Eukaryotic semi-synthetic organisms

Abstract

To provide eukaryotic semi-synthetic organisms that enable the production of unnatural polypeptides useful for important practical applications, such as the generation of a wider range of non-canonical amino acids (ncAAs) and novel therapeutics, as well as methods of using and producing the same.SOLUTION: A eukaryotic cell comprising (a) a messenger RNA (mRNA) having a codon containing a first unnatural base, and (b) a transfer RNA (tRNA) having an anticodon containing a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell, and the mRNA is capable of being translated in the cell to produce a polypeptide comprising at least one unnatural amino acid.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 908,421, filed September 30, 2019.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on September 24, 2020, is named 36271-810_601_SL.txt, and is 19,000 bytes in size.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with U.S. government support under Grant No. GM118178 awarded by the National Institutes of Health (NIH). The U.S. government has certain rights in this invention. [Background technology]

[0004] Every protein ever produced in cells is encoded by a four-letter, two-base-pair genetic alphabet. This generally limits the amino acids from which proteins can be built to the standard 20 proteinogenic amino acids. While this has enabled the diversity of life, many potential functions are unavailable, and expanding it to include non-canonical amino acids (ncAAs), including those selected to provide desired activities, could enable the creation of novel proteins with improved properties for applications ranging from materials to therapeutics. Efforts to incorporate ncAAs have primarily relied on expanding the genetic alphabet through stop codon (UAG) or four-letter codon (quadruple codon) suppression, but in these cases, the incorporation of ncAAs must conflict with the codon's natural function. To circumvent this limitation, efforts have focused on synthesizing genomes in which natural stop codons or rare codons have been eliminated, thus freeing them for reassignment to ncAAs. However, rare codons can play important roles in regulating translation and protein folding, and genome synthesis is impractical as a general strategy, especially with large eukaryotic genomes.

[0005] An alternative approach relies on the use of unnatural base pairs (UBPs), which, in principle, from a practical standpoint, would allow the creation of a virtually unlimited number of novel codons completely unencumbered by natural functions. The pursuit of medicinal chemistry mimicry has led to the development of a family of UBPs, such as dNaM-dTPT3 (Figure 1B), which has been used as the basis for Escherichia coli (E. coli) semisynthetic biosystems (SSOs). E. coli SSOs store UBPs in their genomes or on plasmids, transcribe them into mRNA and tRNA, and translate proteins containing the ncAA using tRNAs charged with the ncAA by orthogonal synthetases. E. coli SSOs have important practical applications, as they are currently being used to generate novel therapeutics.

[0006] The breadth of ncAAs and resulting non-natural polypeptides that can be produced is determined, at least in part, by the SSO used. To date, the use of UBPs such as dNAM-dTPT3 has not been demonstrated in eukaryotic SSOs or systems. Proof-of-concept for the approach summarized herein in eukaryotic cells will enable the production of a broader range of ncAAs and resulting non-natural polypeptides that are useful for important practical applications, such as the generation of novel therapeutics. Summary of the Invention [Means for solving the problem]

[0007] In some embodiments, provided herein are eukaryotic semisynthetic organisms (SSOs) generated by probing the translation of unnatural codons. Protein production was characterized after direct, transient, triple transfection with mRNA containing the unnatural codon, a tRNA containing the cognate unnatural codon, and DNA encoding a synthetase suitable for charging the tRNA with a non-standard amino acid (ncAA).

[0008] Aspects disclosed herein provide a eukaryotic cell comprising (a) a messenger RNA (mRNA) having a codon comprising a first unnatural base and (b) a transfer RNA (tRNA) having an anticodon comprising a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell, and wherein the mRNA can be translated in the cell to produce a polypeptide comprising at least one unnatural amino acid. In some embodiments, the tRNA is charged with an unnatural amino acid. In some embodiments, the eukaryotic cell further comprises a polypeptide translated from the mRNA, wherein the polypeptide comprises at least one unnatural amino acid. In some embodiments, the eukaryotic cell further comprises a ribosome capable of translating the polypeptide comprising at least one unnatural amino acid from the mRNA using the tRNA.

[0009] Embodiments disclosed herein also provide a eukaryotic cell comprising: (a) a first unnatural ribonucleotide comprising a first unnatural base; and (b) a second unnatural ribonucleotide comprising a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell.

[0010] In some embodiments, the first unnatural base or the second unnatural base is (i) 2-thiouracil, 2-thio-thymine, 2′-deoxyuridine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthine-9-yl (I), 5-halouracil, 5-propynyl-uracil, 6-azo-thymine, 6-azo-uracil, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, pseudouracil, uracil-5-oxaacetic acid methyl ester, uracil-5-oxaacetic acid, 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl)uracil, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil or dihydrouracil; (ii) 5-hydroxymethylcytosine, 5-trifluoromethylcytosine, 5-halocytosine, 5-propynylcytosine, 5-hydroxymethyl ... Cytosine, cyclocytosine, cytosine arabinoside, 5,6-dihydrocytosine, 5-nitrocytosine, 6-azocytosine, azacytosine, N4-ethylcytosine, 3-methylcytosine, 5-methylcytosine, 4-acetylcytosine, 2-thiocytosine, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one) (iii) 2-aminoadenine, 2-propyladenine, 2-amino-adenine, 2-F-adenine, 2-amino-propyl-adenine, 2-amino-2'-deoxyadenosine, 3-deazaadenine, 7-methyladenine, 7-deaza-adenine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thio Alkyl and 8-hydroxyl substituted adenines, N6-isopentenyladenine, 2-methyladenine, 2,6-diaminopurine, 2-methylthio-N6-isopentenyladenine or 6-aza-adenine; (iv) 2-methylguanine, 2-propyl and alkyl derivatives of guanine, 3-deazaguanine, 6-thio-guanine, 7-methylguanine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-8-azaguanine, 8-azaguanine, 8-halo, 8-amino , 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted guanines, 1-methylguanine, 2,2-dimethylguanine, 7-methylguanine, or 6-aza-guanine; and (v) hypoxanthine, xanthine, 1-methylinosine, queosine, beta-D-galactosylqueosine, inosine, beta-D-mannosylqueosine, wybutoxosine, hydroxyurea, (acp3)w, 2-aminopyridine, or 2-pyridone. In some embodiments, the first unnatural base and the second unnatural base are each independently selected from the group consisting of: [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is selected from the group consisting of: [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base or the second unnatural base has a modification at the 2' position: OH, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2'-F, 2'-OCH, 2'-O(CH)OCH, (wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 , alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -NH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10; and / or modifications at the 5' position: 5'-vinyl, 5'-methyl (R or S); Modification at position 4: 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of an oligonucleotide, or group for improving the pharmacodynamic properties of an oligonucleotide, and any combination thereof. The modified sugar moiety is selected from the group consisting of:

[0011] In some embodiments, the eukaryotic cell further comprises (a) a transfer RNA (tRNA) having an anticodon comprising a first unnatural base; and (b) a messenger RNA (mRNA) having a codon comprising a second unnatural base, wherein the first and second unnatural bases are capable of forming an unnatural base pair (UBP) in the eukaryotic cell. In some embodiments, the eukaryotic cell further comprises (a) a transfer RNA (tRNA) having an anticodon comprising a second unnatural base; and (b) a messenger RNA (mRNA) having a codon comprising the first unnatural base, wherein the first and second unnatural bases are capable of forming an unnatural base pair (UBP) in the eukaryotic cell. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at a first position (XNN) in the codon of the mRNA. In some embodiments, the mRNA codon comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the middle position (NXN) in the mRNA codon. In some embodiments, the mRNA codon comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the last position (NNX) in the mRNA codon. In some embodiments, the eukaryote further comprises a polypeptide translated from the mRNA, wherein the polypeptide comprises at least one unnatural amino acid. In some embodiments, the at least one unnatural amino acid (a) is a lysine analog; (b) comprises an aromatic side chain; (c) comprises an azide group; (d) comprises an alkyne group; or (e) comprises an aldehyde or ketone group.In some embodiments, the one or more unnatural amino acids are N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl. -L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L- In some embodiments, the at least one unnatural amino acid is selected from the group consisting of 3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, selenocysteine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the at least one unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, the at least one unnatural amino acid is N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, at least one unnatural amino acid is N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, at least one unnatural amino acid is N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a HEK293T cell. In some embodiments, the cell is a hamster cell. In some embodiments, the hamster cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the cell is isolated and purified. In some embodiments, the mRNA and tRNA are stabilized for degradation in the eukaryotic cell.

[0012] Aspects disclosed herein provide a semisynthetic organism comprising a eukaryotic cell described herein.

[0013] Aspects disclosed herein provide a eukaryotic cell line comprising a plurality of eukaryotic cells of the present disclosure.

[0014] Aspects disclosed herein provide methods for producing a polypeptide comprising one or more unnatural amino acids in a eukaryotic cell, the method comprising: (a) introducing into the cell (i) a messenger RNA (mRNA) having a codon comprising a first unnatural base and (ii) a transfer RNA (tRNA) having an anticodon comprising a second unnatural base in the eukaryotic cell, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell; and (b) translating the mRNA using the tRNA to produce a polypeptide comprising one or more unnatural amino acids. In some embodiments, the tRNA is charged with an unnatural amino acid.

[0015] Aspects disclosed herein also provide methods for producing a polypeptide comprising one or more unnatural amino acids in a eukaryotic cell, the method comprising: (a) providing a eukaryotic cell comprising (i) a messenger RNA (mRNA) having a codon comprising a first unnatural base; and (ii) a transfer RNA (tRNA) having an anticodon comprising a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell; and (b) translating the mRNA using the tRNA with a ribosome endogenous to the eukaryotic cell to produce a polypeptide comprising one or more unnatural amino acids. In some embodiments, the polypeptide comprises a eukaryotic glycosylation pattern. The glycosylation pattern can correspond to the cell in which it is produced (e.g., a mammalian glycosylation pattern if the cell is mammalian, a human glycosylation pattern if the cell is human, etc.).

[0016]

[0010] Embodiments disclosed herein also provide a method of producing a polypeptide in a eukaryotic cell, wherein the polypeptide comprises one or more unnatural amino acids, the method comprising: (a) providing a eukaryotic cell, wherein the eukaryotic cell comprises: (i) an mRNA comprising a codon comprising a first unnatural base; (ii) a tRNA comprising an anticodon comprising a second unnatural base, wherein the first and second unnatural bases form a complementary base pair; and (iii) a tRNA synthetase that preferentially aminoacylates the tRNA with the one or more unnatural amino acids relative to natural amino acids; and (b) providing one or more unnatural amino acids to the eukaryotic cell, wherein the eukaryotic cell produces the polypeptide comprising the one or more unnatural amino acids.

[0017] Embodiments disclosed herein also provide methods for producing a polypeptide comprising one or more unnatural amino acids in a eukaryotic cell, the method comprising: (a) providing a eukaryotic cell comprising: (i) a transfer RNA (tRNA) having an anticodon comprising a first unnatural base; and (ii) a messenger RNA (mRNA) having a codon comprising a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell; and (c) translating the polypeptide comprising one or more unnatural amino acids from the mRNA using the tRNA by a ribosome endogenous to the eukaryotic cell.

[0018] In some embodiments, the mRNA codon comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the first position (XNN) in the mRNA codon. In some embodiments, the mRNA codon comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the middle position (NXN) in the mRNA codon. In some embodiments, the mRNA codon comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the last position (NNX) in the mRNA codon.In some embodiments, the first unnatural base or the second unnatural base is (a) 2-thiouracil, 2-thio-thymine, 2′-deoxyuridine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl(I), 5-halouracil; 5-propynyl-uracil, 6-azo-thymine, 6-azo-uracil, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, pseudouracil, uracil-5-oxaacetic acid methyl ester, uracil-5-oxaacetic acid, 5-methyl-2-thiouracil , 3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil or dihydrouracil; (b) 5-hydroxymethylcytosine, 5-trifluoromethylcytosine, 5-halocytosine, 5-propynylcytosine, 5 -Hydroxycytosine, cyclocytosine, cytosine arabinoside, 5,6-dihydrocytosine, 5-nitrocytosine, 6-azocytosine, azacytosine, N4-ethylcytosine, 3-methylcytosine, 5-methylcytosine, 4-acetylcytosine, 2-thiocytosine, phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), phenoxazine cytidine (9-(2-aminoethoxy)-pyrimido[5, (4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one) or pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one); (c) 2-aminoadenine, 2-propyladenine, 2-amino-adenine, 2-F-adenine, 2-amino-propyl-adenine, 2-amino-2'-deoxyadenosine, 3-deazaadenine, 7-methyladenine, 7-deaza-adenine, 8-azaadenine, 8-halo, 8-amino. , 8-thiol, 8-thioalkyl and 8-hydroxyl substituted adenines, N6-isopentenyladenine, 2-methyladenine, 2,6-diaminopurine, 2-methylthio-N6-isopentenyladenine or 6-aza-adenine; (d) 2-methylguanine, 2-propyl and alkyl derivatives of guanine, 3-deazaguanine, 6-thio-guanine, 7-methylguanine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-8-azaguanine, 8-azaguanine, 8-halo , 8-amino, 8-thiol, 8-thioalkyl and 8-hydroxyl substituted guanines, 1-methylguanine, 2,2-dimethylguanine, 7-methylguanine or 6-aza-guanine; and (e) hypoxanthine, xanthine, 1-methylinosine, queosine, beta-D-galactosylqueosine, inosine, beta-D-mannosylqueosine, wybutoxosine, hydroxyurea, (acp3)w, 2-aminopyridine or 2-pyridone. In some embodiments, the first unnatural base or the second unnatural base is selected from the group consisting of: [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is selected from the group consisting of: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. The base or second unnatural base may have a modification at the 2' position: OH, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2'-F, 2'-OCH, 2'-O(CH)OCH, (wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 , alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -NH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10; and / or modifications at the 5' position: 5'-vinyl, 5'-methyl (R or S); Modifications at the 4' position: The modified sugar moiety is selected from the group consisting of 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and any combination thereof.

