Non-natural amino acid and application thereof in construction of fluorescent protein and probe

Abstract

The invention relates to non-natural amino acid and application thereof in construction of fluorescent protein and a probe. Specifically, the invention relates to a brand-new non-natural amino acid, an orthogonal aminoacyl-tRNA synthetase (O-RS), an orthogonal-tRNA (O-tRNA), a translation system, a non-natural polypeptide containing the non-natural amino acid, a kit containing the non-natural amino acid and / or the orthogonal aminoacyl-tRNA synthetase (O-RS) and / or the orthogonal-tRNA (O-tRNA), and a translation method of the non-natural polypeptide containing the non-natural amino acid and / or the orthogonal aminoacyl-tRNA synthetase (O-RS) and / or the orthogonal-tRNA (O-tRNA). The invention also relates to a method for preparing a non-natural polypeptide containing the non-natural amino acid by using the orthogonal aminoacyl-tRNA synthetase (O-RS) and the orthogonal-tRNA (O-tRNA) and a method for modifying the polypeptide. In addition, the invention also relates to the use of the non-natural amino acid as a probe for detecting the presence or amount of a target protein or polypeptide and the use for producing or preparing a non-natural polypeptide.

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, an orthogonal aminoacyl-tRNA synthetase (O-RS), an orthogonal-tRNA (O-tRNA), a translation system, a non-natural polypeptide comprising the non-natural amino acid, a kit comprising the non-natural amino acid and / or an orthogonal aminoacyl-tRNA synthetase (O-RS) and / or an orthogonal-tRNA (O-tRNA), a method for preparing a non-natural polypeptide comprising the non-natural amino acid using the orthogonal aminoacyl-tRNA synthetase (O-RS) and orthogonal-tRNA (O-tRNA) and a method for modifying the polypeptide, and further relates to the use of the non-natural amino acid as a probe to detect the presence or amount of a target protein or polypeptide and the use of the non-natural amino acid for producing or preparing a non-natural polypeptide. Background Art

[0002] Natural fluorescent proteins and subsequent derivative proteins are the mainstream tools for protein fluorescent labeling. Their advantages are simple genetic coding, wide wavelength range selection, and mature transformation strategies, but their disadvantages are also obvious: large protein volume may interfere with the structure and function of the target protein, the labeling site is limited to the end of the protein, and it is difficult to achieve precise modification of the internal site of the protein. The fluorescent labeling method of small molecules faces the disadvantages of reaction selectivity, reaction rate, and difficulty in achieving real-time detection in living cells. On the other hand, protein non-natural amino acid labeling based on genetic code expansion (GCE) technology can achieve flexible modification of any site of the protein, and the extremely small amino acid volume also avoids interference with the structure and function of the target protein as much as possible. However, the key to protein fluorescent labeling using GCE technology lies in the development of better fluorescent non-natural amino acids (FL-ncAAs or FLAAs). Existing FLAAs are essentially traditional fluorescent dyes, with limitations such as inherent fluorescence, fluorescence background problems, short wavelengths, 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 the present application have designed and prepared a new type of fluorescent rotor noncanonical amino acids (FR-ncAAs or FRAAs). The characteristics of these novel fluorescent rotor noncanonical amino acids are that their fluorescence is potential and activatable. For example, in a free state, excited FMRs can undergo free intramolecular rotation and thus do not show fluorescence; however, for another example, when FMRs are combined with structurally complementary macromolecules, intramolecular rotation is restricted, thereby showing fluorescence.

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

[0006] Then, the inventors used the above fluorescent rotor non-natural amino acids as genetically encodable chromophores and introduced them into a variety of natural non-fluorescent proteins using GCE technology, successfully creating a variety of artificial fluorescent proteins with different structures, and expanding the existing fluorescent protein tool library.

[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 Ra , 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 , 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.

[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] Formula I-A1 and Formula I-A2

[0033] 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;

[0034] or,

[0035]

[0036] 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;

[0037] 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;

[0038] More preferably, R1 is selected from: -NH2, -NR a R b , where R a , R b independently selected from methyl;

[0039] R4 is independently selected from: -(CH2) n CH(NH2)(COOH), n is an integer selected from 1 to 3;

[0040] Preferably, R4 is selected from: -(CH2)CH(NH2)(COOH);

[0041] Preferably, Formula IB is selected from:

[0042]

[0043] Formula I-B1 and Formula I-B2

[0044] R1 and R4 in the above formula I-B1 and formula I-B2 are defined as the corresponding variables in the above formula IB.

[0045] In some embodiments, the non-natural amino acid is selected from the following:

[0046] 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;

[0047] 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;

[0048] 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;

[0049] 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;

[0050] Preferably, the O-RS carries the non-natural amino acid described in the first aspect;

[0051] 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:

[0052] 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:

[0053] 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:

[0054] 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;

[0055] Preferably, the O-tRNA is an amber suppressor tRNA;

[0056] 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;

[0057] 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:

[0058] 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:

[0059] 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.

[0060] In a fifth aspect, the present invention provides a vector comprising the isolated nucleic acid of the fourth aspect;

[0061] Preferably, the vector is a cloning vector or an expression vector;

[0062] Preferably, the vector further comprises a polynucleotide encoding an orthogonal tRNA (O-tRNA);

[0063] Preferably, the O-RS is capable of aminoacylating the O-tRNA with the non-natural amino acid described in the first aspect;

[0064] Preferably, the O-tRNA is an amber suppressor tRNA;

[0065] 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;

[0066] 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;

[0067] 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;

[0068] 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.

[0069] 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;

[0070] 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);

[0071] Preferably, the host cell further contains a polynucleotide encoding an orthogonal tRNA (O-tRNA);

[0072] Preferably, the O-RS is capable of aminoacylating the O-tRNA with the non-natural amino acid described in the first aspect;

[0073] 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;

[0074] Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

[0075] 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.

[0076] In an eighth aspect, the present invention provides a translation system, comprising:

[0077] (a) a first non-natural amino acid selected from the non-natural amino acids described in the first aspect;

[0078] (b) a first orthogonal aminoacyl-tRNA synthetase (O-RS); and

[0079] (c) a first orthogonal tRNA (O-tRNA);

[0080] wherein the first O-RS is capable of aminoacylating the first O-tRNA with the first unnatural amino acid;

[0081] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0082] 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;

[0083] 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;

[0084] 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;

[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: 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;

[0086] 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;

[0087] 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;

[0088] 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);

[0089] Preferably, the host cell contains a polynucleotide encoding the first O-RS, a polynucleotide encoding the first O-tRNA, or a combination thereof.

[0090] In a ninth aspect, the present invention provides a cell comprising the translation system described in the eighth aspect;

[0091] 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;

[0092] 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;

[0093] 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;

[0094] 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;

[0095] 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.

