containing the non-natural amino acid; Preferably, the glutamine binding protein containing the non-natural amino acid is capable of undergoing conformational changes after binding to a glutamine molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation); preferably, the non-natural amino acid is located in the following region of the glutamine binding protein or its adjacent position, and the region has different spatial conformations in the glutamine binding protein when it is bound or not bound to glutamine; for example, the non-natural amino acid is located at the following sites of the glutamine binding protein: sites corresponding to sites 51, 74, 117, 118, 138 or 159 of SEQ ID NO:36; Preferably, the non-natural amino acid is located at the following position of the glutamine binding protein: a position corresponding to position 51, 117 or 118 of SEQ ID NO: 36; preferably, the non-natural amino acid is pAPCF; Preferably, the glutamine binding protein containing a non-natural amino acid comprises an amino acid sequence as shown in SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:38; preferably, the non-natural amino acid (e.g., pAPCF) is located at the position of SEQ ID NO:36, SEQ ID NO:37 or SEQ ID NO:

38. The site corresponding to site 51, 117 or 118 of NO:36; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

15. The non-natural polypeptide of claim 12, wherein The non-natural polypeptide comprises or is a G protein domain containing the non-natural amino acid; Preferably, the G protein domain containing the non-natural amino acid can undergo a conformational change after binding to a GTP or GDP molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation); preferably, the non-natural amino acid is located in the following region of the G protein domain or its adjacent position, and the region has different spatial conformations in the G protein domain when bound or not bound to GTP or GDP; for example, the non-natural amino acid is located at the following sites of the G protein domain: sites corresponding to sites 33, 36, 145, 146, 147 or 148 of SEQ ID NO:39; Preferably, the unnatural amino acid is located at the following site of the G protein domain: the site corresponding to site 33 of SEQ ID NO: 39; preferably, the unnatural amino acid is PAPd; Preferably, the G protein domain containing non-natural amino acids comprises the amino acid sequence as shown in SEQ ID NO:39 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:39, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:39; preferably, the non-natural amino acid (e.g., PAPd) is located at a position corresponding to position 33 of SEQ ID NO:39; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, or any combination thereof.

16. The non-natural polypeptide of claim 12, wherein The non-natural polypeptide comprises or is calmodulin containing the non-natural amino acid; Preferably, the calmodulin containing unnatural amino acids is capable of binding to Ca 2+ The non-natural amino acid undergoes a conformational change after binding to the calmodulin molecule, so that the intramolecular rotation state of the non-natural amino acid changes (for example, from restricted intramolecular rotation to free intramolecular rotation, or from free intramolecular rotation to restricted intramolecular rotation); preferably, the non-natural amino acid is located in the following region of calmodulin or its adjacent position, the region is bound or not bound to Calmodulin 2+ In the case of the non-natural amino acid, the non-natural amino acid has a different spatial conformation in the calmodulin; for example, the non-natural amino acid is located at the following sites of calmodulin: sites corresponding to sites 19, 68, 72, 88, 91, 92, 108 or 141 of SEQ ID NO:40; Preferably, the unnatural amino acid is located at the following sites of calmodulin: sites corresponding to sites 19, 68, 92 or 141 of SEQ ID NO:40; preferably, the unnatural amino acid is PAPd; Preferably, the calmodulin containing unnatural amino acids comprises an amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence as shown in SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:45; preferably, the unnatural amino acid (e.g., PAPd) is located at the position of SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:44 or SEQ ID NO:

45. The site corresponding to site 19, 68, 92 or 141 of ID NO:40; Preferably, the non-natural polypeptide further comprises a second peptide, such as a signal peptide, a peptide linker, a tag peptide, a fluorescent protein, an M13 polypeptide or any combination thereof.

17. An isolated nucleic acid comprising a polynucleotide encoding the non-natural polypeptide of any one of claims 1-16, wherein: The unnatural amino acid is encoded by a selector codon (eg, a stop codon, such as an amber codon).

18. A vector comprising the isolated nucleic acid of claim 17; Preferably, the vector is a cloning vector or an expression vector.

19. A host cell comprising the isolated nucleic acid of claim 17 or the vector of claim 18; Preferably, the host cell is a prokaryotic cell (such as E. coli) or a eukaryotic cell (such as a yeast cell, a fungal cell, a plant cell, an animal cell, such as a mammalian cell, such as a human cell); Preferably, the host cell further contains a polynucleotide encoding a first orthogonal aminoacyl-tRNA synthetase (O-RS), a polynucleotide encoding a first orthogonal tRNA (O-tRNA), or a combination thereof; Preferably, the first orthogonal tRNA (O-tRNA) is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell; Preferably, said first orthogonal-tRNA is capable of being aminoacylated by said first O-RS with said unnatural amino acid; Preferably, the first O-tRNA is an amber suppressor tRNA; Preferably, the first O-tRNA is derived from an archaeon, such as Methanococcus jannaschii, Methanogenic archaeon, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof or is encoded by SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof; Preferably, the first O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or a conservative variant thereof or is encoded by SEQ ID NO: 16 or a conservative variant thereof; Preferably, the first O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 14 or a conservative variant thereof or is encoded by SEQ ID NO: 14 or a conservative variant thereof; Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the unnatural amino acid; Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is capable of recognizing the unnatural amino acid described in any one of claims 1 to 3, and aminoacylates the first orthogonal tRNA (O-tRNA) with the unnatural amino acid; Preferably, the first O-RS is derived from an aminoacyl-tRNA synthetase of Methanococcus jannaschii or a Methanogenic archaeon, such as a Methanococcus jannaschii tyrosyl-tRNA synthetase or a Methanogenic archaeon pyrrolysyl-tRNA synthetase; Preferably, the first O-RS is a mutant of tyrosyl-tRNA synthetase (TyrRS); preferably, the TyrRS is derived from an archaea, such as Methanococcus jannaschii; preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, wherein, The conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, or has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions) compared to the amino acid sequence shown in SEQ ID NO: 15, and retains the ability of SEQ ID NO: 15 to aminoacylate the first orthogonal tRNA (O-tRNA) with the non-natural amino acid; preferably, the orthogonal tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or a conservative variant thereof or an O-tRNA encoded by SEQ ID NO: 16 or a conservative variant thereof; further preferably, the host cell is a prokaryotic cell, such as Escherichia coli; Preferably, the first O-RS is a mutant of pyrrolysyl-tRNA synthetase (PylRS); preferably, the PylRS is derived from an archaeon, such as a methanogenic archaeon, Methanosarcina barkeri, Methanosarcina Mazei, or Methanomethylophilus Alvus; preferably, the O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13 or a conservative variant thereof, wherein the conservative variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 13, or has a sequence identity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% with SEQ ID NO: 12 or SEQ ID NO:

13. NO:13 has one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions), and retains the ability of SEQ ID NO:12 or SEQ ID NO:13 to aminoacylate the first orthogonal tRNA (O-tRNA) with the non-natural amino acid; preferably, the orthogonal tRNA comprises a polynucleotide sequence shown in SEQ ID NO:14 or a conservative variant thereof or an O-tRNA encoded by SEQ ID NO:14 or a conservative variant thereof; further preferably, the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 15 or a conservative variant thereof, and is capable of aminoacylating the first orthogonal tRNA (e.g., a first O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 16 or a conservative variant thereof or encoded by SEQ ID NO: 16 or a conservative variant thereof) with an unnatural amino acid selected from the following: Preferably, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 12 or a conservative variant thereof, and is capable of aminoacylating the first orthogonal tRNA (e.g., a first O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 14 or a conservative variant thereof or encoded by SEQ ID NO: 14 or a conservative variant thereof) with a non-natural amino acid selected from the following: Preferably, the first O-RS has an amino acid sequence as shown in SEQ ID NO: 13 or a conservative variant thereof, and is capable of aminoacylating the first orthogonal tRNA (e.g., a first O-tRNA comprising a polynucleotide sequence as shown in SEQ ID NO: 14 or a conservative variant thereof or encoded by SEQ ID NO: 14 or a conservative variant thereof) with a non-natural amino acid selected from the following: Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

20. A method for preparing the non-natural polypeptide of any one of claims 1 to 16, comprising culturing the host cell of claim 19 in a medium containing the non-natural amino acid under conditions that allow expression of the non-natural polypeptide; and, recovering the non-natural polypeptide expressed by the host cell; Preferably, the host cell further expresses a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), or a combination thereof; Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the unnatural amino acid; Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the first O-RS defined in claim 19; Preferably, the first O-tRNA is capable of specifically recognizing a selector codon (e.g., a stop codon, such as an amber codon) in a cell; preferably, the first O-tRNA is an amber suppressor tRNA; preferably, the first O-tRNA is derived from an archaea, such as Methanococcus jannaschii, methanogenic archaea, Methanosarcina barkeri, Methanosarcina mazei, or Methanomethylophilus Alvus; preferably, the first O-tRNA comprises a polynucleotide sequence shown in SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof or is encoded by SEQ ID NO: 16 or SEQ ID NO: 14 or a conservative variant thereof.

21. Use of the non-natural polypeptide of claims 1 to 16 for detecting protein interactions or abnormal protein aggregation or for detecting the presence or amount of a target molecular entity; Preferably, the target molecular entity is selected from glucose, glutamine, GTP, GDP or Ca 2+ .

22. Use of the non-natural polypeptide of claim 12 for detecting the presence or amount of a target molecular entity; wherein, The target molecular entity is a substrate of the non-natural polypeptide; Preferably, the target molecular entity is selected from glucose, glutamine, GTP, GDP or Ca 2+ .

23. A method for detecting the presence or amount of a target molecular entity in a sample to be tested, comprising contacting the non-natural polypeptide of claim 12 with the sample to be tested, and detecting a change in a fluorescent signal emitted by the non-natural amino acid; wherein: The target molecular entity is a substrate of the non-natural polypeptide.

24. Use of the non-natural polypeptide of claim 13 for detecting the presence or amount of glucose.

25. A method for detecting the presence or amount of glucose in a test sample, comprising contacting the non-natural polypeptide of claim 13 with the test sample, and detecting a change in a fluorescent signal emitted by the non-natural amino acid.

26. Use of the non-natural polypeptide of claim 14 for detecting the presence or amount of glutamine.

27. A method for detecting the presence or amount of glutamine in a test sample, comprising contacting the non-natural polypeptide of claim 14 with the test sample, and detecting changes in a fluorescent signal emitted by the non-natural amino acid.

28. Use of the non-natural polypeptide of claim 15 for detecting the presence or amount of GTP or GDP.

29. A method for detecting the presence or amount of GTP or GDP in a test sample, comprising contacting the non-natural polypeptide of claim 15 with the test sample, and detecting changes in a fluorescent signal emitted by the non-natural amino acid.

30. The non-natural polypeptide of claim 16 is used to detect Ca 2+ The existence or quantity of use.

31. A method for detecting Ca 2+ A method for detecting the presence or amount of a non-natural amino acid in a sample to be tested, comprising contacting the non-natural polypeptide of claim 16 with the sample to be tested, and detecting the change in the fluorescent signal emitted by the non-natural amino acid.

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