[0019] In some embodiments, the eukaryotic cell is a human cell. In some embodiments, the human cell is a HEK293T cell. In some embodiments, the cell is a hamster cell. In some embodiments, the hamster cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the unnatural amino acid (a) is a lysine analog; (b) comprises an aromatic side chain; (c) comprises an azide group; (d) comprises an alkyne group; or (e) comprises an aldehyde or ketone group. In some embodiments, the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine , p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino- Selected from the group consisting of 3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, selenocysteine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.In some embodiments, the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, one or more unnatural amino acids are N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the one or more unnatural amino acids are N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the one or more unnatural amino acids are N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0020] Embodiments disclosed herein provide methods of producing a polypeptide in a eukaryotic cell, where the polypeptide comprises one or more unnatural amino acids, the method comprising: (a) providing a eukaryotic cell, the eukaryotic cell comprising: (i) an mRNA comprising a codon comprising one or more unnatural bases; (ii) a tRNA comprising an anticodon comprising one or more unnatural bases, wherein the one or more unnatural bases comprising the codon in the mRNA and the one or more unnatural bases comprising the anticodon in the tRNA form complementary base pairs; and (iii) a tRNA synthetase that preferentially aminoacylates the tRNA with the one or more unnatural amino acids compared to natural amino acids; and (b) providing one or more unnatural amino acids to the eukaryotic cell, where the eukaryotic cell produces the polypeptide comprising the one or more unnatural amino acids. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the first position (XNN) in the codon of the mRNA. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the middle position (NXN) in the codon of the mRNA. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the last position (NNX) in the codon of the mRNA. In some embodiments, the one or more unnatural bases comprising the codon in the mRNA are represented by the formula [ka] or [ka] wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide, and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first non-natural base or the second non-natural base is

[0021] [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is selected from the group consisting of:

[0022] [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. The base [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base is [ka] then the second unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the non-natural nucleotide containing codon in the mRNA is [ka] In some embodiments, the non-natural nucleotide containing codon in the mRNA is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the non-natural nucleotide containing codon in the mRNA is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the non-natural nucleotide containing codon in the mRNA is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the unnatural base (X) is located at the first position (XNN) in the codon of the mRNA, and the unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the unnatural base (X) is located at a central position (NXN) in the codon of the mRNA, and the unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the codon of the mRNA comprises three consecutive nucleobases (NNN), and the unnatural base (X) is located at the last position (NNX) in the codon of the mRNA, and the unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the anticodon of the tRNA comprises three consecutive nucleobases (NNN), and a first unnatural base (X) is located at a first position (XNN) in the anticodon of the tRNA. In some embodiments, the unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the anticodon of the tRNA comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the central position (NXN) in the anticodon of the tRNA. In some embodiments, the unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the anticodon of the tRNA comprises three consecutive nucleobases (NNN), and the first unnatural base (X) is located at the last position (NNX) in the anticodon of the tRNA. In some embodiments, the unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is selected from: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the unnatural base is [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the codon and anticodon each comprise three consecutive nucleobases (NNN), the codon in the mRNA comprises a first unnatural base (X) located at the first position of the codon (XNN), and the anticodon in the tRNA comprises a second unnatural base (Y) located at the last position of the anticodon (NNY). In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are different. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are selected from the group consisting of: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both selected from the group consisting of [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from [ka] where in each case the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] In some embodiments, the codon and anticodon each comprise three consecutive nucleobases (NNN), the codon in the mRNA comprises a first unnatural base (X) located in the central position of the codon (NXN), and the anticodon in the tRNA comprises a second unnatural base (Y) located in the central position of the anticodon (NYN). In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are different. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are selected from the group consisting of: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both selected from the group consisting of [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from [ka] where in each case the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] In some embodiments, the codon and anticodon each comprise three consecutive nucleobases (NNN), the codon in the mRNA comprises a first unnatural base (X) located at the last position of the codon (NNX), and the anticodon in the tRNA comprises a second unnatural base (Y) located at the first position of the anticodon (YNN). In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are different. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are selected from the group consisting of: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both selected from the group consisting of [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from [ka] where in each case the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] In some embodiments, the first unnatural base (X) located in the codon of the mRNA is [ka] In some embodiments, the codon in the mRNA is selected from AXC, GXC, or GXU, where X is an unnatural base. In some embodiments, the codon in the mRNA is AXC, where X is an unnatural base. In some embodiments, the codon in the mRNA is GXC, where X is an unnatural base. In some embodiments, the codon in the mRNA is GXU, where X is an unnatural base. In some embodiments, the codon in the mRNA is selected from AXC, GXC, or GXU, where the anticodon in the tRNA is selected from GYU, GYC, and AYC, where X is a first unnatural base and Y is a second unnatural base. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, the codon in the mRNA is AXC and the anticodon in the tRNA is GYU. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, the codon in the mRNA is GXC and the anticodon in the tRNA is GYC. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, the codon in the mRNA is GXU and the anticodon is AYC. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, the tRNA is derived from Methanococcus jannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.In some embodiments, the aminoacyl-tRNA synthetase (also simply referred to herein as tRNA synthetase) is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. In some embodiments, the tRNA and tRNA synthetase are derived from Methanococcus yannaschii. In some embodiments, the tRNA and tRNA synthetase are derived from Methanosarcina barkeri. In some embodiments, the tRNA and tRNA synthetase are derived from Methanosarcina mazei. In some embodiments, the tRNA and tRNA synthetase are derived from Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanococcus yannaschii and the tRNA synthetase is derived from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanosarcina barkeri and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina mazei, or Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanosarcina acetivorans and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina mazei. In some embodiments, the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanosarcina mazei. The ATPase is derived from Methanosarcina barkeri. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a HEK293T cell. In some embodiments, the cell is a hamster cell. In some embodiments, the hamster cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the unnatural amino acid (a) is a lysine analog; (b) comprises an aromatic side chain; (c) comprises an azide group; (d) comprises an alkyne group; or (e) comprises an aldehyde or ketone group.In some embodiments, the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine , p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino- In some embodiments, the unnatural amino acid is selected from the group consisting of 3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, selenocysteine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, at least one unnatural amino acid is N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, at least one unnatural amino acid is N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine.In some embodiments, the at least one unnatural amino acid is N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the mRNA and tRNA are stabilized for degradation in eukaryotic cells. In some embodiments, the polypeptide is produced by translation of the mRNA using tRNA by ribosomes that are endogenous to the eukaryotic cell.

[0023]

[0003] Embodiments disclosed herein provide a system for expression of an unnatural polypeptide, the system comprising: (a) at least one unnatural amino acid; (b) an mRNA encoding the unnatural polypeptide, the mRNA comprising at least one codon comprising one or more first unnatural bases; (c) a tRNA comprising at least one anticodon comprising one or more second unnatural bases, wherein the one or more first unnatural bases and the one or more second unnatural bases form one or more complementary base pairs; and (d) a eukaryotic ribosome capable of translating the mRNA into a polypeptide comprising the unnatural amino acid using a tRNA and a tRNA synthetase. The tRNA is charged with the unnatural amino acid, and / or the system can further comprise a tRNA synthetase and / or one or more nucleic acid constructs comprising a nucleic acid sequence encoding the tRNA synthetase, where the tRNA synthetase preferentially aminoacylates the tRNA with the at least one unnatural amino acid. The system can be in vitro (e.g., a reconstituted system, either cell-free such as a cell lysate or of purified components) or in a eukaryotic cell. In some embodiments, at least one codon of the mRNA is , three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at a first position (XNN) in at least one codon of the mRNA. In some embodiments, at least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at a middle position (NXN) in the codon of the mRNA. In some embodiments, at least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at a last position (NNX) in at least one codon of the mRNA. In some embodiments, the one or more unnatural bases have the formula [ka] wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide, and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first non-natural bases or the one or more second non-natural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are selected from the group consisting of: [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. The first unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at a first position (XNN) in the codon of the mRNA, and the one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at a central position (NXN) in the codon of the mRNA, and the one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at the last position (NNX) in the codon of the mRNA, and the one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one anticodon of the tRNA comprises three consecutive nucleobases (NNN); and one or more second unnatural bases (X) are located at a first position (XNN) in the anticodon of the tRNA. In some embodiments, the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one anticodon of the tRNA comprises three consecutive nucleobases (NNN); one or more second unnatural bases (X) are located at central positions (NXN) in the anticodon of the tRNA. In some embodiments, one or more second unnatural bases The base is [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one anticodon of the tRNA comprises three consecutive nucleobases (NNN); and one or more second unnatural bases (X) are located at the last position (NNX) in the anticodon of the tRNA. In some embodiments, the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are selected from: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more second unnatural bases are [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (NNN), wherein at least one codon comprises one or more first unnatural bases (X) located at the first position of the codon (XNN), and at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located at the last position of the anticodon (NNY). In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same. In some embodiments, one or more first unnatural bases (X) located in a codon of an mRNA and one or more second unnatural bases (Y) located in the anticodon of a tRNA are different. In some embodiments, one or more first unnatural bases (X) located in a codon of an mRNA and one or more second unnatural bases (Y) located in the anticodon of a tRNA are different. [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from the group consisting of: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both selected from the group consisting of: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, one or more first unnatural bases (X) located in the codon of the mRNA and the antisense oligonucleotide of the tRNA The one or more second unnatural bases (Y) located in the codon are both [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA are: [ka] and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: [ka] and in each case the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA are: [ka] In some embodiments, the one or more first unnatural bases (X) located in a codon of an mRNA are: [ka] In some embodiments, at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (NNN), wherein at least one codon in the mRNA comprises one or more first unnatural bases (X) located in a central position (NXN) of the at least one codon, and at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located in a central position (NYN) of the anticodon. In some embodiments, the one or more first unnatural bases (X) located in a codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different. In some embodiments, the one or more first unnatural bases (X) located in a codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same. In some embodiments, one or more first unnatural bases (X) located in a codon of an mRNA and one or more second unnatural bases (Y) located in the anticodon of a tRNA are different. In some embodiments, one or more first unnatural bases (X) located in a codon of an mRNA and one or more second unnatural bases (Y) located in the anticodon of a tRNA are different. [ka] and the wavy line indicates the bond to the ribosyl moiety. wherein one or more first unnatural bases (X) located in the codon of the mRNA and one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both selected from the group consisting of: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA are: [ka] and one or more second non-naturally occurring nucleotides located in the anticodon of the tRNA selected from The base (Y) is [ka] and in each case the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA are: [ka] In some embodiments, the one or more first unnatural bases (X) located in a codon of an mRNA are: [ka] In some embodiments, at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (NNN), wherein at least one codon in the mRNA comprises one or more first unnatural bases (X) located at the last position (NNX) of the at least one codon, and at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located at the first position (YNN) of the anticodon. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same. In some embodiments, one or more first unnatural bases (X) located in a codon of an mRNA and one or more second unnatural bases (Y) located in the anticodon of a tRNA are different. In some embodiments, one or more first unnatural bases (X) located in a codon of an mRNA and one or more second unnatural bases (Y) located in the anticodon of a tRNA are different. [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from the group consisting of: [ka] and the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both selected from the group consisting of: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both: [ka] where the wavy line indicates the bond to the ribosyl moiety. In some embodiments, the one or more first unnatural bases (X) located in the codon of the mRNA are: [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from: [ka] In some embodiments, the one or more first unnatural bases (X) located in a codon of an mRNA are: [ka] In some embodiments, the one or more first unnatural bases (X) located in a codon of an mRNA are: [ka] In some embodiments, at least one codon in the mRNA is selected from AXC, GXC, or GXU, and X is an unnatural base. In some embodiments, at least one codon in the mRNA is AXC, and X is an unnatural base. In some embodiments, at least one codon in the mRNA is GXC, and X is an unnatural base. In some embodiments, at least one codon in the mRNA is GXU, and X is an unnatural base. In some embodiments, at least one codon in the mRNA is selected from AXC, GXC, or GXU, and at least one anticodon in the tRNA is selected from GYU, GYC, and AYC, and X is one or more first unnatural bases, and Y is one or more second unnatural bases. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, at least one codon in the mRNA is AXC and at least one anticodon in the tRNA is GYU. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, at least one codon in the mRNA is GXC and at least one anticodon in the tRNA is GYC. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, at least one codon in the mRNA is GXU and at least one anticodon is AYC. In some embodiments, X and Y are the same or different. In some embodiments, X and Y are the same. In some embodiments, X and Y are different. In some embodiments, the tRNA is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.In some embodiments, the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. In some embodiments, the tRNA and tRNA synthetase are derived from Methanococcus yannaschii. In some embodiments, the tRNA and tRNA synthetase are derived from Methanosarcina barkeri. In some embodiments, the tRNA and tRNA synthetase are derived from Methanosarcina mazei. In some embodiments, the tRNA and tRNA synthetase are derived from Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanococcus yannaschii and the tRNA synthetase is derived from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanosarcina barkeri and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina mazei, or Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina acetivorans. In some embodiments, the tRNA is derived from Methanosarcina acetivorans and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina mazei. In some embodiments, the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanosarcina barkeri. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a HEK293T cell. In some embodiments, the cell is a hamster cell. In some embodiments, the hamster cell is a Chinese hamster ovary (CHO) cell. In some embodiments, the unnatural amino acid: (a) is a lysine analog; (b) comprises an aromatic side chain; (c) comprises an azide group; (d) comprises an alkyne group; or (e) comprises an aldehyde or ketone group. In some embodiments, the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine , p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino- is selected from the group consisting of 3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenylselanyl)propanoic acid, selenocysteine, N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.In some embodiments, the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK). In some embodiments, at least one unnatural amino acid is N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, at least one unnatural amino acid is N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, at least one unnatural amino acid is N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine. In some embodiments, the mRNA and tRNA are stabilized for degradation in eukaryotic cells. In some embodiments, the polypeptide is produced by translation of the mRNA using tRNA by ribosomes that are endogenous to the eukaryotic cell.

[0024] In one embodiment, the eukaryotic cell contains mRNA encoding enhanced green fluorescent protein (EGFP) with an unnatural codon at position 151 (EGFP151(NXN) (where N refers to one of the natural nucleobases and X refers to NaM), a Methanosarcina mazei tRNAPyl recoded with the cognate unnatural anticodon (tRNAPyl(NYN) (where Y refers to TPT3)), and a chimeric Methanosarcina barkeri pyrrolysyl-tRNA synthetase (ChPylRS) capable of charging the unnatural tRNAPyl with N6-(2-azidoethoxy)-carbonyl-L-lysine (AzK).

[0025] Various aspects of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which: [Brief explanation of the drawings]

[0026] [Figure 1A] 1A-1C illustrate exemplary UBPs and workflows using UBPs according to the present embodiment. Figure 1A shows exemplary unnatural base pairs (UBPs) dNaM and dTPT3. [Figure 1B] 1A-1C illustrate the UBP of the present embodiment and a workflow using the UBP. FIG. 1B illustrates a workflow using the UBP for site-specific incorporation of non-standard amino acids (ncAAs) into proteins using unnatural XY base pairs. The incorporation of three ncAAs into a protein is shown by way of example only; any number of ncAAs may be incorporated. [Figure 1C] 1A-1C illustrate an example of a UBP and a workflow using the UBP according to the present embodiment. FIG. 1C shows an example UBP. [Figure 2] Figure 2 depicts a dXTP analog, with the ribose and phosphate omitted for clarity. [Figure 3A] 3A-3B show exemplary unnatural bases. [Figure 3B] 3A-3B show exemplary unnatural bases. [Figure 4A] Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4B] Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4C]Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4D] Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4E] Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4F] Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 4G] Figures 4A-4G illustrate exemplary unnatural amino acids. These unnatural amino acids (UAAs) are genetically encoded in proteins (Figure 4D - UAA#1-42; Figure 4E - UAA#43-89; Figure 4F - UAA#90-128; Figure 4G - UAA#129-167). Figures 4D-4G are adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69. [Figure 5] Figures 5A-5B illustrate the translation of unnatural codons in HEK293T cells. Figure 5A shows the average EGFP fluorescence signal of HEK293T cells transfected with unnatural codons, with or without their cognate tRNAs, as measured by flow cytometry. Figure 5B shows a protein shift assay of HEK293T cells transfected with the unnatural codon GXC using cell lysates. [Figure 6] Figures 6A-6B illustrate the translation of unnatural codons in CHO cells. Figure 6A shows the average EGFP fluorescence signal of CHO cells transfected with unnatural codons (represented by DNA encoding the unnatural codons) with or without their cognate tRNAs (and self-pairing tRNA for the codon AGX) as measured by flow cytometry. Figure 6B shows a protein shift assay of CHO cells transfected with the unnatural codons AXC, GXC, GXT, GYC, and AGX (represented by DNA encoding the unnatural codons) using purified EGFP. [Figure 7A] Figures 7A-7B show the translation of unnatural codons within the CYBA UTR context in CHO cells. Figure 7A: Mean EGFP fluorescence signal of CHO cells transfected with unnatural codons within the CYBA UTR context, with or without their cognate tRNA (and self-pairing tRNA for the codon AGX), as measured by flow cytometry. *P<0.05, **P<0.005, ***P<0.0005, ****P<0.00005 (paired two-tailed t-test). [Figure 7B] Figures 7A-7B show the translation of unnatural codons within the CYBA UTR context in CHO cells. Figure 7B: Protein shift assay of CHO cells transfected with the unnatural codons GXC and GYC within the CYBA UTR context using purified EGFP. [Figure 7C]Figures 7C-7D show the protein expression ratio between mRNAs containing CYBA UTRs and mRNAs containing CS2 UTRs. Figure 7C shows the EGFP expression level ratios of various unnatural codons within the CYBA UTR and CS2 UTR. Expression levels were measured by flow cytometry. [Figure 7D] Figures 7C-7D show the protein expression ratio between mRNAs with CYBA UTRs and mRNAs with CS2 UTRs. Figure 7D shows mRNA abundance measured at 4 hours post-transcription and 8 hours post-transcription using RT-qPCR. The ratio of mRNA remaining after 8 hours to mRNA remaining after 4 hours is compared across various mRNA constructs. Note that unnatural codons in Figures 7A and 7B are represented by the coding sequence of the DNA encoding the mRNA. DETAILED DESCRIPTION OF THE INVENTION

[0027] Specific Terms Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not limiting of the claimed subject matter. In this application, the use of the singular includes the plural unless expressly stated otherwise. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless expressly stated otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0028] As used herein, ranges and amounts may be expressed as "about" a particular value or range. About includes the exact amount. Thus, "about 5 μL" means "about 5 μL" and also "5 μL." In general, the term "about" includes amounts that are expected to be within experimental error.