[0096] In a tenth aspect, the present invention provides a kit comprising

[0097] (i): a first unnatural amino acid selected from the unnatural amino acids described in the first aspect; or

[0098] (ii): a first orthogonal aminoacyl-tRNA synthetase (O-RS), or a first polynucleotide encoding the first O-RS; or,

[0099] (iii) : a combination of (i) and (ii);

[0100] Preferably, the kit further comprises, (iv): a first orthogonal tRNA (O-tRNA), or, a second polynucleotide encoding the first O-tRNA;

[0101] Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the first unnatural amino acid;

[0102] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0103] 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;

[0104] 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);

[0105] 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;

[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: 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;

[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: 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;

[0108] 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;

[0109] Preferably, the kit further comprises a second unnatural amino acid; further preferably, the kit further comprises:

[0110] (a): a second orthogonal aminoacyl-tRNA synthetase (O-RS), or a first polynucleotide encoding the second O-RS; or,

[0111] (b): a second orthogonal tRNA (O-tRNA), or a second polynucleotide encoding the second O-tRNA; or,

[0112] (c): a combination of (a) and (b);

[0113] 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;

[0114] 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;

[0115] 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;

[0116] 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).

[0117] In an eleventh 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;

[0118] Optionally, the non-natural polypeptide is further linked (covalently linked or fused) to another polypeptide,

[0119] Preferably, the additional polypeptide is linked to the N-terminus or C-terminus of the polypeptide or variant thereof, optionally via a linker;

[0120] Preferably, the additional polypeptide is selected from a protein tag, a targeting moiety or any combination thereof.

[0121] In some embodiments, for the above-mentioned non-natural polypeptide, wherein the intramolecular rotation of the non-natural amino acid in the spatial structure of the non-natural polypeptide is restricted;

[0122] Preferably, the non-natural polypeptide further comprises a mutation at a position adjacent to the non-natural amino acid; preferably, the mutation can enhance or weaken the restriction on the intramolecular rotation of the non-natural amino acid;

[0123] Preferably, the non-natural polypeptide is derived from a polypeptide selected from the following: GFP protein, ThRS protein, FNIII protein, Z protein, α or β tubulin, AAV virus capsid protein, VHH nanobody protein, Nanofitin protein, cytochrome b562 protein, and contains the non-natural amino acid, and the intramolecular rotation of the non-natural amino acid in the spatial structure of the non-natural polypeptide is restricted;

[0124] Preferably, the non-natural polypeptide comprises or is a GFP protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following positions of the GFP protein: a position corresponding to position 150, 152, 62 or 203 of SEQ ID NO:6;

[0125] Preferably, the non-natural amino acid is located at the following positions of the GFP protein: the position corresponding to position 150 or 152 of SEQ ID NO:4 or SEQ ID NO:6; preferably, the non-natural amino acid is pAPCF; preferably, the GFP protein further comprises a mutation at one or more positions selected from the following positions: the positions corresponding to positions 65, 66, 69, 201, 222, 224 of SEQ ID NO:4; or, preferably, the GFP protein further comprises a mutation at one or more positions selected from the following positions: the positions corresponding to positions 66, 201, 61, 65, 222, 224 of SEQ ID NO:6;

[0126] Preferably, the GFP protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:4 or SEQ ID NO:6 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:4 or SEQ ID NO:6, 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:4 or SEQ ID NO:6; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 150 or 152 of SEQ ID NO:4 or SEQ ID NO:6;

[0127] or

[0128] Preferably, the non-natural polypeptide comprises or is a ThRS protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following sites of the ThRS protein: a site corresponding to site 11, 40, 79 or 125 of SEQ ID NO:9;

[0129] Preferably, the non-natural amino acid is located at the following site of the ThRS protein: the site corresponding to site 11 of SEQ ID NO:9; preferably, the non-natural amino acid is pAPCF; preferably, the ThRS protein further comprises a mutation at one or more sites selected from the following: sites corresponding to sites 13, 79, 121, 123 of SEQ ID NO:9;

[0130] Preferably, the ThRS protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO: 9 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: 9, 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: 9, and the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 11 of SEQ ID NO: 9;

[0131] or

[0132] Preferably, the non-natural polypeptide comprises or is a FNIII protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following positions of the FNIII protein: a position corresponding to position 36, 20, 22, 38, 61, 72 or 74 of SEQ ID NO: 11;

[0133] Preferably, the unnatural amino acid is located at the following position of the FNIII protein: the position corresponding to position 36 of SEQ ID NO: 11; preferably, the unnatural amino acid is pAPCF; preferably, the FNIII protein further comprises a mutation at one or more positions selected from the following: the positions corresponding to positions 20, 22, 72, 94 of SEQ ID NO: 11;

[0134] Preferably, the FNIII protein containing a non-natural amino acid comprises an amino acid sequence as shown in 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: 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: 11, and the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 36 of SEQ ID NO: 11;

[0135] or

[0136] Preferably, the non-natural polypeptide comprises or is a Z protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following positions of the Z protein: a position corresponding to position 9, 16, 34 or 44 of SEQ ID NO: 18;

[0137] Preferably, the non-natural amino acid is located at the following position of the Z protein: the position corresponding to position 9 of SEQ ID NO: 18; preferably, the non-natural amino acid is pAPCF; preferably, the Z protein further comprises a mutation at one or more positions selected from the following: positions corresponding to positions 9, 32, 35 of SEQ ID NO: 18;

[0138] Preferably, the Z protein containing non-natural amino acids comprises the amino acid sequence as shown in SEQ ID NO: 18 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: 18, 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: 18, and the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 9 of SEQ ID NO: 18;

[0139] 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.

[0140] In a twelfth aspect, the present invention provides an isolated nucleic acid comprising a polynucleotide encoding the non-natural polypeptide of the eleventh aspect, wherein the non-natural amino acid is encoded by a selector codon (eg, a stop codon, eg, an amber codon).

[0141] In a thirteenth aspect, the present invention provides a vector comprising the isolated nucleic acid of the twelfth aspect;

[0142] Preferably, the vector is a cloning vector or an expression vector.

[0143] In a fourteenth aspect, the present invention provides a host cell comprising the isolated nucleic acid of the twelfth aspect or the vector of the thirteenth aspect;

[0144] 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);

[0145] 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;

[0146] Preferably, the first O-RS is capable of aminoacylating the first O-tRNA with the non-natural amino acid of the first aspect;

[0147] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0148] 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;

[0149] Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

[0150] In a fifteenth aspect, the present invention provides a method for preparing the non-natural polypeptide of the eleventh aspect, comprising culturing the host cell of the fourteenth aspect in a culture 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;

[0151] Preferably, the host cell further expresses a first orthogonal aminoacyl-tRNA synthetase (O-RS), a first orthogonal tRNA (O-tRNA), or a combination thereof;

[0152] Preferably, the first O-RS is capable of aminoacylating the first O-tRNA with the non-natural amino acid of the first aspect;

[0153] Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS described in the second aspect;

[0154] 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 archaea, such as Methanococcus jannaschii, methanogenic archaea, 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.

[0155] In a sixteenth aspect, the present invention provides a method for modifying a polypeptide, comprising:

[0156] 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.

[0157] In a seventeenth aspect, the present invention provides use of the non-natural amino acid described in the first aspect as a probe to detect the presence or amount of a target protein or polypeptide.