[0029] As used herein, phrases such as "under conditions suitable to provide" or "under conditions sufficient to produce" in the context of synthetic methods refer to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within the ordinary skill of an experimenter to vary, that provide a useful amount or yield of reaction product. The desired reaction product need not be the only reaction product, nor need the starting material be completely consumed, provided that the desired reaction product can be isolated or otherwise further utilized.

[0030] "Chemically feasible" means a bonding arrangement or compound that does not violate the generally understood rules of organic structure. For example, it is understood that structures within the definitions of the claims that contain a pentavalent carbon atom that does not occur in nature in certain circumstances are not within the scope of the claims. The structures disclosed herein, in all their embodiments, are intended to include only "chemically feasible" structures; for example, any recited structure that is not chemically feasible in a structure shown with variable atoms or groups is not intended to be disclosed or claimed herein.

[0031] An "analog" of a chemical structure, as the term is used herein, refers to a chemical structure that may not be readily synthetically derived from the parent structure, but that retains substantial similarity to the parent structure. In some embodiments, a nucleotide analog is a non-natural nucleotide. In some embodiments, a nucleoside analog is a non-natural nucleoside. Related chemical structures that are readily synthetically derived from the parent chemical structure are referred to as "derivatives."

[0032] Thus, polynucleotide, as the term is used herein, refers to DNA- or RNA-like polymers such as DNA, RNA, peptide nucleic acids (PNAs), locked nucleic acids (LNAs), phosphorothioates, unnatural bases, etc., which are well known in the art. Polynucleotides can be synthesized, for example, on an automated synthesizer using phosphoramidite chemistry or other chemical approaches adapted for use with a synthesizer.

[0033] DNA includes, but is not limited to, cDNA and genomic DNA. DNA is attached to other biomolecules, including, but not limited to, RNA and peptides, by covalent or non-covalent means. RNA also includes coding RNA, such as messenger RNA (mRNA). In some embodiments, the RNA is rRNA, RNAi, snoRNA, microRNA, siRNA, snRNA, exRNA, piRNA, long ncRNA, or any combination or hybrid thereof. In some cases, the RNA is a component of a ribozyme. DNA and RNA can be in any form, including, but not limited to, linear, circular, supercoiled, single-stranded, and double-stranded.

[0034] Peptide nucleic acids (PNAs) are synthetic DNA / RNA analogs in which a peptide-like backbone replaces the sugar-phosphate backbone of DNA or RNA. PNA oligomers exhibit higher binding strength and greater specificity in binding to complementary DNA, and PNA / DNA base mismatches are more destabilizing than similar mismatches in DNA / DNA duplexes. This binding strength and specificity also applies to PNA / RNA duplexes. PNAs are not readily recognized by either nucleases or proteases, making them resistant to enzymatic degradation. PNAs are also stable over a wide pH range. Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991) "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide." ted polyamide," Science 254(5037):1497-500. doi:10.1126 / science.1962210. PMID 1962210; and Egholm M, Buchardt O, Christensen L, Behrens C, Freier SM, Driver DA, Berg RH, Kim SK, Norden B, and Nielsen PE (1993), "PNA Hybridizes to Complementary Oligonucleotides Obeying the Watson-Crick Hydrogen Bonding Rules." Nature 365(6446):566-8. doi:10.1038 / 365566a0. PMID 7692304.

[0035] Locked nucleic acids (LNA) are modified RNA nucleotides in which the ribose moiety of the LNA nucleotide is modified with an additional bridge connecting the 2' oxygen and 4' carbon. This bridge "locks" the ribose in the 3'-endo (north) conformation, which is commonly found in A-form duplexes. LNA nucleotides are mixed with DNA or RNA residues of oligonucleotides as needed. Such oligomers can be chemically synthesized or are commercially available. The locked ribose conformation enhances base stacking and backbone preorganization. See, e.g., Kaur, H; Arora, A; Wengel, J; Maiti, S (2006), "Thermodynamic, Counterion, and Hydration Effects for the Incorporation of Locked Nucleic Acid Nucleotides into DNA Duplexes," Biochemistry 45(23):7347-55. doi:10.1021 / bi060307w. PMID 16752924; Owczarzy R.; You Y., Groth CL, Tataurov AV (2011), "Stability and mismatch discrimination of locked nucleic acid-DNA duplexes." 50(43):9352-9367.doi:10.1021 / bi200904e.PMC3201676.PMID21928795;Alexei A.Koshkin;Sanjay K.Singh, Poul Nielsen, Vivek K.Rajwanshi, Ravindra Kumar, Michael Meldgaard, Carl Erik Olsen, Jesper Wengel (1998), "LNA (Locked Nucleic Acids): Synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine, and uracil bicyclonucleoside monomers, oligomerization, and unprecedented nucleic acid recognition," Tetrahedron 54(14):3607-30. doi:10.1016 / S0040-4020(98)00094-5; and Satoshi Obika; Daishu Nanbu, Yoshiyuki. Hari, Ken-ichiro Morio, Yasuko In, Toshimasa Ishida, Takeshi Imanishi (1997), "Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3'-endo sugar puckering" endo sugar puckering,” Tetrahedron Lett. 38(50):8735-8. doi:10.1016 / S0040-4039(97)10322-7.

[0036] Molecular beacons or molecular beacon probes are oligonucleotide hybridization probes that can detect the presence of specific nucleic acid sequences in homogeneous solutions. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore, which regains fluorescence upon binding to the target nucleic acid sequence. See, e.g., Tyagi S, Kramer FR (1996), "Molecular beacons: probes that fluoresce upon hybridization." See also "Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes," Nat Biotechnol. 14(3):303-8. PMID 9630890; Tapp I, Malmberg L, Rennel E, Wik M, Syvanen AC (April 2000), "Homogeneous scoring of single-nucleotide polymorphisms: comparison of the 5'-nuclease TaqMan assay and Molecular Beacon probes," Biotechniques 28(4):732-8. PMID 10769752; and Akimitsu Okamoto (2011), "ECHO probes: a concept of fluorescence control for practical nucleic acid sensing," Chem. Soc. Rev. 40:5815-5828.

[0037] In some embodiments, a nucleobase is generally the heterocyclic base moiety of a nucleoside. A nucleobase may be naturally occurring, may be modified, may not have similarity to a natural base, and may be synthesized, for example, by organic synthesis. In certain embodiments, a nucleobase comprises any atom or group of atoms that can interact with a base of another nucleic acid, with or without the use of hydrogen bonds. In certain embodiments, a non-natural nucleobase is not derived from a natural nucleobase. It should be noted that a non-natural nucleobase does not necessarily possess basic properties, but is referred to as a nucleobase for simplicity. In some embodiments, when referring to a nucleobase, "(d)" indicates that the nucleobase may be attached to deoxyribose or ribose.

[0038] In some embodiments, nucleosides are compounds that contain a nucleic acid base portion and a sugar portion.Nucleosides include, but are not limited to, naturally occurring nucleosides (found in DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides with mimetic bases and / or sugar groups.Nucleosides include nucleosides that contain any of a variety of substituents.Nucleosides can be glycosidic compounds formed by the glycosidic linkage between the nucleic acid base and the reducing group of the sugar.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0040] Methods, systems and compositions involving unnatural base pairs in eukaryotic cells In certain embodiments, disclosed herein are in vivo methods and compositions for generating nucleic acids with an expanded genetic alphabet in eukaryotic cells (Figures 1A-3B). In some instances, the nucleic acid encodes a non-naturally occurring protein, wherein the non-naturally occurring protein comprises at least one non-naturally occurring amino acid. In some cases, the in vivo methods or compositions described herein utilize or include semi-synthetic organisms. In some instances, the method includes incorporating at least one unnatural base pair (UBP) into one or more nucleic acids. Such a base pair is formed by pairing between the nucleobases of two nucleosides. In the exemplary workflow provided in FIG. 1B, DNA 101 encoding a protein 102 and a tRNA 103 containing complementary unnatural nucleobases (X, Y) is transcribed 104 to produce tRNA 106 and mRNA 107. After charging the tRNA with an unnatural amino acid 105, the mRNA 107 is translated 108 to produce a protein 110 containing one or more unnatural amino acids 109. The methods and compositions described herein, in some instances, enable the site-specific incorporation of unnatural amino acids with high fidelity and yield. Also described herein are methods for using semisynthetic organisms to generate protein products, including those containing at least one unnatural amino acid residue, that contain an expanded genetic alphabet.

[0041] The selection of unnatural nucleobases allows for the optimization of one or more steps in the methods described herein. For example, nucleobases are selected for high efficiency replication, transcription, and / or translation. In some instances, one or more unnatural nucleobase pairs are utilized for the methods described herein. For example, a first set of nucleobases containing deoxyribonucleotides is used for DNA replication (e.g., a first nucleobase and a second nucleobase configured to form a first base pair), and a second set of nucleobases (e.g., a third nucleobase and a fourth nucleobase, where the third and fourth nucleobases are bound to ribose and configured to form a second base pair) is used for transcription / translation. Complementary pairing between the first set of nucleobases and the second set of nucleobases, in some instances, allows transcription of a gene to produce a tRNA or protein from a DNA template containing nucleobases from the first set. Complementary pairing (second base pairs) between the second set of nucleobases, in some instances, allows translation by matching tRNA and mRNA containing unnatural nucleic acids. In some cases, the nucleobases in the first set are bound to a deoxyribose moiety. In some cases, the nucleobases in the first set are bound to a ribose moiety. In some cases, the nucleobases in both sets are unique. In some cases, at least one nucleobase is the same in both sets. In some cases, the first nucleobase and the third nucleobase are the same. In some embodiments, the first base pair and the second base pair are not the same. In some cases, the first base pair, the second base pair and the third base pair are not the same.

[0042] Eukaryotic engineered organisms In some embodiments, the methods and plasmids disclosed herein are further used to generate eukaryotic engineered organisms, such as organisms that incorporate and replicate unnatural nucleotides or unnatural nucleic acid base pairs (UBPs), and can also use nucleic acids containing unnatural nucleotides to transcribe mRNAs and transgenic RNAs that are used to translate proteins containing unnatural amino acid residues. In some examples, the organism is a semisynthetic organism (SSO). In some examples, the SSO is not a prokaryote. In some examples, the SSO is a mammal. In some examples, the mammalian SSO is a human. In some examples, the mammalian SSO is a hamster. In some examples, the human SSO is derived from HEK293T cells. In some examples, the human SSO is derived from Chinese hamster ovary (CHO) cells.

[0043] In some examples, the cells used are genetically transformed with an expression cassette encoding a heterologous protein, e.g., a tRNA synthetase. In some embodiments, the tRNA synthetase preferentially aminoacylates a tRNA containing an anticodon containing an unnatural base with an unnatural amino acid. In some embodiments, the cells are The enzyme includes a tRNA synthetase that preferentially aminoacylates a tRNA containing an anticodon with an unnatural amino acid.

[0044] The cell may be a eukaryotic cell, and the non-natural mutually base-pairing nucleotide pair may be TPT3 and NaM or CNMO.

[0045] Compositions and methods are described herein that involve the use of two or more unnatural base-pairing nucleotides. In some cases, such base-pairing nucleotides enter cells by standard nucleic acid transformation methods known in the art (e.g., electroporation, chemical transformation, or other methods that can introduce nucleic acids containing unnatural nucleotides into cells). In some cases, three or more unnatural base-pairing nucleotides are used. In some cases, the base-pairing unnatural nucleotides enter cells as part of a polynucleotide such as mRNA and / or tRNA. One or more base-pairing unnatural nucleotides that enter cells as part of a polynucleotide (RNA) do not themselves need to be replicated in vivo.

[0046] In some cases, genetically engineered cells are generated by the introduction of nucleic acids, e.g., heterologous nucleic acids, into cells. Any cell described herein is a host cell and can include an expression vector. In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell (e.g., a HEK293T cell). In some embodiments, the mammalian cell is a hamster cell (e.g., a CHO cell). In some embodiments, the cell comprises one or more heterologous polynucleotides. Nucleic acid reagents can be introduced into microorganisms using various techniques. Non-limiting examples of methods used to introduce heterologous nucleic acids into various organisms include transformation, transfection, transduction, electroporation, ultrasound-mediated transformation, conjugation, particle bombardment, etc. In some examples, the addition of a carrier molecule (e.g., a bis-benzimidazolyl compound, see, e.g., U.S. Pat. No. 5,595,899) can increase DNA uptake in cells typically considered difficult to transform by conventional methods. Conventional methods of transformation are readily available to those skilled in the art and can be found in Maniatis, T., E. Fritsch and J. Sambrook (1982) Molecular Cloning: a Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

[0047] In some examples, genetic transformation can be achieved by using, but not limited to, direct introduction of expression cassettes in plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, and artificial chromosomes, or by introducing genetic material into cells or carriers such as cationic liposomes.Such methods are available in the art and can be easily adapted for use in the methods described herein.Transfer vectors can be any nucleotide constructs (e.g., plasmids) used to deliver genes to cells, or can be part of a general strategy for delivering genes, such as recombinant retroviruses or adenoviruses (Ram et al., Cancer Res. 53:83-88, (1993)). Suitable means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described, for example, in Wolff, JA et al., Science, Vol. 247, pp. 1465-1468, (1990); and Wolff, JA, Nature, Vol. 352, pp. 815-818, (1991).

[0048] nucleic acid molecule In some embodiments, the nucleic acid (e.g., also referred to herein as a nucleic acid molecule of interest) is from any source or composition, e.g., RNA, siRNA (short inhibitory RNA), RNAi, tRNA, mRNA, or rRNA (ribosomal RNA), and is in any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). In some embodiments, the nucleic acid comprises a nucleotide, nucleoside, or polynucleotide. In some cases, the nucleic acid includes natural nucleic acids and non-natural nucleic acids. In some cases, the nucleic acid also includes non-natural nucleic acids, such as RNA analogs (e.g., containing base analogs, sugar analogs, and / or non-native backbones, etc.). It is understood that the term "nucleic acid" does not refer to or infer a specific length of a polynucleotide chain, and thus, polynucleotides and oligonucleotides are also included within its definition. Exemplary natural nucleotides include, but are not limited to, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural deoxyribonucleotides include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural ribonucleotides include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP. For natural RNA, the uracil base is uridine. Nucleic acid can be vector, plasmid, phagemid, autonomously replicating sequence (ARS), centromere, artificial chromosome, yeast artificial chromosome (e.g., YAC), or other nucleic acid that can replicate in host cell or replicates in host cell.In some cases, non-natural nucleic acid is nucleic acid analog.In additional cases, non-natural nucleic acid is derived from extracellular source.In other cases, non-natural nucleic acid can be available in the intracellular space of organisms provided herein, for example, genetically modified organisms.In some embodiments, a non-natural nucleotide is not a naturally occurring nucleotide. In some embodiments, a nucleotide that does not contain a natural base comprises a non-natural nucleobase.

[0049] unnatural nucleic acid Nucleotide analogs or non-natural nucleotides include nucleotides containing some type of modification in either the base, sugar, or phosphate moiety. In some embodiments, the modification comprises a chemical modification. In some cases, the modification occurs in the 3'OH or 5'OH group, the backbone, the sugar moiety, or the nucleotide base. In some examples, the modification optionally comprises a non-naturally occurring linker molecule and / or an inter- or intra-strand crosslink. In one aspect, the modified nucleic acid comprises one or more modifications of the 3'OH or 5'OH group, the backbone, the sugar moiety, or the nucleotide base, and / or the addition of a non-naturally occurring linker molecule. In one aspect, the modified backbone comprises a backbone other than a phosphodiester backbone. In one aspect, the modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (in modified RNA). In one embodiment, the modified base comprises a base other than adenine, guanine, cytosine or thymine (in modified DNA) or a base other than adenine, guanine, cytosine or uracil (in modified RNA).

[0050] In some embodiments, the nucleic acid comprises at least one modified base. In some instances, the nucleic acid comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more modified bases. In some cases, modifications to the base moiety include natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases. In some embodiments, the modifications include modified forms of adenine, guanine, cytosine, or thymine (in modified DNA), or modified forms of adenine, guanine, cytosine, or thymine. modified forms of cytosine, cytosine or uracil (modified RNA).

[0051] Modified bases of non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and Included are cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, include 2-aminopropyladenine, 5-propynyluracil, and 5-methylcytosine. cytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CH3)uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines , phenoxazine cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrimido[3',2':4 ,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or pyrimidine base is replaced by other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine Tosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and compounds described in U.S. Pat. Nos. 3,687,808; 4,845,205; 4,910,303, No. 0; No. 4,948,882; No. 5,093,232; No. 5,130,302; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272 No. 5,457,187; No. 5,459,255; No. 5,484,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469 ; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096; WO 99 / 62923; Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, JI (ed.), John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613; and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and Lebleu (eds.), CRC Press, 1993, pp. 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991, Vol. 30, p. 613. In some instances, the non-natural nucleic acid comprises the nucleobase of Figure 2. In some instances, the non-natural nucleic acid comprises the nucleobase of Figure 3A. In some instances, the non-natural nucleic acid comprises the nucleobase of Figure 3B.

[0052] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and in some cases, nucleic acids contain one or several heterocyclic bases other than the five main base components of naturally occurring nucleic acids. For example, heterocyclic bases in some cases include uracil-5-yl, cytosin-5-yl, adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo[2.3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2,3-d]pyrimidin-5-yl, 2-amino-4-oxopyrrolo[2.3-d]pyrimidin-3-yl groups, where the purine is linked to the sugar moiety of the nucleic acid through the 9-position, the pyrimidine through the 1-position, the pyrrolopyrimidine through the 7-position, and the pyrazolopyrimidine through the 1-position.

[0053] In some embodiments, modified bases of non-natural nucleic acids are represented below, where the wavy line identifies the point of attachment to the deoxyribose or ribose: [ka] [ka] [ka] [ka]

[0054] In some embodiments, nucleotide analogues are also modified in the phosphate moiety.Modified phosphate moieties include, but are not limited to, those with modifications in the linkage between two nucleotides, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters and boranophosphates.These phosphates or modified phosphates are linked between two nucleotides by 3'-5' or 2'-5' linkage, and the linkage can be in reverse direction, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. It is understood that the term "nucleotide" includes various salts, mixed salts, and free acid forms. Various salts, mixed salts, and free acid forms are also included. Numerous U.S. patents teach how to make and use nucleotides containing modified phosphates, including, but not limited to, U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,286,718; 5,286,720; 5,286,730; 5,286,740; 5,286,750; 5,286,760; 5,286,770; 5,286,780; 5,286,718; 5,286,720; 5,286,740; 5,286,750; 5,286,760; 5,286,770; 5,286,718; 5,286,720; 5,286,740; 5,286,750; 5,286,718; 5,286,720; 5,286,720; 5,286,74 ... ,321,131; No. 5,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0055] In some embodiments, the non-naturally occurring nucleic acids are 2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT / US2002 / 006460), 5'-substituted DNA and RNA derivatives (PCT / US2011 / 033961; Saha et al., J. Org Chem., 1995, Vol. 60, pp. 788-789; Wang et al., Bioorganic & Medicinal Chemistry Letters, 1999, Vol. 9, pp. 885-890; and Mikhailov et al., Nucleosides & Nucleotides, 1991, Vol. 10(Is. 1-3), pp. 339-343; Leonid et al., 1995, Vol. 14(Is. 3-5), pp. 901-905; and Eppacher et al., Helvetica Chimica Acta, 2004, Vol. 87, pp. 3004-3020; PCT / JP2000 / 004720; PCT / JP2003 / 002342; PCT / JP2004 / 013216; PCT / JP2005 / 020435; PCT / JP2006 / 315479; PCT / JP2006 / 324484; PCT / JP2009 / 056718; PCT / JP2010 / 067560), or 5'-substituted monomers made as monophosphates with modified bases (Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, Vol. 23(1 & 2), pp. 317-337).

[0056] In some embodiments, non-natural nucleic acids contain modifications at the 5' and 2' positions of the sugar ring (PCT / US94 / 02993), such as 5'-CH2-substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, Vol. 83, pp. 1127-1143 and Wu et al., Bioconjugate Chem. 1999, Vol. 10, pp. 921-924). In some cases, non-natural nucleic acids contain amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleoside (5' to 3') in the dimer contains 2'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, pp. 1287-1290). Non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al., Tet. Lett., 1993, Vol. 34, pp. 2723-2726; Collingwood et al., Synlett, 1995, Vol. 7, pp. 703-705; and Hutter et al., Helvetica Chimica Acta, 2002, Vol. 85, pp. 2777-2806). Non-natural nucleic acids can include 5'-phosphonate monomers with 2'-substitutions (U.S. Patent Application Publication No. 2006 / 0074035) and other modified 5'-phosphonate monomers (WO 1997 / 35869). Non-natural nucleic acids can include 5'-modified methylene phosphonate monomers (EP 614907 and EP 629633). Non-natural nucleic acids can include 5'- or 6'-phosphonate ribonucleoside analogs containing hydroxyl groups at the 5' and / or 6' positions (Chen et al., Phosphorus, Sulfur and Sulfur Silicon, 2002, Vol. 777, pp. 1783-1786; Jung et al., Bioorg. Med. Chem., 2000, Vol. 8, pp. 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, pp. 925-933; and Hampton et al., J. Med. Chem., 1976, Vol. 19(8), pp. 1029-1033). Non-naturally occurring nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et al., Oligonucleotides, 2006, Vol. 16(1), pp. 68-82). Non-natural nucleic acids can include nucleosides having a 6'-phosphonate group, where the 5' and / or 6' positions are unsubstituted or contain a thio-tert-butyl group (SC(CH3)3) (and its analogs); a methyleneamino group (CH2NH2) (and its analogs) or a cyano group (CN) (and its analogs) (Fairhurst et al., Synlett, 2001, Vol. 4, pp. 467-472; Kappler et al., J. Med. Chem., 1986, Vol. 29, pp. 1030-1038; Ka ppler et al., J. Med. Chem., 1982, Vol. 25, pp. 1179-1184; Vrudhula et al., J. Med. Chem., 1987, Vol. 30, pp. 888-894; Hampton et al., J. Med. Chem., 1976, Vol. 19, pp. 1371-1377; Geze et al., J. Am. Chem. Soc., 1983, Vol. 105 (No. 26), pp. 7638-7640; and Hampton et al., J. Am. Chem. Soc., 1973, Vol. 95 (No. 13), pp. 4404-4414).

[0057] In some embodiments, non-natural nucleic acids also include modifications to the sugar moiety. In some cases, the nucleic acid contains one or more nucleosides with modified sugar groups. Such sugar-modified nucleosides may be conferred enhanced nuclease stability, increased binding affinity, or some other advantageous biological property. In certain embodiments, the nucleic acid includes a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, but are not limited to, the addition of substituents (5' and / or 2' substituents; bridging of two ring atoms to form bicyclic nucleic acids (BNAs); S, N(R), or C(R1)(R2) (R = H, C1-C) at the oxygen atom of the ribosyl ring. 12 Examples of chemically modified sugars can be found in WO 2008 / 101157, U.S. Patent Application Publication No. 2005 / 0130923, and WO 2007 / 134181.

[0058] In some instances, modified nucleic acids contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be a pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a cyclopentyl group of a sugar "analog." The sugar can be in the form of a pyranosyl or furanosyl. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar can be linked to the respective heterocyclic base in either the [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA / DNA chimeras. For example, sugar modifications can include 2'-O-methyl-uridine or 2'-O-methyl-cytidine. Sugar modifications include 2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethylene glycol-like ribonucleosides. The preparation of these sugars or sugar analogs and the respective "nucleosides" in which such sugars or sugar analogs are linked to heterocyclic bases (nucleobases) is known. Sugar modifications can also be made using and combined with other modifications.

[0059] Modifications to the sugar moiety include natural modifications of ribose and deoxyribose, as well as non-natural modifications. Sugar modifications include, but are not limited to, modifications at the 2-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 It may be alkyl or C2-C10 alkenyl and alkynyl. 2' sugar modifications include, but are not limited to, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2)n NH2, -O(CH2) n CH3, -O(CH2) n ONH2 and -O(CH2) n ON[(CH2) n CH3)]2 (wherein n and m are from 1 to about 10).

[0060] Other modifications at the 2' position include, but are not limited to, C1 to C 10Examples of suitable sugars include lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic or pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar in the 3'-terminal nucleotide or in 2'-5'-linked oligonucleotides, and the 5' position of the 5'-terminal nucleotide. Modified sugars also include those containing modifications at the bridging ring oxygens, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics, such as cyclobutyl moieties, in place of the pentofuranosyl sugar.U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,42 No. 7; No. 5,591,722; No. 5,597,909; No. 5,610,300; No. 5,627,053; No. 5,639,873; No. 5 , 646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,06 There are numerous U.S. patents that teach the preparation of such modified sugar structures and detail and describe the range of base modifications, such as U.S. Patents Nos. 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,681,941; and 5,700,920, each of which is incorporated herein by reference in its entirety.

[0061] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, O-(C1-C2). 1O alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ) and O-CH2-C(=O)-N(R m )(R n ) (wherein each R m and R n are independently H or substituted or unsubstituted C1-C 10 It may also be selected from the group consisting of alkyl.

[0062] In certain embodiments, the nucleic acids described herein comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acids comprise a bridge between the 4' and 2' ribosyl ring atoms. In certain embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, wherein the bridge is a 4'-2' bicyclic nucleic acid. Examples of such 4'-2' bicyclic nucleic acids include, but are not limited to, those of the formula: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CHOCH3)-O-2', and analogs thereof (see U.S. Pat. No. 7,399,845); 4'-C(CH3)(C H3)-O-2' and analogs thereof (see WO 2009 / 006478, WO 2008 / 150729, U.S. Patent Application Publication No. 2004 / 0171570, U.S. Patent No. 7,427,672, Chattopadhyaya et al., J. Org. Chem., Vol. 209, No. 74, pp. 118-134, and WO 2008 / 154401).See, for example, Singh et al., Chem. Commun., 1998, Vol. 4, pp. 455-456; Koshkin et al., Tetrahedron, 1998, Vol. 54, pp. 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA, 2000, Vol. 97, pp. 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, Vol. 8, pp. 2219-2222; Singh et al., J. Org. Chem., 1998, Vol. 63, pp. 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, Vol. 129 (No. 26), pp. 8362-8379; Elayadi et al., Curr. Opinion Invens. Drugs, 2001, Vol. 2, pp. 558-561; Braasch et al., Chem. Biol., 2001, Vol. 8, pp. 1-7; Oram et al., Curr. Opinion Mol. Ther., 2001, Vol. 3, pp. 239-243; U.S. Patent Nos. 4,849,513; 5,015,733; 5,118,800; 5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; 6,525,191; 6,670,461; and 7,399,845; WO 2004 / 106356, WO 1994 / 14226, WO 2005 / 02 1570, WO 2007 / 090071 and WO 2007 / 134181; U.S. Patent Application Publication Nos. 2004 / 0171570, 2007 / 0287831 and 2008 / 0039618; U.S. Provisional Application Nos. 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787 and 61 / 099,844; and International Application Nos. PCT / US2008 / 064591, PCT See also US2008 / 066154, PCT US2008 / 068922 and PCT / DK98 / 00393.

[0063] In certain embodiments, nucleic acids include linked nucleic acids. Nucleic acids can be linked together using any internucleic acid linkage. Two major classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus-containing internucleic acid linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N * -dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, internucleic acid linkages having chiral atoms can be prepared as racemic mixtures or as separate enantiomers, such as alkylphosphonates and phosphorothioates. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within one moiety or between different moieties.

[0064] Backbone phosphate modifications to nucleic acids include, but are not limited to, methylphosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phospho Triesters, phosphorodithioates, phosphodithioates, and boranophosphates can be used in any combination. Other non-phosphate linkages can also be used.

[0065] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity to the modified nucleic acids and / or enhance their stability in vivo.

[0066] In some examples, the phosphorus derivative (or modified phosphate group) is attached to the sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, etc. Exemplary polynucleotides containing modified phosphate or non-phosphate linkages are described in Peyrottes et al., 1996, Nucleic Acids Res. 24:1841-1848; Chaturvedi et al., 1996, Nucleic Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids Res. 24:2966-2973; Matteucci, 1997, "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (Chadwick and Cardew, eds.) John Wiley and Sons, New York, NY; Zon, 1993, "Oligonucleoside Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis and Properties, Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et al., 1988, Biochem. 27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No. 5,453,496; and Micklefield, 2001, Curr. Med. Chem. 8:1157-1179.