[0158] In an eighteenth aspect, the present invention provides use of the non-natural amino acid described in the first aspect for producing or preparing a non-natural polypeptide.

[0159] Definition of terms

[0160] 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.

[0161] 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."

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] 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.

[0170] As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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).

[0185] 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.

[0186] 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.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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).

[0192] 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".

[0193] 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.

[0194] 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.

[0195] 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).

[0196] 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.

[0197] 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.

[0198] 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.

[0199] 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 9 of SEQ ID NO:18" refers to a site in the amino acid sequence of protein Z that is located at an equivalent position to site 9 of SEQ ID NO:18 when protein Z is aligned with SEQ ID NO:18 to obtain the highest percentage identity.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] As used herein, the term "antigen-antibody molecule pair" refers to an antigen and an antibody that can specifically bind to each other.

[0204] 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.

[0205] 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.

[0206] 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).

[0207] Beneficial Effects

[0208] The present invention develops for the first time genetically encodable fluorescent rotor non-natural amino acids (FR-nc AAs); for the first time genetically encodable artificial fluorescent proteins with a variety of structural types and sizes; and realizes precise, low-interference, background-free fluorescent labeling of proteins and the development of fluorescent probes with high signal-to-noise ratio and high versatility. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figure 1 Shown are the (a) absorption spectra and (b) fluorescence emission spectra of pAPCF in different solvents.

[0210] 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.

[0211] Figure 3 Shows the fluorescence intensity changes of pAPCF in glycerol / methanol mixed systems with different viscosities.

[0212] 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.

[0213] Figure 5 Shown are: (a) fluorescence measurement of sfGFP fluorescent protein reporter element; (b) mass spectrometry identification results of sfGFP (151pAPCF) expressed using MjFmrRS.

[0214] Figure 6 Fluorescence flow cytometry screening of the PylRS library: (a) positive screening flow cytometry analysis; (b) negative screening flow cytometry analysis.

[0215] 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.

[0216] Figure 8 Fluorescence photographs and fluorescence intensity comparison of GFP mutants introduced into pAPCF at different sites using GFP (Y66G) as the structural basis.

[0217] Fig. 9 Fluorescence photographs showing GFP-152-B6 protein and the fluorescence intensity comparison with sfGFP-152pAPCF.

[0218] Fig.10 Shown: (a) fluorescence spectrum measurement of Th-A12; (b) fluorescence intensity comparison of Th-A12 and Th-11pAPCF before screening and optimization.

[0219] Fig.11 Shown: (a) fluorescence spectrum measurement of FN-E9; (b) fluorescence intensity comparison of FN-E9 and FN-36pAPCF before screening optimization.

[0220] Fig.12 Shown: (a) fluorescence spectrum measurement of Z-H5; (b) fluorescence intensity comparison of Z-H5 and Z-9pAPCF before screening and optimization. DETAILED DESCRIPTION

[0221] 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.

[0222] 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.

[0223] Sequence information

[0224] The following provides information on some of the sequences involved in this application.

[0225] SEQ ID NO: 1 (nucleotide sequence of the T7 RNA polymerase gene that controls RFP transcription)

[0226]

[0227] SEQ ID NO:2 (amino acid sequence of sfGFP-Y66-mut)

[0228] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTXGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNANSGNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSVQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0229] X stands for pAPCF

[0230] SEQ ID NO:3 (nucleotide sequence encoding sfGFP-Y66-mut)

[0231] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTAGGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACGCTAACTCAGGCAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGGTGCAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA

[0232] SEQ ID NO:4 (Amino acid sequence of sfGFP-152-mut)

[0233] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLGGGVGCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYXTADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYGSTQSVLSKDPNEKRDHMVLLDFATAAGITHGMDELYK

[0234] X represents pAPCF

[0235] SEQ ID NO:5 (Coding nucleotides of sfGFP-152TAG-mut)

[0236] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCACTACTCTGGGCGGCGGTGTTGGATGCTTTTCCCGTTATCCGGATCACATGAAACGGCACGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTGGAGTACAACTTTAACTCACACAATGTATACTAGACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACGGTTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGACTTTGCAACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA

[0237] SEQ ID NO:6 (Amino acid sequence of sfGFP-152-B6)

[0238] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLCGTLSGGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYXTADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYGSTQSVLSKDPNEKRDHMVLLQFSTAAGITHGMDELYK

[0239] X represents pAPCF

[0240] SEQ ID NO:7 (Nucleotide sequence of sfGFP-152-B6)

[0241] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTTGTGGTACTCTGAGTGGCGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCACGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTGGAGTACAACTTTAACTCACACAATGTATACTAGACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACGGTTCGACACAATCTGTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTCAGTTTAGTACTGCTGCTGGGATTACACATGGCATGGATGAGCTCTACAAA

[0242] SEQ ID NO:8 (Amino acid sequence of the initial ThRS protein)

[0243] MRVLLIHSDYIEYEVKDKALKNPEPISEDMKRGRMEEVLVAFISVEKVDEKNPEEVSLKAIEEISKVAEQVKAENVFVIPFAHLSSELAKPSVAMDILNRVYQGLKERGFNVGKAPFGYYKAAKISCKGHPLAELSRTIVPEE

[0244] SEQ ID NO:9 (Amino acid sequence of Th-A12)

[0245] MRVLLIHSDYXECEVKDKALKNPEPISEDMKRGRMEEVLVAFISVEKVDEKNPEEVSLKAIEEISKVAEQVKAENVFVVPFAHLSSELAKPSVAMDILNRVYQGLKERGFNVGKAPFGYYAATKISCKGHPLAELSRTIVPEE

[0246] X stands for pAPCF

[0247] SEQ ID NO: 10 (amino acid sequence of the original FNIII protein)

[0248] MRVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISG LKPGVDYTITVYAVTGRGDSPASSKPISINYRT

[0249] SEQ ID NO: 11 (amino acid sequence of FN-E9)

[0250] MRVSDVPRDLEVVAATPTSVLVSWDAPAVTVRYYRXTYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTATVYAVTGRGDSPASSKPISINHRT

[0251] X stands for pAPCF

[0252] SEQ ID NO: 12 (amino acid sequence of C-C9 PylRS mutant)

[0253] MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIANPRNDLTDLENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALRPMHAMNVIKVSRELRDHTK GPVKIFEIGSCFRKESKSSTHLEEFTMLALAEMGPDGDPMEHLKMYIGDIMDAVGVEYTTSREESDVWVETLDVEINGTEVASGAVGPHKLDPAHDVHEPWAGIGFGLERLLMLKNGKSNARKTGKSITYLNGYKLD

[0254] SEQ ID NO: 13 (amino acid sequence of AC2 PylRS mutant)

[0255] MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIANPRNDLTDLENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALRPMHAMNLKKVGRELRDHTKGPVKIFEIGSCFRKESKSSAHLEEFTMLALAEMGPDGDPMEHLKMYIGDIMDAVGVEYTTSREESDVYVETLDVEINGTEVASGAVGPHKLDPAHDVHEPWAGIGFGLERLLMLKNGKSNARKTGKSITYLNGYKLD