[0067] In some cases, backbone modifications include replacing phosphodiester linkages with alternative moieties, such as anionic, neutral, or cationic groups. Examples of such modifications include anionic internucleoside linkages; N3'-P5' phosphoramidate modifications; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages, such as methylphosphonate; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligos (Micklefield, 2001, Current Medicinal Chemistry 8:1157-1179). Modified nucleic acids can include chimeric or mixed backbones containing one or more modifications, such as a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

[0068] Alternatives to phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyl These include imino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S, and CH moieties. Numerous U.S. patents disclose how to make and use these types of phosphate replacements, including, but not limited to, U.S. Patent Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,47 0,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.It is also understood that in nucleotide substitutes, both the sugar and phosphate moieties of nucleotide can be replaced, for example, by amide-type linkage (aminoethylglycine) (PNA). U.S. Patent No. 5,539,082; U.S. Patent No. 5,714,331; and U.S. Patent No. 5,719,262 teach the preparation and use of PNA molecules, each of which is incorporated herein by reference.See also Nielsen et al., Science, 1991, vol. 254, pp. 1497-1500.For example, to enhance cellular uptake, other types of molecules can be linked (conjugated) to nucleotide or nucleotide analogue.Conjugate can be chemically linked to nucleotide or nucleotide analogue.Such conjugates include, but are not limited to, lipid moieties such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, Vol. 86, pp. 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, Vol. 4, pp. 1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, Vol. 660, pp. 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, Vol. 3, pp. 2765-2770), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, Vol. 20, pp. 533-538), aliphatic chains, for example, dodecanediol or undecyl residues (Saison-Behmoaras et al., EM5OJ, 1991, Vol. 10, pp. 1111-1118; Kabanov et al., FEBS. Lett., 1990, vol. 259, pp. 327-330; Svinarchuk et al., Biochimie, 1993, vol. 75, pp. 49-54), phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, vol. 36, pp. 3651-3654; Shea et al., Nucl. Acids Res., 1990, vol. 18, pp. 3777-3783), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, vol. 14, pp. 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Numerous United States patents teach the preparation of such conjugates, including, but not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,591,584; 5,109,124; Same No. 5,118,802; Same No. 5,138,045; Same No. 5,414,077; Same No. 5,486,603; Same No. 5,512,439; Same No. 5,578 ,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876,335; No. 4,904,582; No. 4,958,0 No. 13; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5, No. 258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241 No. 5,391,723; No. 5,416,203, No. 5,451,463; No. 5,510,475; No. 5,512,667; No. 5,51 Nos. 4,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0069] Described herein are nucleobases used in compositions and methods for the replication, transcription, translation, and incorporation of unnatural amino acids into proteins. In some embodiments, the nucleobases described herein have the structure: [ka] [In the formula, each X is independently carbon or nitrogen; R2 is optionally, and when present, independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, or azido group; each Y is independently sulfur, oxygen, selenium, or a secondary amine; each E is independently oxygen, sulfur, or selenium; The wavy line indicates the point of attachment to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or analog thereof, which is in free form, optionally attached to a monophosphate, diphosphate, or triphosphate group, including an α-thiotriphosphate, β-thiotriphosphate, or γ-thiotriphosphate group, or contained in RNA or DNA, or an RNA or DNA analog. In some embodiments of the nucleobases described herein, R2 is lower alkyl (e.g., C1-C6), hydrogen, or halogen. In some embodiments of the nucleobases described herein, R2 is fluoro. In some embodiments of the nucleobases described herein, X is carbon. In some embodiments of the nucleobases described herein, E is sulfur. In some embodiments of the nucleobases described herein, Y is sulfur. In some embodiments of the nucleobases described herein, the nucleobase has the structure: [ka] In some embodiments of the nucleobases described herein, E is sulfur and Y is sulfur. In some embodiments of the nucleobases described herein, the wavy line indicates the point of attachment to the ribosyl or deoxyribosyl moiety. In some embodiments of the nucleobases described herein, the wavy line indicates the point of attachment to the ribosyl or deoxyribosyl moiety that is connected to the triphosphate group. In some embodiments of the nucleobases described herein, the nucleobase is a component of a nucleic acid polymer. In some embodiments of the nucleobases described herein, the nucleobase is a component of a tRNA. In some embodiments of the nucleobases described herein, the nucleobase is a component of an anticodon in a tRNA. In some embodiments of the nucleobases described herein, the nucleobase is a component of an mRNA. In some embodiments of the nucleobases described herein, the nucleobase is a component of a codon in an mRNA. In some embodiments of the nucleobases described herein, the nucleobase is a component of RNA or DNA. In some embodiments of the nucleobases described herein, the nucleobase is a component of a codon in DNA. In some embodiments of the nucleobases described herein, the nucleobase forms a nucleobase pair with another complementary nucleobase.

[0070] Unnatural deoxyribonucleic acid (DNA) is, in some cases, transcribed into messenger RNA (mRNA) containing the unnatural bases described herein (e.g., d5SICS, dNaM, dTPT3, dMTMO, dCNMO, dTAT1). Exemplary mRNA codons are encoded by exemplary regions of unnatural DNA containing three consecutive deoxyribonucleotides (NNN) containing TTX, TGX, CGX, AGX, GAX, CAX, GXT, CXT, GXG, AXG, GXC, AXC, GXA, CXC, TXC, ATX, CTX, TTX, GTX, TAX, or GGX (where X is an unnatural base attached to a 2' deoxyribosyl moiety). Exemplary mRNA codons resulting from transcription of exemplary unnatural DNAs each contain three consecutive ribonucleotides (NNN) including UUX, UGX, CGX, AGX, GAX, CAX, GXU, CXU, GXG, AXG, GXC, AXC, GXA, CXC, UXC, AUX, CUX, UUX, GUX, UAX, or GGX (where X is an unnatural base attached to a ribosyl moiety). In some embodiments, the unnatural base is at the first position (XNN) in the codon sequence. In some embodiments, the unnatural base is at the second (or middle) position (NXN) in the codon sequence. In some embodiments, the unnatural base is at the third (last) position (NNX) in the codon sequence.

[0071] In some cases, mRNAs containing the codons described herein are translated in vivo in cells (e.g., eukaryotic cells). Translation of mRNAs containing the unnatural bases described herein is mediated by transfer RNAs (tRNAs) containing anticodon sequences that are reverse complements of the mRNA codon sequences described herein. In some embodiments, the tRNA anticodons contain unnatural bases, including YAA, XAA, YCA, XCA, YCG, XCG, YCU, XCU, YUC, XUC, YUG, XUG, AYC, AYG, CYC, CYU, GYC, GYU, UYC, GYG, GYA, YAU, XAU, XAG, YAG, XAC, YAC, XUA, YUA, XCC, or YCC (wherein X and Y each represent an unnatural base, and X and Y are not identical). In some embodiments, In some embodiments, the unnatural base is at the first position (X / YNN) in the anticodon sequence. In some embodiments, the unnatural base is at the second (or middle) position (NX / YN) in the anticodon sequence. In some embodiments, the unnatural base is at the third (last) position (NNX / Y) in the anticodon sequence.

[0072] Nucleobase pairing In some embodiments, an unnatural nucleotide forms a base pair (unnatural base pair; UBP) with another unnatural nucleotide, for example, during translation. For example, a first unnatural nucleic acid can base pair with a second unnatural nucleic acid. For example, one pair of unnatural nucleoside triphosphates that can base pair during translation includes a nucleotide containing (d) 5SICS and a nucleotide containing (d) NaM. Other examples include, but are not limited to: a nucleotide containing (d) CNMO and a nucleotide containing (d) TPT3. Such unnatural nucleotides can have a ribose or deoxyribose sugar moiety (indicated by "(d)"). For example, one pair of unnatural nucleoside triphosphates that can base pair when incorporated into a nucleic acid includes a nucleotide containing TAT1 and a nucleotide containing NaM. In some embodiments, one pair of unnatural nucleoside triphosphates that can base pair when incorporated into a nucleic acid includes a nucleotide containing dCNMO and a nucleotide containing TAT1. In some embodiments, one pair of unnatural nucleoside triphosphates that can form base pairs when incorporated into a nucleic acid includes a nucleotide containing dTPT3 and a nucleotide containing NaM. In some embodiments, the unnatural nucleic acid does not substantially form base pairs with natural nucleic acids (A, T, G, C). In some embodiments, the unnatural nucleic acid can form base pairs with natural nucleic acids.

[0073] In some embodiments, the non-natural (deoxy)ribonucleotide is a non-natural (deoxy)ribonucleotide that can form a UBP but does not substantially base pair with any of the respective natural (deoxy)ribonucleotides. In some embodiments, the non-natural (deoxy)ribonucleotide is a non-natural (deoxy)ribonucleotide that can form a UBP but does not substantially base pair with one or more natural nucleic acids. For example, the non-natural nucleic acid is substantially unable to base pair with A, T, and C, but can base pair with G. For example, the non-natural nucleic acid is substantially unable to base pair with A, T, and G, but can base pair with C. For example, the non-natural nucleic acid is substantially unable to base pair with C, G, and A, but can base pair with T. For example, the non-natural nucleic acid is substantially unable to base pair with C, G, and T, but can base pair with A. For example, the non-natural nucleic acid is substantially unable to base pair with A and T, but can base pair with C and G. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with A and C, but can base pair with T and G. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with A and G, but can base pair with C and T. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with C and T, but can base pair with A and G. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with C and G, but can base pair with T and G. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with T and G, but can base pair with A and G. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with G, but can base pair with A, T, and C. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with A, but can base pair with G, T, and C. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with T, but can base pair with G, A, and C. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with G, but can base pair with G, A, and C. For example, a non-naturally occurring nucleic acid is substantially unable to base pair with C. It cannot base pair with G, T, and A, but can base pair with A.

[0074] Exemplary unnatural nucleotides capable of forming unnatural base pairs (UBPs) under in vivo conditions (e.g., in RNA, such as between tRNA and mRNA) include, but are not limited to, 5SICS, d5SICS, NaM, dNaM, dTPT3, dMTMO, dCNMO, TAT1, and combinations thereof. In some embodiments, unnatural nucleotide base pairs include, but are not limited to: [ka] and its corresponding ribonucleic acid (RNA).

[0075] An unnatural base pair (UBP) is formed between the codon sequence of an mRNA and the anticodon sequence of a tRNA, facilitating the translation of the mRNA into an unnatural polypeptide. The codon-anticodon UBP, in some instances, comprises a codon sequence comprising three consecutive nucleic acids (e.g., UUX) read from 5' to 3' of the mRNA and an anticodon sequence comprising three consecutive nucleic acids (e.g., YAA or XAA) read from 5' to 3' of the tRNA. In some embodiments, when the mRNA codon is UUX, the tRNA anticodon is YAA or XAA. In some embodiments, when the mRNA codon is UGX, the tRNA anticodon is YCA or XCA. In some embodiments, when the mRNA codon is CGX, the tRNA anticodon is YCG or XCG. In some embodiments, when the mRNA codon is AGX, the tRNA anticodon is YCU or XCU. In some embodiments, when the mRNA codon is GAX, the tRNA anticodon is YUC or XUC. In some embodiments, when the mRNA codon is CAX, the tRNA anticodon is YUG or XUG. In some embodiments, when the mRNA codon is GXU, the tRNA anticodon is AYC. In some embodiments, when the mRNA codon is CXU, the tRNA anticodon is AYG. In some embodiments, when the mRNA codon is GXG, the tRNA anticodon is CYC. In some embodiments, when the mRNA codon is AXG, the tRNA anticodon is CYU. In some embodiments, when the mRNA codon is GXC, the tRNA anticodon is GYC. In some embodiments, when the mRNA codon is AXC, the tRNA anticodon is GYU. In some embodiments, when the mRNA codon is GXA, the tRNA anticodon is UYC. In some embodiments, when the mRNA codon is CXC, the tRNA anticodon is GYG. In some embodiments, when the mRNA codon is UXC, the tRNA anticodon is GYA.In some embodiments, when the mRNA codon is AUX, the tRNA anticodon is YAU or XAU. In some embodiments, when the mRNA codon is CUX, the tRNA anticodon is XAG or YAG. In some embodiments, when the mRNA codon is UUX, the tRNA anticodon is YAU or XAU. The tRNA anticodon is XAA or YAA. In some embodiments, when the mRNA codon is GUX, the tRNA anticodon is XAC or YAC. In some embodiments, when the mRNA codon is UAX, the tRNA anticodon is XUA or YUA. In some embodiments, when the mRNA codon is GGX, the tRNA anticodon is XCC or YCC.

[0076] Natural and Unnatural Amino Acids As used herein, amino acid residue may refer to a molecule that contains both an amino group and a carboxyl group.Suitable amino acids include, but are not limited to, both the D-isomer and the L-isomer of naturally occurring amino acids, and non-naturally occurring amino acids prepared by organic synthesis or any other method.As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, unnatural amino acids, and amino acid analogs.

[0077] The term "α-amino acid" may refer to a molecule that contains both an amino group and a carboxyl group attached to a carbon, referred to as the α-carbon. For example: [ka]

[0078] The term "β-amino acid" can refer to a molecule that contains both an amino group and a carboxyl group in the β configuration.

[0079] A "naturally occurring amino acid" can refer to any one of the 20 amino acids commonly found in peptides synthesized in nature, and are known by the single letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.

[0080] The following table provides a summary of the properties of natural amino acids.

[0081] [Table 1]

[0082] "Hydrophobic amino acids" include small and large hydrophobic amino acids. "Small hydrophobic amino acids" may be glycine, alanine, proline, and analogs thereof. "Large hydrophobic amino acids" may be valine, leucine, isoleucine, phenylalanine, methionine, tryptophan, and analogs thereof. "Polar amino acids" may be serine, threonine, asparagine, glutamine, cysteine, tyrosine, and analogs thereof. "Charged amino acids" may be lysine, arginine, histidine, aspartic acid, glutamate, and analogs thereof.

[0083] An "amino acid analog" can be a molecule that is structurally similar to an amino acid and can be substituted for an amino acid in the formation of a peptidomimetic macrocycle. Amino acid analogs include, but are not limited to, beta-amino acids and amino acids in which the amino or carboxy group has been replaced with a similarly reactive group (e.g., replacement of a primary amine with a secondary or tertiary amine, or replacement of the carboxy group with an ester).

[0084] A "non-standard amino acid (ncAA)" or "unnatural amino acid" can be an amino acid that is not one of the 20 amino acids commonly found in peptides synthesized in nature and is known by the one-letter abbreviations A, R, N, C, D, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In some cases, non-natural amino acids are a subset of non-standard amino acids.

[0085] Amino acid analogs may include β-amino acid analogs. Examples of β-amino acid analogs include: Examples include, but are not limited to, cyclic beta amino acid analogs; beta-alanine; (R)-beta-phenylalanine; (R)-1,2,3,4-tetrahydro-isoquinoline-3-acetic acid; (R)-3-amino-4-(1-naphthyl)-butyric acid; (R)-3-amino-4-(2,4-dichlorophenyl)butyric acid; (R)-3-amino-4-(2-chlorophenyl)-butyric acid; (R)-3-amino-4-(2-cyanophenyl)-butyric acid; (R)-3-amino-4-(2-fluorophenyl)-butyric acid; (R)-3-amino-4-(2-furyl)-butyric acid ;(R)-3-Amino-4-(2-methylphenyl)-butyric acid;(R)-3-Amino-4-(2-naphthyl)-butyric acid;(R)-3-Amino-4-(2-thienyl)-butyric acid;(R)-3-Amino-4-(2-trifluoromethylphenyl)-butyric acid;(R)-3-Amino-4-(3,4-dichlorophenyl)butyric acid;(R)-3-Amino-4-(3,4-difluorophenyl)butyric acid;(R)-3-Amino-4-(3-benzothienyl)-butyric acid;(R)-3-Amino-4-(3-chlorophenyl)-butyric acid;(R)-3-Amino-4-(3-cyanophenyl)-butyric acid Acid;(R)-3-Amino-4-(3-fluorophenyl)-butyric acid;(R)-3-Amino-4-(3-methylphenyl)-butyric acid;(R)-3-Amino-4-(3-pyridyl)-butyric acid;(R)-3-Amino-4-(3-thienyl)-butyric acid;(R)-3-Amino-4-(3-trifluoromethylphenyl)-butyric acid;(R)-3-Amino-4-(4-bromophenyl)-butyric acid;(R)-3-Amino-4-(4-chlorophenyl)-butyric acid;(R)-3-Amino-4-(4-cyanophenyl)-butyric acid;(R)-3-Amino-4-(4-fluorophenyl)-butyric acid; (R)-3-Amino-4-(4-iodophenyl)-butyric acid;(R)-3-Amino-4-(4-methylphenyl)-butyric acid;(R)-3-Amino-4-(4-nitrophenyl)-butyric acid;(R)-3-Amino-4-(4-pyridyl)-butyric acid;(R)-3-Amino-4-(4-trifluoromethylphenyl)-butyric acid;(R)-3-Amino-4-pentafluoro-phenylbutyric acid;(R)-3-Amino-5-hexenoic acid;(R)-3-Amino-5-hexynoic acid;(R)-3-Amino-5-phenylpentanoic acid;(R)-3-Amino-6-phenyl-5-hexenoic acid(S)-1,2,3,4-Tetrahydro-isoquinoline-3-acetic acid;(S)-3-amino-4-(1-naphthyl)-butyric acid;(S)-3-amino-4-(2,4-dichlorophenyl)butyric acid;(S)-3-amino-4-(2-chlorophenyl)-butyric acid;(S)-3-amino-4-(2-cyanophenyl)-butyric acid;(S)-3-amino-4-(2-fluorophenyl)-butyric acid;(S)-3-amino-4-(2-furyl)-butyric acid;(S)-3-amino-4-(2-methylphenyl)-butyric acid;(S)-3-amino-4-(2-naphthyl)-butyric acid;(S)-3 -Amino-4-(2-thienyl)-butyric acid;(S)-3-Amino-4-(2-trifluoromethylphenyl)-butyric acid;(S)-3-Amino-4-(3,4-dichlorophenyl)butyric acid;(S)-3-Amino-4-(3,4-difluorophenyl)butyric acid;(S)-3-Amino-4-(3-benzothienyl)-butyric acid;(S)-3-Amino-4-(3-chlorophenyl)-butyric acid;(S)-3-Amino-4-(3-cyanophenyl)-butyric acid;(S)-3-Amino-4-(3-fluorophenyl)-butyric acid;(S)-3-Amino-4-(3-methylphenyl)-butyric acid ;(S)-3-Amino-4-(3-pyridyl)-butyric acid;(S)-3-Amino-4-(3-thienyl)-butyric acid;(S)-3-Amino-4-(3-trifluoromethylphenyl)-butyric acid;(S)-3-Amino-4-(4-bromophenyl)-butyric acid;(S)-3-Amino-4-(4-chlorophenyl)butyric acid;(S)-3-Amino-4-(4-cyanophenyl)-butyric acid;(S)-3-Amino-4-(4-fluorophenyl)butyric acid;(S)-3-Amino-4-(4-iodophenyl)-butyric acid;(S)-3-Amino-4-(4-methylphenyl)-butyric acid;(S )-3-Amino-4-(4-nitrophenyl)-butyric acid;(S)-3-Amino-4-(4-pyridyl)-butyric acid;(S)-3-Amino-4-(4-trifluoromethylphenyl)-butyric acid;(S)-3-Amino-4-pentafluoro-phenylbutyric acid;(S)-3-Amino-5-hexenoic acid;(S)-3-Amino-5-hexynoic acid;(S)-3-Amino-5-phenylpentanoic acid;(S)-3-Amino-6-phenyl-5-hexenoic acid;1,2,5,6-Tetrahydropyridine-3-carboxylic acid;1,2,5,6-Tetrahydropyridine-4-carboxylic acid;3-Amino-3-(2-chlorophenyl)-propionic acid;3-; Amino-3-(2-thienyl)-propionic acid;3-Amino-3-(3-bromophenyl)-propionic acid;3-Amino-3-(4-chlorophenyl)-propionic acid;3-Amino-3-(4-methoxyphenyl)-propionic acid;3-Amino-4,4,4-trifluorobutyric acid;3-Aminoadipic acid;D-β-Phenylalanine;β-Leucine;L-β-Homoalanine;L-β-Homoaspartic acid γ-benzyl ester;L-β-Homoglutamic acid δ-benzyl ester;L-β-Homoisoleucine;L-β-Homoleucin;L-β-Homomethionine;L-β-Homophenylalanine;L-β-Homoproline;L-β-Homotryptophan;L-β-Homovaline;L-Nω-Benzyloxycarbonyl-β-Homolysine;Nω-L-β-Homoarginine;O-Benzyl-L-β -homohydroxyproline; O-benzyl-L-β-homoserine; O-benzyl-L-β-homothreonine; O-benzyl-L-β-homotyrosine; γ-trityl-L-β-homoasparagine; (R)-β-phenylalanine; L-β-homoaspartic acid γ-t-butyl ester; L-β-homoglutamic acid δ-t-butyl ester; L-Nω-β-homolysine; Nδ-trityl-L-β-homoglutamine; Nω-2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl-L-β-homoarginine; Ot-butyl-L-β-homohydroxyproline; Ot-butyl-L-β-homoserine; Ot-butyl-L-β-homothreonine; Ot-butyl-L-β-homotyrosine; 2-aminocyclopentanecarboxylic acid; and 2-aminocyclohexanecarboxylic acid.