[0256] SEQ ID NO:14 (tRNA pyl 's nucleotide sequence)

[0257] GGGGGACGGTCCGGCGACCAGCGGGTCTCTAAAACCTAGCCAGCGGGGTTCGACGCCCCGGTCTCTCGCCA

[0258] SEQ ID NO:15 (Amino acid sequence of MjFmrRS)

[0259] MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHLGHYLQIKKMIDLQNAGFDIIIYLADLHAYLNQKGELDEIRKIGDYNKKVFEAMGLKAKYVYGSEHGLDKDYTLNVYRLALKTTLKRARRSMELIAREDENPKVAEVIYPIMQVNGIHYEGVDVAVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMRLKNAVAEELIKILEPIRKRL

[0260] SEQ ID NO:16 (tRNA tyr 's nucleotide sequence)

[0261] CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAATCCGCATGGCAGGGGTTCAAATCCCCTCCGCCGGACCA

[0262] SEQ ID NO: 17 (original Z protein amino acid sequence)

[0263] VDNKFNKEMRNAYWEIALLPNLNNQQKRAFIRSLYDDPSQSANLLAEAKKLNDAQAPK

[0264] SEQ ID NO: 18 (amino acid sequence of Z-H5)

[0265] VDNKFNKEXRNAYWEIALLPNLNNQQKRAFICSLTDDPSQSANLLAEAKKLNDAQAPK

[0266] X stands for pAPCF

[0267] SEQ ID NO: 19 (amino acid sequence of sfGFP)

[0268] MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLV

[0269] TTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIEL

[0270] KGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIG

[0271] DGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK

[0272] SEQ ID NO:20 (nucleotide sequence of GFP)

[0273] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGGT

[0274] GATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGAAA

[0275] ACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGTCAC

[0276] TACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATGACTT

[0277] TTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGG

[0278] GACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCGAGT

[0279] TAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTACAACTTTA

[0280] ACTCACACAATGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAACTTCAAAA

[0281] TTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAA

[0282] TTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGA

[0283] AAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGATT

[0284] ACACATGGCATGGATGAGCTCTACAAATAA

[0285] SEQ ID NO:21 (Nucleotide sequence of RFP)

[0286] ATGGTGAGCGAGCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGGCACCGT

[0287] GAACAACCACCACTTCAAGTGCACATCCGAGGGCGAAGGCAAGCCCTACGAGGGCACCCAG

[0288] ACCATGAGAATCAAGGCGGTCGAGGGCGGCCCTCTCCCCTTCGCCTTCGACATCCTGGCTAC

[0289] CAGCTTCATGTACGGCAGCAAAACCTTCATCAACCACACCCAGGGCATCCCCGACTTCTTTA

[0290] AGCAGTCCTTCCCCGAGGGCTTCACATGGGAGAGAGTCACCACATACGAAGACGGGGGCGT

[0291] GCTGACCGCTACCCAGGACACCAGCCTCCAGGACGGCTGCCTCATCTACAACGTCAAGATCA

[0292] GAGGGGTGAACTTCCCATCCAACGGCCCTGTGATGCAGAAGAAAACACTCGGCTGGGAGGC

[0293] CTCCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTG

[0294] AAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGAAGACCACATACAGATCCAAGAAAC

[0295] CCGCTAAGAACCTCAAGATGCCCGGCGTCTACTATGTGGACAGAAGACTGGAAAGAATCAAG

[0296] GAGGCCGACAAAGAGACCTACGTCGAGCAGCACGAGGTGGCTGTGGCCAGATACTGCGACC

[0297] TCCCTAGCAAACTGGGGCACAGATAA

[0298] SEQ ID NO:22(Nucleotide sequence of sfGFP(151TAG) reporter gene)

[0299] ATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAATTAGATGG

[0300] TGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGTGATGCTACAAACGGA

[0301] AAACTCACCCTTAAATTTATTTGCACTACTGGAAAACTACCTGTTCCGTGGCCAACACTTGT

[0302] CACTACTCTGACCTATGGTGTTCAATGCTTTTCCCGTTATCCGGATCACATGAAACGGCATG

[0303] ACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGAT

[0304] GACGGGACCTACAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTA

[0305] TCGAGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACTCGAGTAC

[0306] AACTTTAACTCACACAATGTATAGATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAA

[0307] CTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGTTCAACTAGCAGACCATTATCAACAAA

[0308] ATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTCGACACAATCT

[0309] GTCCTTTCGAAAGATCCCAACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGC

[0310] TGCTGGGATTACACATGGCATGGATGAGCTCTACAAAGGATCCCACCACCACCACCACCAC

[0311] TAA

[0312] SEQ ID NO:23 (Amino acid sequence of G1pylRS)

[0313] MVVKFTDSQIQHLMEYGDNDWSEAEFEDAAARDKEFSSQFSKLKSANDKGLKDVIANPRND

[0314] LTDLENKIREKLAARGFIEVHTPIFVSKSALAKMTITEDHPLFKQVFWIDDKRALRPMHAMNLY

[0315] KVMRELRDHTKGPVKIFEIGSCFRKESKSSTHLEEFTMLNLVEMGPDGDPMEHLKMYIGDIMD

[0316] AVGVEYTTSREESDVYVETLDVEINGTEVASGAVGPHKLDPAHDVHEPWAGIGFGLERLLMLKN

[0317] GKSNARKTGKSITYLNGYKLD

[0318] SEQ ID NO:24 (Amino acid sequence of mCherry-GFP fusion protein)

[0319] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLP

[0320] FAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFI

[0321] YKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTY

[0322] KAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKHRSQPWVPRARDPP

[0323] VATLVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATXGKLTLKFICTTGKLPVPWPTL

[0324] VTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRI

[0325] ELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPI

[0326] GDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

[0327] X stands for pAPCF or PAPd

[0328] Example 1: Synthesis of unnatural amino acid pAPCF

[0329] 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.

[0330]

[0331] (1) tert-butyl(S)-3-(4-(bromomethyl)phenyl)-2-((diphenylmethylene)amino)propanoate

[0332] 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%.

[0333] H NMR characterization:

[0334] 1 H 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).

[0335] C NMR characterization:

[0336] 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.

[0337] Mass spectrometry characterization:

[0338] LC-MS(ESI)calcd for C 27 H 28 BrNO2(M+1):478.1;obsd:478.1

[0339] (2) tert-butyl(S)-3-(4-(cyanomethyl)phenyl)-2-((diphenylmethylene)amino)propanoate

[0340] 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%.

[0341] H NMR characterization:

[0342] 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).

[0343] C NMR characterization:

[0344] 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.

[0345] Mass spectrometry characterization:

[0346] LC-MS(ESI)calcd for C 28 H 28 N2O2(M+1):425.2; obsd:425.2

[0347] (3) tert-butyl(S,Z)-3-(4-(2-(4-((tert-butoxycarbonyl)amino)phenyl)-1-cyanovinyl)phenyl)-2-((diphenylmethylene)amino)propanoate

[0348] 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%.

[0349] H NMR characterization:

[0350] 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).

[0351] C NMR characterization:

[0352] 13C 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.

[0353] Mass spectrometry characterization:

[0354] LC-MS(ESI)calcd for C 40 H 41 N3O4(M+1):628.3; obsd:628.3

[0355] (4) pAPCF amino acid

[0356] 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%.

[0357] H NMR characterization:

[0358] 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).

[0359] C NMR characterization:

[0360] 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.

[0361] Mass spectrometry characterization:

[0362] LC-MS(ESI)calcd for C 18 H 17 N3O2(M+1):308.14; obsd:308.14

[0363] Example 2 Synthesis of unnatural amino acids mPAPF and mDMPF

[0364] 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.

[0365] 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.

[0366]

[0367] The comprehensive yield of mPAPF was 29.2%.

[0368] Mass spectrometry characterization: (M+1) + calcd:308.14;obsd:308.15

[0369] NMR characterization of mPAPF:

[0370] 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).

[0371] 13 C 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.

[0372] The comprehensive yield of mDMPF was 31.9%.

[0373] Mass spectrometry characterization: (M+1) + calcd:336.17;obsd:336.18

[0374] NMR characterization of mDMPF:

[0375] 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).

[0376] 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.

[0377] Example 3: Synthesis of unnatural amino acids PAPd and DMPd

[0378] 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.

[0379] 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.

[0380]

[0381] (1) Synthesis of 2,6-bis(bromomethyl)pyridine

[0382] 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.

[0383] NMR characterization:

[0384] 1H NMR (400MHz, DMSO): δ7.83 (t, J = 7.7Hz, 1H), 7.48 (d, J = 7.7Hz, 2H), 4.67 (s, 4H).

[0385] 13 C NMR (101MHz, DMSO): δ156.56,138.45,123.13,34.40.

[0386] Mass spectrometry characterization:

[0387] LC-MS(ESI)calcd for C7H7Br2N(M+1):265.9;obsd:265.

[0388] (2) PAPd amino acid. PAPd amino acid was prepared as a reddish brown solid with a comprehensive yield of 28.8%.

[0389] (3) DMPd amino acid. DMPd amino acid was prepared as a dark red solid with a comprehensive yield of 31.4%.

[0390] Mass spectrometry characterization of PAPd:

[0391] LC-MS(ESI)calcd for C 17 H 16 N4O2(M+1):309.14;obsd:309.14

[0392] NMR characterization of PAPd:

[0393] 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).

[0394] 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.

[0395] Mass spectrometry characterization of DMPd: LC-MS (ESI) calcd for (M+1) + :337.17; obsd:337.17

[0396] NMR Characterization of DMPd:

[0397] 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).

[0398] 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.

[0399] Example 4 Synthesis of unnatural amino acid PAFCN

[0400] 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.

[0401] 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.

[0402]

[0403] Synthesis of 2,6-Bis(bromomethyl)benzonitrile

[0404] 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%

[0405] Mass spectrometry characterization of PAFCN: LC-MS (ESI) calcd for (M+1) + :333.14; obsd:333.14

[0406] NMR characterization of PAFCN:

[0407] 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).

[0408] 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.

[0409] Example 5: Performance Test

[0410] 1. Fluorescence spectroscopy measurement

[0411] (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.

[0412] (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.

[0413] The inventors dissolved pAPCF in different solvents and then measured the corresponding absorption and fluorescence emission spectra ( Figure 1 and Table 1).

[0414] Table 1 Maximum absorption wavelength and emission wavelength of pAPCF in various solvents

[0415]

[0416] 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.

[0417] 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.

[0418] Table 2 Maximum fluorescence excitation, emission wavelength and fluorescence activation multiples of various unnatural amino acids in glycerol solvent

[0419]

[0420]

[0421] 2. Characterization of Viscosity Dependence of Fluorescence

[0422] 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.

[0423] 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.

[0424] 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 ).

[0425] Example 6 Screening for mutants of the MjTyrRS system that can be used in prokaryotic cells

[0426] 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.

[0427] Screening method: Screening and identification of specific aaRS / tRNA pairs in E. coli

[0428] 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.

[0429] 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.

[0430] Example 7 Screening for pyrrolysyl tRNA synthetase mutants

[0431] 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.

[0432] Step 1: Construction of PylRS mutant library

[0433] 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.

[0434] Step 2: Fluorescence flow cytometry screening of the PylRS mutant library

[0435] 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.

[0436] Step 3: Verify the efficiency of PylRS mutants

[0437] 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.

[0438] 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 ).

[0439] Briefly, to verify the efficiency of unnatural amino acid introduction in eukaryotic cells, a gene encoding C-C9 PylRS or AC2 PylRS and tRNA pyl A plasmid containing the nucleotide sequence of pyl Plasmid) and pCDNA3.1-mCherry-EGFP (39TAG) plasmid were simultaneously transfected into HEK293T cells, and then the cell culture medium was replaced with fresh culture medium containing 50-500uM of the specified non-natural amino acid (such as pAPCF or PAPd), and the 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 the introduction of non-natural amino acids (such as pAPCF or PAPd). The results showed that C-C9PyRS can simultaneously and efficiently recognize amino acids such as pAPCF, PAPd, PAFCN, 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, DMPd, and introduce them into polypeptides expressed using a eukaryotic cell expression system ( Figure 7 ).

[0440] About 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 (whose nucleotide sequence is shown in SEQ ID NO: 14)

[0441] Regarding the pCDNA3.1-mCherry-EGFP (39TAG) plasmid, it encodes the mCherry-EGFP fusion protein (its amino acid sequence is shown in SEQ ID NO: 24), wherein the mCherry fluorescent protein is fused and expressed at the N-terminus of EGFP, and the amino acid at the 39th position 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.

[0442] Example 8 Construction and verification of an artificial fluorescent protein derived from GFP protein containing pAPCF

[0443] This embodiment and the embodiments below introduce pAPCF into a specific site of the target protein, thereby constructing genetically encoded artificial fluorescent proteins containing pAPCF. Since the pAPCF in these artificial fluorescent proteins is in a restricted intramolecular rotation state, these artificial fluorescent proteins emit the expected fluorescence under the excitation of excitation light.

[0444] Step 1 Design and preparation of the non-fluorescent sfGFP-Y66G mutant

[0445] In this example, in order to destroy the chromophore of sfGFP, the key amino acid Tyr 66 that forms the chromophore was mutated to Gly using point mutation PCR to obtain a GFP mutant that does not produce fluorescence.

[0446] In brief, a point mutation base of Y66G was set on the primer DNA, and PCR amplification was performed using the sfGFP gene as a template according to conventional PCR conditions. After the PCR reaction was completed, DNA agarose gel electrophoresis was performed, the DNA fragment was purified and the concentration was determined. Subsequently, the PCR product containing the target point mutation verified by sequencing was cloned into a plasmid to construct a pET-sfGFP (Y66G) plasmid (the nucleotide sequence of sfGFP-Y66-mut is shown in SEQ ID NO: 3), and the plasmid was transferred into BL21 (DE3) cells for protein expression and purification, thereby obtaining a fluorescent silent sfGFP (Y66G) mutant (the amino acid sequence is shown in SEQ ID NO: 2).