[0086] Amino acid analogs may include analogs of alanine, valine, glycine, or leucine. Examples of amino acid analogs of alanine, valine, glycine, and leucine include, but are not limited to, α-methoxyglycine, α-allyl-L-alanine, α-aminoisobutyric acid, α-methyl-leucine, β-(1-naphthyl)-D-alanine, β-(1-naphthyl)-L-alanine, β-(2-naphthyl)-D-alanine, β-(2-naphthyl)-L-alanine, β-(2-pyridyl)-D-alanine, β-(2-pyridyl)-L-alanine. β-(2-Thienyl)-D-alanine;β-(2-Thienyl)-L-alanine;β-(3-Benzothienyl)-D-alanine;β-(3-Benzothienyl)-L-alanine;β-(3-Pyridyl)-D-alanine;β-(3-Pyridyl)-L-alanine;β-(4-Pyridyl)-D-alanine;β-(4-Pyridyl)-L-alanine;β-Chloro-L-alanine;β-Cyano-L-alanine;β-Cyclohexyl-D-alanine;β-Cyclohexyl-L- Alanine; β-Cyclopenten-1-yl-alanine; β-Cyclopentyl-alanine; β-Cyclopropyl-L-Ala-OH; Dicyclohexylammonium salt; β-t-Butyl-D-alanine; β-t-Butyl-L-alanine; γ-Aminobutyric acid; L-α,β-Diaminopropionic acid; 2,4-Dinitrophenylglycine; 2,5-Dihydro-D-phenylglycine; 2-Amino-4,4,4-trifluorobutyric acid; 2-Fluoro-phenylglycine; 3-Amino 4,4,4-Trifluorobutyric acid; 3-Fluorovaline; 4,4,4-Trifluorovaline; 4,5-Dehydro-L-leu-OH; Dicyclohexylammonium salt; 4-Fluoro-D-phenylglycine; 4-Fluoro-L-phenylglycine; 4-Hydroxy-D-phenylglycine; 5,5,5-Trifluoroleucine; 6-Aminohexanoic acid; Cyclopentyl-D-Gly-OH; Dicyclohexylammonium salt; Cyclopentyl-Gly-OH.Dicyclohexylammonium salt;D-α,β-Diaminopropionic acid;D-α-Aminobutyric acid;D-α-t-Butylglycine;D-(2-Thienyl)glycine;D-(3-Thienyl)glycine;D-2-Aminocaproic acid;D-2-Indanylglycine;D-Allylglycine-dicyclohexylammonium salt;D-Cyclohexylglycine;D-Norvaline;D-Phenylglycine;β-Aminobutyric acid ;β-Aminoisobutyric acid;(2-Bromophenyl)glycine;(2-Methoxyphenyl)glycine;(2-Methylphenyl)glycine;(2-Thiazolyl)glycine;(2-Thienyl)glycine;2-Amino-3-(dimethylamino)-propionic acid;L-α,β-Diaminopropionic acid;L-α-Aminobutyric acid;L-α-t-Butylglycine;L-(3-Thienyl)glycine;L-2-Amino-3-(di. Methylamino)-propionic acid;L-2-aminocaproic acid dicyclohexylammonium salt;L-2-indanylglycine;L-allylglycine dicyclohexylammonium salt;L-cyclohexylglycine;L-phenylglycine;L-propargylglycine;L-norvaline;N-α-aminomethyl-L-alanine;D-α,γ-diaminobutyric acid;L-α,γ-diaminobutyric acid;β-cyclopropyl-L-alanine;(N-β-(2, (4-Dinitrophenyl))-L-α,β-diaminopropionic acid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-D-α,β-diaminopropionic acid;(N-β-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,β-diaminopropionic acid;(N-β-4-Methyltrityl)-L-α,β-diaminopropionic acid;(N-β-Allyloxycarboxamide) (N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyric acid;(N-γ-1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl)-L-α,γ-diaminobutyric acid;(N-γ-4-methyltrityl)-D-α,γ-diaminobutyric acid;(N-γ-4-methyltrityl)-L-α,γ-diaminobutyric acid -allyloxycarbonyl)-L-α,γ-diaminobutyric acid; D-α,γ-diaminobutyric acid; 4,5-dehydro-L-leucine; cyclopentyl-D-Gly-OH; cyclopentyl-Gly-OH; D-allylglycine; D-homocyclohexylalanine; L-1-pyrenylalanine; L-2-aminocaproic acid; L-allylglycine; L-homocyclohexylalanine; and N-(2-hydroxy-4-methoxy-Bzl)-Gly-OH.

[0087] The amino acid analogs may include arginine or lysine analogs. Examples of arginine and lysine amino acid analogs include, but are not limited to, citrulline; L-2-amino-3-guanidinopropionic acid; L-2-amino-3-ureidopropionic acid; L-citrulline; Lys(Me)-OH; Lys(N)-OH; Nδ-benzyloxycarbonyl-L-ornithine; Nω-nitro-D-arginine; Nω-nitro-L-arginine; α-methylornithine; 2,6-diaminoheptanedioic acid; L-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohex-1-ylidene)-methyl)-2,6-diaminoheptanedioic acid; )ethyl)-D-ornithine; (Nδ-1-(4,4-dimethyl-2,6-dioxo-cyclohexen-1-ylidene)ethyl)-L-ornithine; (Nδ-4-methyltrityl)-D-ornithine; (Nδ-4-methyltrityl)-L-ornithine; D-ornithine; L-ornithine; Arg(Me)(Pbf)-OH; Arg(Me)-OH (asymmetric); Arg(Me)-OH (symmetric); Lys(ivDde)-OH; Lys(Me)-OH.HCl; Lys(Me)-OH chloride; Nω-nitro-D-arginine; and Nω-nitro-L-arginine.

[0088] Amino acid analogs may include analogs of aspartic acid or glutamic acid. Examples of amino acid analogs of aspartic acid and glutamic acid include, but are not limited to, α-methyl-D-aspartic acid; α-methyl-glutamic acid; α-methyl-L-aspartic acid; γ-methylene-glutamic acid; (N-γ-ethyl)-L-glutamine; [N-α-(4-aminobenzoyl)]-L-glutamic acid; 2,6-diaminopimelic acid; L-α-aminosuberic acid; D-2-aminoadipic acid; D-α-aminosuberic acid; α-aminopimelic acid; iminodiacetic acid; L-2-aminoadipic acid; threo-β-methyl-aspartic acid; γ-carboxy-D-glutamic acid γ,γ-di-t-butyl ester; γ-carboxy-L-glutamic acid γ,γ-di-t-butyl ester; Glu(OAll)-OH; L-Asu(OtBu)-OH; and pyroglutamic acid.

[0089] Amino acid analogs may include analogs of cysteine and methionine. Examples of amino acid analogs of cysteine and methionine include, but are not limited to: Cys( Farnesyl)-OH, Cys(farnesyl)-OMe, α-methyl-methionine, Cys(2-hydroxyethyl)-OH, Cys(3-aminopropyl)-OH, 2-amino-4-(ethylthio)butyric acid, buthionine, buthionine sulfoximine, ethionine, methionine methylsulfonium chloride, selenomethionine, cysteic acid, [2-(4-pyridyl)ethyl]-DL-penicillamine, [2-(4-pyridyl)ethyl]-L-cysteine, 4-methoxybenzyl-D-penicillamine, 4-methoxybenzyl-L-penicillamine, 4-methylbenzyl-D-penicillamine, 4-methylbenzyl-L-penicillamine cysteine, benzyl-D-cysteine, benzyl-L-cysteine, benzyl-DL-homocysteine, carbamoyl-L-cysteine, carboxyethyl-L-cysteine, carboxymethyl-L-cysteine, diphenylmethyl-L-cysteine, ethyl-L-cysteine, methyl-L-cysteine, t-butyl-D-cysteine, trityl-L-homocysteine, trityl-D-penicillamine, cystathionine, homocystine, L-homocystine, (2-aminoethyl)-L-cysteine, seleno-L-cystine, cystathionine, Cys(StBu)-OH, and acetamidomethyl-D-penicillamine.

[0090] Amino acid analogs may include analogs of phenylalanine and tyrosine. Examples of amino acid analogs of phenylalanine and tyrosine include: β-methyl-phenylalanine, β-hydroxyphenylalanine, α-methyl-3-methoxy-DL-phenylalanine, α-methyl-D-phenylalanine, α-methyl-L-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 2,4-dichloro-phenylalanine, 2-(trifluoromethyl)-D-phenylalanine, 2-(trifluoromethyl)-L-phenylalanine, 2-bromo-D-phenylalanine, 2-bromo-L-phenylalanine, 2-chloro-D-phenylalanine, 2-chloro-L-phenylalanine, 2-cyano-D-phenylalanine, 2-cyano-L-phenylalanine, and 2-fluoro-D-phenylalanine. , 2-fluoro-L-phenylalanine, 2-methyl-D-phenylalanine, 2-methyl-L-phenylalanine, 2-nitro-D-phenylalanine, 2-nitro-L-phenylalanine, 2,4,5-trihydroxy-phenylalanine, 3,4,5-trifluoro-D-phenylalanine, 3,4,5-trifluoro-L-phenylalanine, 3,4-dichloro-D-phenylalanine, 3,4-dichloro-L-phenylalanine, 3,4-difluoro-D-phenylalanine, 3,4-difluoro-L-phenylalanine, 3,4-dihydroxy-L-phenylalanine, 3,4-dimethoxy-L-phenylalanine, 3,5,3'-triiodo-L-thyronine, 3,5-diiodo-D-tyrosine, 3,5-diiodo-L-tyrosine, 3,5-Diiodo-L-thyronine, 3-(trifluoromethyl)-D-phenylalanine, 3-(trifluoromethyl)-L-phenylalanine, 3-amino-L-tyrosine, 3-bromo-D-phenylalanine, 3-bromo-L-phenylalanine, 3-chloro-D-phenylalanine, 3-chloro-L-phenylalanine, 3-chloro-L-tyrosine, 3-cyano-D-phenylalanine, 3-cyano-L-phenylalanine, 3-fluoro-D-phenylalanine, 3-fluoro-L-phenylalanine, 3-fluoro-tyrosine, 3-iodo-D-phenylalanine, 3-iodo-L-phenylalanine, 3-iodo-L-tyrosine, 3-methoxy-L-tyrosine, 3-methyl-D-phenylalanine, 3-methyl 4-(trifluoromethyl)-L-phenylalanine, 3-nitro-D-phenylalanine, 3-nitro-L-phenylalanine, 3-nitro-L-tyrosine, 4-(trifluoromethyl)-D-phenylalanine, 4-(trifluoromethyl)-L-phenylalanine, 4-amino-D-phenylalanine, 4-amino-L-phenylalanine, 4-benzoyl-D-phenylalanine, 4-benzoyl-L-phenylalanine, 4-bis(2-chloroethyl)amino-L-phenylalanine, 4-bromo-D-phenylalanine, 4-bromo-L-phenylalanine, 4-chloro-D-phenylalanine, 4-chloro-L-phenylalanine, 4-cyano-D-phenylalanine, 4-cyano-L-phenylalanine, 4-fluoro-D-, These include phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-D-phenylalanine, 4-iodo-L-phenylalanine, homophenylalanine, thyroxine, 3,3-diphenylalanine, thyronine, ethyl-tyrosine, and methyltyrosine.

[0091] Amino acid analogs may include analogs of proline. Examples of amino acid analogs of proline include, but are not limited to: 3,4-dehydro-proline, 4-fluoro-proline, cis-4-hydroxy-proline, thiazolidine-2-carboxylic acid, and trans-4-fluoro-proline.

[0092] Amino acid analogues can include serine and threonine analogues.The example of serine and threonine amino acid analogues includes but is not limited to: 3-amino-2-hydroxy-5-methylhexanoic acid, 2-amino-3-hydroxy-4-methylpentanoic acid, 2-amino-3-ethoxybutanoic acid, 2-amino-3-methoxybutanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-benzyloxypropionic acid, 2-amino-3-ethoxypropionic acid, 4-amino-3-hydroxybutanoic acid and α-methylserine.

[0093] Amino acid analogs may include analogs of tryptophan. Examples of tryptophan amino acid analogs include, but are not limited to, α-methyl-tryptophan, β-(3-benzothienyl)-D-alanine, β-(3-benzothienyl)-L-alanine, 1-methyl-tryptophan, 4-methyl-tryptophan, 5-benzyloxy-tryptophan, 5-bromo-tryptophan, 5-chloro-tryptophan, 5-fluoro-tryptophan, 5-hydroxytryptophan, 5-hydroxy-L-tryptophan, 5-methoxy-tryptophan, 5-methoxy-L-tryptophan, 5-methyl-tryptophan, 6 -Bromo-tryptophan; 6-chloro-D-tryptophan; 6-chloro-tryptophan; 6-fluoro-tryptophan; 6-methyl-tryptophan; 7-benzyloxy-tryptophan; 7-bromo-tryptophan; 7-methyl-tryptophan; D-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 6-methoxy-1,2,3,4-tetrahydronorharman-1-carboxylic acid; 7-azatryptophan; L-1,2,3,4-tetrahydro-norharman-3-carboxylic acid; 5-methoxy-2-methyl-tryptophan; and 6-chloro-L-tryptophan.

[0094] The amino acid analog may be racemic. In some cases, the D-isomer of the amino acid analog is used. In some cases, the L-isomer of the amino acid analog is used. In some cases, the amino acid analog contains a chiral center in the R or S configuration. Sometimes, the amino group of the β-amino acid analog is substituted with a protecting group, such as tert-butyloxycarbonyl (BOC group), 9-fluorenylmethyloxycarbonyl (FMOC), tosyl, etc. Sometimes, the carboxylic acid functional group of the β-amino acid analog is protected, for example, as its ester derivative. In some cases, a salt of the amino acid analog is used.

[0095] In some embodiments, the unnatural amino acid is an unnatural amino acid described in Liu CC, Schultz, PG Annu. Rev. Biochem. 2010, 79, 413. In some embodiments, the unnatural amino acid comprises N6((2-azidoethoxy)-carbonyl)-L-lysine.

[0096] In some embodiments, an amino acid residue described herein (e.g., in a protein) is mutated to a non-natural amino acid prior to attachment to a conjugate moiety. Mutation to an unnatural amino acid prevents or minimizes the immune system's autoantigen response. As used herein, the term "unnatural amino acid" refers to an amino acid other than the 20 amino acids that naturally occur in proteins. Non-limiting examples of unnatural amino acids include: p-acetyl-L-phenylalanine, p-iodo-L-phenylalanine, p-methoxyphenylalanine, O-methyl-L-tyrosine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanines, and the like. , isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-boronophenylalanine, O-propargyltyrosine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, selenocysteine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, N6-((azidoethoxy)-carbonyl)-L-lysine, AzK), N6-(( N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine, non-natural analogs of tyrosine amino acid; non-natural analogs of glutamine amino acid; non-natural analogs of phenylalanine amino acid; non-natural analogs of serine amino acid; non-natural analogs of threonine amino acid; alkyl, aryl, acyl, azido, cyano, halo, hydrazine, hydra dido, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino substituted amino acids, or combinations thereof; amino acids with photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; metal-binding amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids;Biotin or biotin analog containing amino acids; keto containing amino acids; polyethylene glycol or polyether containing amino acids; heavy atom substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with elongated side chains; amino acids containing toxic groups; sugar-substituted amino acids; carbon-linked sugar-containing amino acids; redox-active amino acids; α-hydroxy-containing acids; aminothioacids; α,α-disubstituted amino acids; β-amino acids; cyclic amino acids other than proline or histidine, and aromatic amino acids other than phenylalanine, tyrosine, or tryptophan;

[0097] In some embodiments, the unnatural amino acid comprises a selectively reactive group or a reactive group for site-selective labeling of a target protein or polypeptide. In some cases, the chemical reaction is a biorthogonal reaction (e.g., a biocompatible and selective reaction). In some cases, the chemical reaction is a Cu(I)-catalyzed or "copper-free" alkyne azide triazole-forming reaction, Staudinger ligation, inverse electron demand Diels-Alder (IEDDA) reaction, "photoclick" chemistry, or a metal-mediated process such as olefin metathesis and Suzuki-Miyaura or Sonogashira cross-coupling. In some embodiments, the unnatural amino acid comprises a photoreactive group that crosslinks upon irradiation, e.g., with UV light. In some embodiments, the unnatural amino acid comprises a photocaged amino acid. In some cases, the unnatural amino acid is a para-substituted, meta-substituted, or ortho-substituted amino acid derivative.

[0098] In some cases, the unnatural amino acid is p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, O-methyl-L-tyrosine, p-methoxyphenylalanine, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcp-serine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-bromophenylalanine, p-amino-L-phenylalanine, or isopropyl-L-phenylalanine.

[0099] In some cases, the unnatural amino acid is 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxy-phenylalanine, or 3-iodotyrosine. In some cases, the unnatural amino acid is phenylselenocysteine. In some cases, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide (including a phenylalanine derivative). In some cases, the unnatural amino acid is a benzophenone, ketone, iodide, methoxy, acetyl, benzoyl, or azide-containing lysine derivative. In some cases, the unnatural amino acid comprises an aromatic side chain. In some cases, the unnatural amino acid does not comprise an aromatic side chain. In some cases, the unnatural amino acid comprises an azide group. In some cases, the unnatural amino acid comprises a Michael acceptor group. In some cases, the Michael acceptor group comprises an unsaturated moiety capable of forming a covalent bond via a 1,2-addition reaction. In some cases, the Michael acceptor group comprises an electron-deficient alkene or alkyne. In some cases, the Michael acceptor group includes, but is not limited to, alpha, beta unsaturated: ketone, aldehyde, sulfoxide, sulfone, nitrile, imine, or aromatic. In some cases, the unnatural amino acid is dehydroalanine. In some cases, the unnatural amino acid comprises an aldehyde or ketone group. In some cases, the unnatural amino acid is a lysine derivative comprising an aldehyde or ketone group. In some cases, the unnatural amino acid is a lysine derivative comprising one or more O, N, Se, or S atoms at the beta, gamma, or delta position. In some cases, the unnatural amino acid is a lysine derivative comprising an O, N, Se, or S atom at the gamma position. In some cases, the unnatural amino acid is a lysine derivative in which the epsilon N atom is replaced with an oxygen atom. In some cases, the unnatural amino acid is a lysine derivative that is a non-naturally occurring post-translationally modified lysine.

[0100] In some cases, the unnatural amino acid is an amino acid that includes a side chain and the sixth atom from the alpha position includes a carbonyl group. In some cases, the unnatural amino acid is an amino acid that includes a side chain and the sixth atom from the alpha position includes a carbonyl group and the fifth atom from the alpha position is nitrogen. In some cases, the unnatural amino acid is an amino acid that includes a side chain and the seventh atom from the alpha position is an oxygen atom.

[0101] In some cases, the unnatural amino acid is a serine derivative that includes selenium. In some cases, the unnatural amino acid is selenoserine (2-amino-3-hydroselenopropanoic acid). In some cases, the unnatural amino acid is 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid. In some cases, the unnatural amino acid is 2-amino-3-(phenylselanyl)propanoic acid. In some cases, the unnatural amino acid includes selenium, and oxidation of the selenium results in the formation of an alkene-containing unnatural amino acid.

[0102] In some cases, the unnatural amino acid comprises a cyclooctynyl group. In some cases, the unnatural amino acid comprises a transcycloctenyl group. In some cases, the unnatural amino acid comprises a norbornenyl group. In some cases, the unnatural amino acid comprises a cyclopropenyl group. In some cases, the unnatural amino acid comprises a diazirine group. In some cases, the unnatural amino acid comprises a tetrazine group.

[0103] In some instances, the unnatural amino acid is a lysine derivative in which the side chain nitrogen is carbamylated. In some instances, the unnatural amino acid is a lysine derivative in which the side chain nitrogen is acylated. In some instances, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)amino]- In some instances, the unnatural amino acid is 2-amino-6-{[(tert-butoxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-Boc-N6-methyllysine. In some instances, the unnatural amino acid is N6-acetyllysine. In some instances, the unnatural amino acid is pyrrolysine. In some instances, the unnatural amino acid is N6-trifluoroacetyllysine. In some instances, the unnatural amino acid is 2-amino-6-{[(benzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(p-iodobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(p-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-prolyl lysine. In some instances, the unnatural amino acid is 2-amino-6-{[(cyclopentyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-(cyclopentanecarbonyl)lysine. In some instances, the unnatural amino acid is N6-(tetrahydrofuran-2-carbonyl)lysine. In some instances, the unnatural amino acid is N6-(3-ethynyltetrahydrofuran-2-carbonyl)lysine. In some instances, the unnatural amino acid is N6-((prop-2-yn-1-yloxy)carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino-6-{[(2-azidocyclopentyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-((2-azidoethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino-6-{[(2-nitrobenzyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is 2-amino-6-{[(2-cyclooctynyloxy)carbonyl]amino}hexanoic acid. In some instances, the unnatural amino acid is N6-(2-aminobut-3-ynoyl)lysine. In some instances, the unnatural amino acid is 2-amino-6-((2-aminobut-3-ynoyl)oxy)hexanoic acid.In some instances, the unnatural amino acid is N6-(allyloxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-(butenyl-4-oxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-(pentenyl-5-oxycarbonyl)lysine. In some instances, the unnatural amino acid is N6-((but-3-yn-1-yloxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6-((pent-4-yn-1-yloxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6-(thiazolidine-4-carbonyl)lysine. In some instances, the unnatural amino acid is 2-amino-8-oxononanoic acid. In some instances, the unnatural amino acid is 2-amino-8-oxooctanoic acid. In some instances, the unnatural amino acid is N6-(2-oxoacetyl)lysine.

[0104] In some instances, the unnatural amino acid is N6-propionyl lysine. In some instances, the unnatural amino acid is N6-butyryl lysine. In some instances, the unnatural amino acid is N6-(but-2-enoyl) lysine. In some instances, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-yloxy)carbonyl) lysine. In some instances, the unnatural amino acid is N6-((spiro[2.3]hex-1-en-5-ylmethoxy)carbonyl) lysine. In some instances, the unnatural amino acid is N6-(((4-(1-(trifluoromethyl)cycloprop-2-en-1-yl)benzyl)oxy)carbonyl) lysine. In some instances, the unnatural amino acid is N6-((bicyclo[2.2.1]hept-5-en-2-ylmethoxy)carbonyl) lysine. In some instances, the unnatural amino acid is cysteinyl lysine. In some instances, the unnatural amino acid is N6-((1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((2-(3-methyl-3H-diazirin-3-yl)ethoxy)carbonyl)lysine. In some instances, the unnatural amino acid is N6-((3-(3- In some instances, the unnatural amino acid is N6-((metanitrobenyloxy)N6-methylcarbonyl)lysine. In some instances, the unnatural amino acid is N6-((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)-lysine. In some instances, the unnatural amino acid is N6-((cyclohept-3-en-1-yloxy)carbonyl)-L-lysine.

[0105] In some embodiments, the unnatural amino acid is incorporated into the protein by an unnatural codon that comprises an unnatural nucleotide.

[0106] In some cases, incorporation of unnatural amino acids into proteins is mediated by orthogonal engineered synthetase / tRNA pairs. Such orthogonal pairs include a natural or mutant synthetase that can charge a particular unnatural amino acid to the unnatural tRNA, often while minimizing a) charging of other endogenous or alternative unnatural amino acids to the unnatural tRNA and b) charging of any other (including endogenous) tRNAs. Such orthogonal pairs include a tRNA that can be charged by the synthetase while avoiding charging of other endogenous amino acids by the endogenous synthetase. In some embodiments, such pairs are identified from various organisms, such as bacteria, yeast, archaea, or human sources. In some embodiments, the orthogonal synthetase / tRNA pair includes components from a single organism. In some embodiments, the orthogonal synthetase / tRNA pair includes components from two different organisms. In some embodiments, the orthogonal synthetase / tRNA pair includes components that facilitate translation of different amino acids prior to modification. In some embodiments, the orthogonal synthetase is an engineered alanine synthetase. In some embodiments, the orthogonal synthetase is an engineered arginine synthetase. In some embodiments, the orthogonal synthetase is an engineered asparagine synthetase. In some embodiments, the orthogonal synthetase is an engineered aspartate synthetase. In some embodiments, the orthogonal synthetase is an engineered cysteine synthetase. In some embodiments, the orthogonal synthetase is an engineered glutamine synthetase. In some embodiments, the orthogonal synthetase is an engineered glutamate synthetase. In some embodiments, the orthogonal synthetase is an engineered alanine synthetase. In some embodiments, the orthogonal synthetase is an engineered histidine synthetase. In some embodiments, the orthogonal synthetase is an engineered leucine synthetase. In some embodiments, the orthogonal synthetase is an engineered isoleucine synthetase. In some embodiments, the orthogonal synthetase is an engineered lysine synthetase.In some embodiments, the orthogonal synthetase is an engineered methionine synthetase. In some embodiments, the orthogonal synthetase is an engineered phenylalanine synthetase. In some embodiments, the orthogonal synthetase is an engineered proline synthetase. In some embodiments, the orthogonal synthetase is an engineered serine synthetase. In some embodiments, the orthogonal synthetase is an engineered threonine synthetase. In some embodiments, the orthogonal synthetase is an engineered tryptophan synthetase. In some embodiments, the orthogonal synthetase is an engineered tyrosine synthetase. In some embodiments, the orthogonal synthetase is an engineered valine synthetase. In some embodiments, the orthogonal synthetase is an engineered phosphoserine synthetase. In some embodiments, the orthogonal tRNA is an engineered alanine tRNA. In some embodiments, the orthogonal tRNA is an engineered arginine tRNA. In some embodiments, the orthogonal tRNA is a modified asparagine tRNA. In some embodiments, the orthogonal tRNA is a modified aspartic acid tRNA. In some embodiments, the orthogonal tRNA is a modified cysteine tRNA. In some embodiments, the orthogonal tRNA is modified. In some embodiments, the orthogonal tRNA is a modified glutamine tRNA. In some embodiments, the orthogonal tRNA is a modified glutamate tRNA. In some embodiments, the orthogonal tRNA is a modified alanine glycine tRNA. In some embodiments, the orthogonal tRNA is a modified histidine tRNA. In some embodiments, the orthogonal tRNA is a modified leucine tRNA. In some embodiments, the orthogonal tRNA is a modified isoleucine tRNA. In some embodiments, the orthogonal tRNA is a modified lysine tRNA. In some embodiments, the orthogonal tRNA is a modified methionine tRNA. In some embodiments, the orthogonal tRNA is a modified phenylalanine tRNA. In some embodiments, the orthogonal tRNA is a modified proline tRNA. In some embodiments, the orthogonal tRNA is a modified serine tRNA. In some embodiments, the orthogonal tRNA is a modified threonine tRNA. In some embodiments, the orthogonal tRNA is a modified tryptophan tRNA. In some embodiments, the orthogonal tRNA is a modified tyrosine tRNA. In some embodiments, the orthogonal tRNA is a modified valine tRNA. In some embodiments, the orthogonal tRNA is a modified phosphoserine tRNA.

[0107] In some embodiments, unnatural amino acids are incorporated into proteins by aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pairs. Exemplary aaRS-tRNA pairs include, but are not limited to, the Methanococcus jannaschii (Mj-Tyr) aaRS / tRNA pair, the E. coli TyrRS (Ec-Tyr) / B. stearothermophilus tRNACUA pair, the E. coli LeuRS (Ec-Leu) / B. stearothermophilus tRNACUA pair, and a pyrrolysyl-tRNA pair. In some cases, unnatural amino acids are incorporated into proteins by the Mj-TyrRS / tRNA pair. Exemplary unnatural amino acids (UAA) that can be incorporated by the Mj-TyrRS / tRNA pair include, but are not limited to, para-substituted phenylalanine derivatives, such as p-aminophenylalanine and p-methyphenylalanine; meta-substituted tyrosine derivatives, such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-boronophenylalanine; and o-nitrobenzyltyrosine.

[0108] In some cases, unnatural amino acids are incorporated into proteins via Ec-Tyr / tRNACUA or Ec-Leu / tRNACUA pairs. Exemplary UAAs that can be incorporated via Ec-Tyr / tRNACUA or Ec-Leu / tRNACUA pairs include, but are not limited to, phenylalanine derivatives containing benzophenone, ketone, iodide, or azide substitutions; O-propargyl tyrosine; α-aminocaprylic acid, O-methyl tyrosine, O-nitrobenzyl cysteine; and 3-(naphthalen-2-ylamino)-2-amino-propanoic acid.