[0447] Step 2 Construction of sfGFP(Y66G)-152TAG saturation mutation library

[0448] Based on the fluorescence-silent sfGFP (Y66G) mutant, appropriate sites were selected to introduce pAPCF. The crystal structure of sfGFP (PDB: 1QYO) was analyzed using Pymol software (https: / / pymol.org / 2 / ), and sites 150 or 152 near the original HBI chromophore were selected, and the coding nucleotides of the sites were mutated by TAG to introduce pAPCF at the sites. The results showed that the obtained GFP mutants (sfGFP-150pAPCF and sfGFP-152pAPCF) could produce weak fluorescence.

[0449] Next, based on structural analysis, the sfGFP-152-mut mutant (whose amino acid sequence is shown in SEQ ID NO: 4, containing the following multiple point mutations, including I152TAG, T65G, Y66G, Q69G, L201G, E222D, V224A) was further constructed to enhance the fluorescence of the mutant.

[0450] Next, a saturation mutation library was established based on sfGFP-152-mut to further improve the fluorescence intensity of the mutant. Using the pET-sfGFP-152TAG-mut plasmid (the nucleotides of sfGFP-152TAG-mut are shown in SEQ ID NO: 5) as a template, while retaining the original mutations Y66G and L201G, the coding nucleotides of sites 61, 65, 222, and 224 near pAPCF were subjected to NNK saturation mutation to construct a saturation mutation library plasmid of pET-sfGFP-152TAG-61 / 65 / 222 / 224NNK. The saturation mutation library plasmid and pUltra-MjFmrRS-tRNA tyr Transfer them into BL21(DE3) together, culture and collect the bacteria for subsequent use.

[0451] Step 3 Screening, identification, expression and purification of the target fluorescent protein mutant sfGFP-152-B6

[0452] The E. coli containing the mutant library plasmid was revived and cultured. 0.2mM IPTG and 0.8mM pAPCF were added to the positive group, and then cultured in a shaker at 25°C for 16 hours to induce the expression of sfGFP protein mutants. During fluorescence sorting, bacteria with fluorescence intensity in the top 0.1% were selected, enriched and cultured. Single clones were picked for identification, and the ratio of fluorescence intensity / OD600 was used to evaluate the relative fluorescence intensity of each protein mutant. After identifying clones that emit stronger fluorescence, the bacterial solution was transferred for amplification and plasmid extraction, and sent for sequencing to determine the specific amino acid point mutation. Finally, a sfGFP-152-B6 mutant was identified and screened. The screened fluorescent protein mutant sfGFP-152-B6 was expressed and purified. The operation process mainly includes:

[0453] (1) Plasmid encoding the sfGFP-152-B6 mutant (SEQ ID NO: 7) and pUltra-MjFmrRS-tRNA tyr The plasmid was transformed into BL21 (DE3) (purchased from Thermo Fisher), and single clones were selected for culture.

[0454] (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, 0.2 mM IPTG (purchased from INALCO) and 0.8 mM pAPCF were added to induce expression at 25°C for 16 h.

[0455] (3) After harvesting the bacteria, discard the supernatant, wash once with PBS, and then resuspend the bacteria in 10 ml of lysis buffer (25 mM tris, 500 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mM PMSF, pH 8.0).

[0456] (4) Incubate the bacterial solution on ice and ultrasonically lyse it for 20 min.

[0457] (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 mix and incubate on ice for 1.5 hours.

[0458] (6) The protein solution was transferred to a gravity-type protein purification column and the column was washed with 20 mM and 40 mM imidazole wash buffer (25 mM tris, 500 mM NaCl, 20 or 40 mM imidazole, 10% glycerol, pH 8.0) in sequence.

[0459] (7) Finally, the target mutant protein was washed off with elution buffer (25 mM tris, 500 mM NaCl, 10% glycerol, 250 mM imidazole, pH 7.4).

[0460] (8) Finally, the protein eluate was concentrated using a 10K ultrafiltration tube and replaced with PBS three times.

[0461] The experimental results showed that the mutants in which the amino acids at positions 150 and 152 of sfGFP-Y66G were replaced with pAPCF respectively all produced blue fluorescence, preliminarily proving that pAPCF can be used as a chromophore molecule after being introduced into the protein. The sfGFP-152-mut (amino acid sequence as shown in SEQ ID NO: 4) showed significantly enhanced fluorescence, and its brightness was 4.6 times higher than that of the mutant sfGFP-152pAPCF in which only the amino acid at position 152 was replaced with pAPCF ( Figure 8). By constructing a library through saturation mutagenesis based on the sfGFP-152-mut mutant, a consensus mutant sequence with high enrichment was finally identified, namely 152TAG, Y66G, L201G, V61C, T65S, ​​E222N, V224S, named sfGFP-152-B6 (its amino acid sequence is shown in SEQ ID NO:6). The experimental results showed that sfGFP-152-B6 could emit bright blue fluorescence after being excited. Quantitative analysis of fluorescence intensity showed that the fluorescence intensity of sfGFP-152-B6 was 68 times that of sfGFP-152pAPCF ( Fig. 9 ). This shows that saturation mutation library construction based on structural analysis combined with fluorescence flow sorting is an effective strategy to optimize the fluorescence properties of artificial fluorescent proteins. The non-natural amino acids (such as pAPCF) of the present application can be used to develop artificial fluorescent proteins.

[0462] Example 9 Construction and verification of artificial fluorescent protein derived from ThRS protein

[0463] The properties of artificial fluorescent proteins have been successfully verified in Example 8. In order to verify the versatility of this method, the following examples will select a variety of proteins with different structures to construct similar artificial fluorescent proteins. The threonyl-tRNA synthetase (ThRS) of Pyrococcus abyssi bacteria has a molecular weight of 17kDa (amino acid sequence as shown in SEQ ID NO: 8). This example is based on the ThRS protein to construct and verify an artificial fluorescent protein derived from ThRS.

[0464] A DNA fragment encoding ThRS protein was synthesized to construct a pET-ThRS plasmid expressing ThRS protein. The crystal structure of ThRS protein was analyzed (PDB: 1Y2Q), and a site inside the protein with a certain cavity was selected as the introduction site of pAPCF. Site 11 of ThRS was selected to introduce pAPCF to obtain the ThRS-11pAPCF mutant. Then, referring to the aforementioned embodiment, key sites around pAPCF (including sites Y13, I79, K121, and A123) were selected for saturation mutation to establish a saturation mutation library. Subsequently, the saturation mutation library was subjected to high-throughput screening using fluorescence-activated cell flow sorting to obtain mutants with improved fluorescence properties.

[0465] The experimental results showed that the ThRS-11pAPCF protein can produce fluorescence. Referring to the method in Example 8, a saturated mutant library was established and after flow screening and secondary verification, a mutant producing bright blue-green fluorescence was identified, which has point mutations 11TAG, Y13C, I79V, K121A, A123T, and is named Th-A12 (amino acid sequence as shown in SEQ ID NO: 9), and the fluorescence intensity is 42 times that of ThRS-11pAPCF ( Fig.10 ). Compared with the fluorescence wavelength of sfGFP-152-B6, the fluorescence wavelength of Th-A12 has a certain red shift, indicating that although pAPCF is also introduced as a fluorescent chromophore, different protein environments can still have a certain effect on the fluorescence properties. Because the structure of ThRS is significantly different from the α-helix and β-folded structure of GFP, this shows that the methods and strategies for developing artificial fluorescent proteins based on the non-natural amino acids (such as pAPCF) of the present application can be extended to other proteins that do not have the β-barrel structure of natural fluorescent proteins.

[0466] Example 10 Construction and verification of artificial fluorescent protein derived from FNIII protein

[0467] Human fibronectin type III (FNIII) is a small protein consisting of only 7 β-folds and contains 95 amino acids (the amino acid sequence is shown in SEQ ID NO: 10). Due to its good stability, mutation tolerance and similarity to antibody domains, FNIII is widely used to develop various antibody analogs. This example will develop a smaller artificial fluorescent protein based on pAPCF based on FNIII.

[0468] A DNA fragment encoding FNIII protein was synthesized to construct a pET-FNIII plasmid expressing FNIII protein. The crystal structure of FNIII protein (PDB: 1TTG) was analyzed, and a site inside the protein with a certain cavity was selected as the introduction site of pAPCF. Site 36 of FNIII was selected to introduce pAPCF to obtain the FN-36pAPCF mutant. Next, referring to the above examples, key sites around pAPCF (including sites L20, I22, I72, Y94, T96) were selected for saturation mutation to establish a saturation mutation library. Subsequently, the saturation mutation library was subjected to high-throughput screening using fluorescence-activated cell flow sorting to obtain mutants with improved fluorescence properties.

[0469] The experimental results show that the FN-36pAPCF protein can produce fluorescence. Referring to the above examples, after the above optimization of the FN-36pAPCF mutant, a mutant with bright blue fluorescence was determined, which was named FN-E9 (its amino acid sequence is shown in SEQ ID NO: 11), comprising point mutations 36TAG, L20V, I22V, I72A, Y94H, and its fluorescence intensity was increased by 49 times compared with FN-36pAPCF ( Fig.11 ). These experimental results show that the methods and strategies for developing artificial fluorescent proteins based on the non-natural amino acids (such as pAPCF) of the present application can be applied to smaller protein sizes and more special structures.

[0470] Example 11 Construction and verification of artificial fluorescent protein derived from Z protein

[0471] The Z domain of protein A (Z protein) of staphylococcal bacteria was selected as the research object (amino acid sequence as shown in SEQ ID NO: 17), which has a molecular weight of 7 kDa, contains 58 amino acids, and has a protein conformation of 3 simple α-helices, with good stability, point mutation tolerance and high expression. In this example, the Z protein will be used as the basis for constructing a minimal fluorescent protein.

[0472] Synthesize a DNA fragment encoding the Z protein and construct a pET-Z plasmid expressing the Z protein. Analyze the crystal structure of the Z protein (PDB: 1LP1), and select a site inside the protein with a certain space as the introduction site of pAPCF. Select site 9 of the Z protein to introduce pAPCF to obtain the Z-9pAPCF mutant. Then, referring to the aforementioned embodiment, select key sites around pAPCF (including sites R32, Y35) for saturation mutation to establish a saturation mutation library. Subsequently, use fluorescence-activated cell flow sorting to perform high-throughput screening on the saturation mutation library to obtain mutants with improved fluorescence properties.

[0473] The results showed that the Z-9pAPCF mutant was able to produce a certain amount of blue fluorescence. This indicates that the α-helical structure can also constrain the chromophore molecule, enabling it to excite fluorescence. Referring to the aforementioned embodiment, after the above optimization of the Z-9pAPCF mutant, a brighter fluorescent mutant was identified, which was named Z-H5 (its amino acid sequence is shown in SEQ ID NO: 18), comprising 9TAG, R32C, and Y35T mutations, and its fluorescence intensity was increased by 3 times compared to Z-9pAPCF ( Fig.12). The above results show that the method and strategy for developing artificial fluorescent proteins based on the non-natural amino acids (such as pAPCF) of the present application can even be applied to a protein size of only 7 kDa and a simple conformation of three α helices, thereby developing the smallest artificial fluorescent protein.

Claims

1. An unnatural amino acid having a structure of formula I: in, 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 amino acid according to claim 1, which has a structure of Formula IA or Formula IB: in, 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 amino acid according to any one of claims 1 to 2, which is selected from the following:

4. An orthogonal aminoacyl-tRNA synthetase (O-RS), wherein the O-RS is capable of recognizing the unnatural amino acid of any one of claims 1 to 3 and aminoacylates the orthogonal tRNA (O-tRNA) with the unnatural amino acid; 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; 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 an orthogonal tRNA (O-tRNA) with the non-natural amino acid; 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; Preferably, the O-RS carries the unnatural amino acid according to any one of claims 1 to 3; 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: 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: 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:

5. An orthogonal-tRNA (O-tRNA) that can specifically recognize a selector codon (e.g., a stop codon, such as an amber codon) in a cell and is aminoacylated by the unnatural amino acid of any one of claims 1 to 3; Preferably, the O-tRNA is an amber suppressor tRNA; 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; 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: 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:

6. An isolated nucleic acid comprising a polynucleotide encoding the orthogonal aminoacyl-tRNA synthetase (O-RS) of claim 4.

7. A vector comprising the isolated nucleic acid of claim 6; Preferably, the vector is a cloning vector or an expression vector; Preferably, the vector further comprises a polynucleotide encoding an orthogonal tRNA (O-tRNA); Preferably, the O-RS is capable of aminoacylating the O-tRNA with the unnatural amino acid of any one of claims 1 to 3; Preferably, the O-tRNA is an amber suppressor tRNA; 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; 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; 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; 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.

8. A host cell comprising the isolated nucleic acid of claim 6 or the vector of claim 7; 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 an orthogonal tRNA (O-tRNA); Preferably, the O-RS is capable of aminoacylating the O-tRNA with the unnatural amino acid of any one of claims 1 to 3; 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; Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

9. A method for preparing the orthogonal aminoacyl-tRNA synthetase (O-RS) of claim 4, comprising culturing the host cell of claim 8 under conditions that allow expression of the O-RS; and recovering the O-RS expressed by the host cell.

10. A translation system, comprising: (a) a first unnatural amino acid selected from the unnatural amino acids of any one of claims 1 to 3; (b) a first orthogonal aminoacyl-tRNA synthetase (O-RS); and (c) a first orthogonal tRNA (O-tRNA); in, The first O-RS is capable of aminoacylation of the first O-tRNA with the first unnatural amino acid; Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS of claim 4; 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; 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; 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; 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; 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; 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; 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); Preferably, the host cell contains a polynucleotide encoding the first O-RS, a polynucleotide encoding the first O-tRNA, or a combination thereof.

11. A cell comprising the translation system of claim 10; 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; 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; 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; 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; 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.

12. A kit comprising (i): a first unnatural amino acid selected from the unnatural amino acids of any one of claims 1 to 3; or (ii): a first orthogonal aminoacyl-tRNA synthetase (O-RS), or a first polynucleotide encoding the first O-RS; or, (iii) : a combination of (i) and (ii); Preferably, the kit further comprises, (iv): a first orthogonal tRNA (O-tRNA), or, a second polynucleotide encoding the first O-tRNA; Preferably, the first O-RS is capable of aminoacylation of the first O-tRNA with the first unnatural amino acid; Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS of claim 4; 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; 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); 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; 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; 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; 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; Preferably, the kit further comprises a second unnatural amino acid; further preferably, the kit further comprises: (a): a second orthogonal aminoacyl-tRNA synthetase (O-RS), or a first polynucleotide encoding the second O-RS; or, (b): a second orthogonal tRNA (O-tRNA), or a second polynucleotide encoding the second O-tRNA; or, (c): A combination of (a) and (b); 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; 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; 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; 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).

13. A non-natural polypeptide, wherein The non-natural polypeptide comprises the non-natural amino acid according to any one of claims 1 to 3; Optionally, the non-natural polypeptide is further linked (covalently linked or fused) to another polypeptide, Preferably, the additional polypeptide is linked to the N-terminus or C-terminus of the polypeptide or variant thereof, optionally via a linker; Preferably, the additional polypeptide is selected from a protein tag, a targeting moiety or any combination thereof.

14. The non-natural polypeptide of claim 13, wherein The intramolecular rotation of the non-natural amino acid in the spatial structure of the non-natural polypeptide is restricted; Preferably, the non-natural polypeptide further comprises a mutation at a position adjacent to the non-natural amino acid; preferably, the mutation can enhance or weaken the restriction on the intramolecular rotation of the non-natural amino acid; Preferably, the non-natural polypeptide is derived from a polypeptide selected from the following: GFP protein, ThRS protein, FNIII protein, Z protein, α or β tubulin, AAV virus capsid protein, VHH nanobody protein, Nanofitin protein, cytochrome b562 protein, and contains the non-natural amino acid, and the intramolecular rotation of the non-natural amino acid in the spatial structure of the non-natural polypeptide is restricted; Preferably, the non-natural polypeptide comprises or is a GFP protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following positions of the GFP protein: a position corresponding to position 150, 152, 62 or 203 of SEQ ID NO:6; Preferably, the non-natural amino acid is located at the following sites of the GFP protein: the site corresponding to site 150 or 152 of SEQ ID NO:4 or SEQ ID NO:6; preferably, the non-natural amino acid is pAPCF; preferably, the GFP protein further comprises a mutation at one or more sites selected from the following sites: the sites corresponding to sites 65, 66, 69, 201, 222, 224 of SEQ ID NO:4; or, preferably, the GFP protein further comprises a mutation at one or more sites selected from the following sites: the sites corresponding to sites 66, 201, 61, 65, 222, 224 of SEQ ID NO:6; Preferably, the GFP protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO:4 or SEQ ID NO:6 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:4 or SEQ ID NO:6, 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:4 or SEQ ID NO:6; preferably, the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 150 or 152 of SEQ ID NO:4 or SEQ ID NO:6; or Preferably, the non-natural polypeptide comprises or is a ThRS protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following sites of the ThRS protein: a site corresponding to site 11, 40, 79 or 125 of SEQ ID NO:9; Preferably, the non-natural amino acid is located at the following site of the ThRS protein: the site corresponding to site 11 of SEQ ID NO:9; preferably, the non-natural amino acid is pAPCF; preferably, the ThRS protein further comprises a mutation at one or more sites selected from the following: sites corresponding to sites 13, 79, 121, 123 of SEQ ID NO:9; Preferably, the ThRS protein containing non-natural amino acids comprises an amino acid sequence as shown in SEQ ID NO: 9 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: 9, 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: 9, and the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 11 of SEQ ID NO: 9; or Preferably, the non-natural polypeptide comprises or is a FNIII protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following positions of the FNIII protein: a position corresponding to position 36, 20, 22, 38, 61, 72 or 74 of SEQ ID NO: 11; Preferably, the unnatural amino acid is located at the following position of the FNIII protein: the position corresponding to position 36 of SEQ ID NO: 11; preferably, the unnatural amino acid is pAPCF; preferably, the FNIII protein further comprises a mutation at one or more positions selected from the following: the positions corresponding to positions 20, 22, 72, 94 of SEQ ID NO: 11; Preferably, the FNIII protein containing a non-natural amino acid comprises an amino acid sequence as shown in 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: 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: 11, and the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 36 of SEQ ID NO: 11; or Preferably, the non-natural polypeptide comprises or is a Z protein containing the non-natural amino acid: for example, the non-natural amino acid is located at the following positions of the Z protein: a position corresponding to position 9, 16, 34 or 44 of SEQ ID NO: 18; Preferably, the non-natural amino acid is located at the following position of the Z protein: the position corresponding to position 9 of SEQ ID NO: 18; preferably, the non-natural amino acid is pAPCF; preferably, the Z protein further comprises a mutation at one or more positions selected from the following: positions corresponding to positions 9, 32, 35 of SEQ ID NO: 18; Preferably, the Z protein containing non-natural amino acids comprises the amino acid sequence as shown in SEQ ID NO: 18 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: 18, 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: 18, and the non-natural amino acid (e.g., pAPCF) is located at a position corresponding to position 9 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.

15. An isolated nucleic acid comprising a polynucleotide encoding the non-natural polypeptide of any one of claims 13-14, wherein: The unnatural amino acid is encoded by a selector codon (eg, a stop codon, such as an amber codon).

16. A vector comprising the isolated nucleic acid of claim 15; Preferably, the vector is a cloning vector or an expression vector.

17. A host cell comprising the isolated nucleic acid of claim 15 or the vector of claim 16; 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 O-RS is capable of aminoacylating the first O-tRNA with the unnatural amino acid according to any one of claims 1 to 3; Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS of claim 4; 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; Preferably, the host cell is cultured in a medium containing the unnatural amino acid.

18. A method for preparing the non-natural polypeptide of any one of claims 13-14, comprising culturing the host cell of claim 17 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 aminoacylating the first O-tRNA with the unnatural amino acid according to any one of claims 1 to 3; Preferably, the first orthogonal aminoacyl-tRNA synthetase (O-RS) is selected from the O-RS of claim 4; 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 archaea, such as Methanococcus jannaschii, methanogenic archaea, 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.

19. A method for modifying a polypeptide, comprising: A non-natural amino acid, preferably a non-natural amino acid as described in any one of claims 1 to 3, 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 a non-natural amino acid as described in any one of claims 1 to 3; 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.

20. Use of the non-natural amino acid according to any one of claims 1 to 3 as a probe to detect the presence or amount of a target protein or polypeptide.

21. Use of the non-natural amino acid according to any one of claims 1 to 3 for producing or preparing a non-natural polypeptide.

Images