[0109] In some cases, unnatural amino acids are incorporated into proteins via pyrrolysyl-tRNA pairs. In some cases, PylRS is obtained from archaeal species such as methanogens. In some cases, PylRS is obtained from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans. Exemplary UAAs that can be incorporated by pyrrolysyl-tRNA pairs include, but are not limited to, amide and carbamate substituted lysines, such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N-ε-D-prolyl-L-lysine, and N-ε-cyclopentyloxycarbonyl-L-lysine; N-ε-acryloyl-L-lysine; N-ε-[(1-(6-nitrobenzo[d][1,3]dioxol-5-yl)ethoxy)carbonyl]-L-lysine; and N-ε-(1-methylcyclopro-2-enecarboxamido)lysine. Examples include:

[0110] In some cases, unnatural amino acids are incorporated into proteins described herein by synthetases disclosed in U.S. Patent Nos. 9,988,619 and 9,938,516. Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl unnatural amino acids. In some embodiments, such UAAs comprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycles. In some embodiments, such amino acids comprise other chemical groups that can be conjugated to coupling partners, such as azides, tetrazines, or water-soluble moieties. In some embodiments, such synthetases are expressed and used to incorporate UAAs into proteins in vivo. In some embodiments, such synthetases are used to incorporate UAAs into proteins using cell-free translation systems, such as reconstituted systems of cell lysates or purified components. tRNA can be charged with an unnatural amino acid in a cell-free system or in a separate reaction beforehand (so that the charged tRNA is added directly to a system containing ribosomes, mRNA, and other components, without the need to add a synthetase or a construct encoding the synthetase to the system).

[0111] Systems for in vitro translation are described, for example, in Zeenko et al., RNA 14:593-602 (2008); Spirin, Trends Biotechnol. 2004:538-545 (2004); and Endo et al., Curr. Opin. Biotechnol. 17:373-380 (2006). Systems can be prepared from cell lysates (e.g., extracts) or reconstituted from purified components. In addition to ribosomes, tRNAs, and other components described herein, systems can include one or more translation initiation factors; ATP; and one or more translation termination factors. In some embodiments, systems further include one or more molecular chaperones, which can assist in folding of the nascent polypeptide during and / or after translation.

[0112] In some cases, unnatural amino acids are incorporated into proteins described herein by naturally occurring synthetases. In some embodiments, unnatural amino acids are incorporated into proteins by organisms that are auxotrophic for one or more amino acids. In some embodiments, a synthetase corresponding to the auxotrophic amino acid can charge the corresponding tRNA with the unnatural amino acid. In some embodiments, the unnatural amino acid is selenocysteine or a derivative thereof. In some embodiments, the unnatural amino acid is selenomethionine or a derivative thereof. In some embodiments, the unnatural amino acid is an aromatic amino acid, where the aromatic amino acid comprises an aryl halide, such as an iodide. In embodiments, the unnatural amino acid is structurally similar to the auxotrophic amino acid.

[0113] In some cases, the unnatural amino acid includes an unnatural amino acid shown in Figure 4A.

[0114] In some cases, the unnatural amino acid comprises a lysine or phenylalanine derivative or analog. In some cases, the unnatural amino acid comprises a lysine derivative or lysine analog. In some cases, the unnatural amino acid comprises pyrrolysine (Pyl). In some cases, the unnatural amino acid comprises a phenylalanine derivative or phenylalanine analog. In some cases, the unnatural amino acid comprises a ...yl-tRNA synthetase as described by Wan et al., "Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an excellent genetic code expansion tool." "An outstanding genetic code expansion tool," Biochem Biophys Aceta 1844(6):1059-4070 (2014). In some cases, the unnatural amino acids include the unnatural amino acids shown in Figure 4B and Figure 4C.

[0115] In some embodiments, the unnatural amino acids include the unnatural amino acids shown in Figures 4D-4G (adapted from Table 1 in Dumas et al., Chemical Science 2015, 6, 50-69).

[0116] In some embodiments, the unnatural amino acids incorporated into the proteins described herein are disclosed in U.S. Patent No. 9,840,493; U.S. Patent No. 9,682,934; U.S. Patent Application Publication No. 2017 / 0260137; U.S. Patent No. 9,938,516; or U.S. Patent Application Publication No. 2018 / 0086734. Exemplary UAAs that can be incorporated by such synthetases include para-methylazido-L-phenylalanine, aralkyl, heterocyclyl, and heteroaralkyl, and lysine-derived unnatural amino acids. In some embodiments, such UAAs comprise pyridyl, pyrazinyl, pyrazolyl, triazolyl, oxazolyl, thiazolyl, thiophenyl, or other heterocycles. In some embodiments, such amino acids comprise azides, tetrazines, or other chemical groups that can be attached to coupling partners such as water-soluble moieties. In some embodiments, the UAAs comprise an azide linked to an aromatic moiety via an alkyl linker. In some embodiments, the alkyl linker is a C1-C10 linker. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an alkyl linker. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an amino group. In some embodiments, the UAA comprises a tetrazine linked to the aromatic moiety via an alkylamino group. In some embodiments, the UAA comprises an azide linked to the terminal nitrogen of an amino acid side chain via an alkyl chain (e.g., N6 of a lysine derivative, or N5, N4, or N3 of a derivative containing a shorter alkyl side chain). In some embodiments, the UAA comprises a tetrazine linked to the terminal nitrogen of an amino acid side chain via an alkyl chain. In some embodiments, the UAA comprises an azide or tetrazine linked to an amide via an alkyl linker. In some embodiments, the UAA is an azide- or tetrazine-containing carbamate or an amide of 3-aminoalanine, serine, lysine, or a derivative thereof. In some embodiments, such a UAA is incorporated into a protein in vivo. In some embodiments, such a UAA is incorporated into a protein in a cell-free system.

[0117] cell type In some embodiments, many types of cells / microorganisms are used, for example, for transformation or genetic engineering. In some embodiments, the cells are eukaryotic cells. In some cases, the cells are eukaryotic cells, such as cultured animal, plant, or human cells. In additional cases, the cells are present in an organism, such as a plant or animal.

[0118] In some embodiments, the engineered microorganism is a unicellular organism, capable of dividing and growing frequently. The microorganism may comprise one or more of the following characteristics: aerobic, anaerobic, filamentous, non-filamentous, haploid, diploid, auxotrophic, and / or non-auxotrophic. In certain embodiments, the engineered microorganism is a non-prokaryotic microorganism. In some embodiments, the engineered microorganism is a eukaryotic microorganism (e.g., yeast, fungus, amoeba). In some embodiments, the engineered microorganism is a fungus. In some embodiments, the engineered organism is a yeast.

[0119] Any suitable yeast may be used as a host microorganism, an engineered microorganism, a genetically modified organism, or a heterologous organism. or may be selected as a source of modified polynucleotides. Yeasts include, but are not limited to, Yarrowia yeasts (e.g., Y. lipolytica (formerly classified as Candida lipolytica)), Candida yeasts (e.g., C. revkaufi, C. viswanathii, C. pulcherrima, C. tropicalis, C. utilis), Rhodotorula yeasts (e.g., R. glutinus, R. graminis), Rhodosporidium yeasts (e.g., R. toruloides), Saccharomyces yeasts (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Cryptococcus yeasts, Trichosporon yeasts (e.g., T. pullans, T. cutaneum), Pichia yeasts (e.g., P. pastoris), and Lipomyces yeasts (e.g., L. starkeyii, L. lipoferus). In some embodiments, a suitable yeast is a yeast of the genus Arachniotus, Aspergillus, Aureobasidium, Auxarthron, Blastomyces, Candida, Chrysosporium, Debaryomyces, Coccidiodes, Cryptococcus, Gymnoascus, Hansenula, Histoplasma, Issatchenkia, Kluyveromyces, Lipomyces, Lssatchenkia, Microsporum, Myxotrichum, Myxozyma, Oidiodendron, Pachysolen, Penicillium, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Scopulariopsis, Sepedonium, Trichosporon, or Yarrowia.Arachniotus flavoluteus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aureobasidium pullulans, Auxarthron. thaxteri、Blastomyces dermatitidis、Candida albicans、Candida dubliniensis、Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida lustitaniae, Candida parapsilosis, Candida pulcherrima, Candida revkaufi, Candida rugosa, Candida tropicalis, Candida utilis, Candida viswanathii, Candida xestobii, Chrysosporuim keratinophilum, Coccidiodes immitis, Cryptococcus albidus var. diffluens, Cryptococcus laurentii, Cryptococcus neofomans, Debaryomyces hansenii, Gymnoascus dugwayensis, Hansenula anomala, Histoplasma capsulatum, Issatchenkia occidentalis, Isstachenkia orientalis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Kluyveromyces waltii, Lipomyces lipoferus, Lipomyces starkeyii, Microsporum gypseum, Myxotrichum deflexum, Oidiodendron echinulatum, Pachysolen tannophilis, Penicillium notatum, Pichia anomala, Pichia pastoris, Pichia stipitis, Rhodosporidium toruloides, Rhodotorula glutinus , Rhodotorula graminis, Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Scopulariopsis acremonium, Sepedonium chrysospermum, Trichosporon cutaneum, Trichosporon pullans, Yarrowia lipolytica, or Yarrowia lipolytica (formerly classified as Candida lipolytica). In some embodiments, the yeast is a Y. lipolytica strain, including, but not limited to, ATCC20362, ATCC8862, ATCC18944, ATCC20228, ATCC76982, and LGAM S(7)1 strains (Papanikolaou S. and Aggelis G., Bioresort. Technol. 82(1):43-9 (2002)). In certain embodiments, the yeast is a Candida species (i.e., Candida genus) yeast. Any suitable Candida species may be used and / or may be genetically modified for the production of fatty dicarboxylic acids (e.g., octanedioic acid, decanedioic acid, dodecanedioic acid, tetradecanedioic acid, hexadecanedioic acid, octadecanedioic acid, eicosanedioic acid). In some embodiments, suitable Candida species include, but are not limited to, Candida albicans, Candida dubliniensis, Candida famata, Candida glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lambica, Candida lipolytica, Candida Candida species include Candida lustitaniae, Candida parapsilosis, Candida pulcherrima, Candida revkaufi, Candida rugosa, Candida tropicalis, Candida utilis, Candida viswanathii, Candida xestobii, and any other Candida species yeasts described herein. Non-limiting examples of Candida species strains include, but are not limited to, strains sAA001 (ATCC20336), sAA002 (ATCC20913), sAA003 (ATCC20962), sAA496 (US Patent Application Publication No. 2012 / 0077252), sAA106 (US Patent Application Publication No. 2012 / 0077252), SU-2 (ura3- / ura3-), and H5343 (beta-oxidation blocked; U.S. Patent No. 5,648,247). Any suitable strain of yeast derived from a Candida species can be utilized as the parent strain for genetic recombination.

[0120] Yeast genera, species, and strains are often so closely related in genetic content that they can be difficult to distinguish, classify, and / or name. In some cases, it can be difficult to distinguish, classify, and / or name strains of C. lipolytica and Y. lipolytica, and in some cases, they can be considered the same organism. In some cases, it can be difficult to distinguish, classify, and / or name various strains of C. tropicalis and C. viswanathii (see, e.g., Arie et al., J. Gen. Appl. Microbiol., 46, 257-262 (2000)). Some C. tropicalis and C. viswanathii strains obtained from ATCC and other commercial or academic sources can be considered equivalent and equally suitable for the embodiments described herein. In some embodiments, some parent strains of C. tropicalis and C. viswanathii are considered to differ only in name.

[0121] Any suitable fungus may be selected as a host microorganism, engineered microorganism, or source of a heterologous polynucleotide. Non-limiting examples of fungi include, but are not limited to, Aspergillus fungi (e.g., A. parasiticus, A. nidulans), Thraustochytrium fungi, Schizochytrium fungi, and Rhizopus fungi (e.g., R. arrhizus, R. oryz). In some embodiments, the fungus is an A. parasiticus strain, including but not limited to, ATCC 24690, and in particular embodiments, the fungus is an A. nidulans strain, including but not limited to, ATCC 38163.

[0122] Cells of non-microbial origin can be utilized as host microorganisms, engineered microorganisms, or sources of heterologous polynucleotides. Examples of such cells include, but are not limited to, insect cells (e.g., Drosophila (e.g., D. melanogaster), Spodoptera (e.g., S. frugiperda Sf9 or Sf21 cells), and Trichoplusa (e.g., High-Five cells); nematode cells (e.g., C. elegans cells); avian cells; amphibian cells (e.g., Xenopus laevis cells); reptilian cells; mammalian cells (e.g., NIH3T3, 293, CHO, COS, VERO, C127, BHK, Per-C6, Bowes melanoma, and HeLa cells); and plant cells (e.g., Arabidopsis thaliana, Nicotania tabacum, Cuphea acinifolia, Cuphea aequipetala, Cuphea angustifolia, Cuphea appendiculata, Cuphea avigera, Cuphea avigera var. pulcherrima, Cuphea axilliflora, Cuphea bahiensis, Cuphea baillonis, Cuphea brachypoda, Cuphea bustamanta, Cuphea calcarata, Cuphea calophylla, Cuphea calophylla subsp. mesostemon, Cuphea carthagenensis, Cuphea circaeoides, Cuphea confertiflora, Cuphea cordata, Cuphea crassiflora, Cupphea cyanea, Cupphea decandra, Cuphea denticulata, Cuphea disperma, Cupphea epilobiifolia, Cupphea ericoides, Cupphea flava、Cuphea flavisetula、Cuphea fuchsiifolia、Cuphea gaumeri、Cuphea glutinosa、Cuphea heterophylla、Cuphea hookeriana、Cuphea hyssopifolia (Mexican-heather)、Cuphea hyssopoides, Cuphea ignea, Cuphea ingrata, Cuphea jorullensis, Cuphea lanceolata, Cuphea linarioides, Cuphea llavea, Cuphea lophostoma, Cuphea lutea, Cuphea lutescens, Cuphea melanium, Cuphea melvilla, Cuphea micrantha, Cuphea micropetala, Cuphea mimuloides, Cuphea nitidula, Cuphea palustris, Cuphea parsonsia, Cuphea pascuorum, Cuphea paucipetala, Cuphea procumbens, Cuphea pseudosilene, Cuphea pseudovaccinium, Cuphea pulchra, Cuphea racemosa, Cuphea repens, Cuphea salicifolia, Cuphea salvadorensis, Cuphea schumannii, Cuphea sessiliflora, Cuphea sessilifolia, Cuphea setosa, Cuphea spectabilis, Cuphea spermacoce, Cuphea splendida, Cuphea splendida var. viridiflava, Cuphea strigulosa, Cuphea subuligera, Cuphea teleandra, Cuphea thymoides, Cuphea tolucana, Cuphea urens, Cuphea utriculosa, Cuphea viscosissima, Cupphea watsoniana, Cupphea wrightii, Cupphea lanceolata).

[0123] Microorganisms or cells used as host organisms or sources of heterologous polynucleotides are commercially available. The microorganisms and cells described herein, as well as other suitable microorganisms and cells, are available, for example, from Invitrogen Corporation (Carlsbad, CA), the American Type Culture Collection (Manassas, Virginia), and the Agricultural Research Culture Collection (NRRL; Peoria, Illinois). Host microorganisms and engineered microorganisms can be provided in any suitable form. For example, such microorganisms can be provided in liquid or solid culture (e.g., agar-based media), which may be primary cultures or may have been passaged one or more times (e.g., diluted and cultured). Microorganisms can also be provided in frozen or dried form (e.g., lyophilized). Microorganisms can be provided at any suitable concentration.

[0124] Nucleic Acid Reagents and Tools Nucleotide and / or nucleic acid reagents (or polynucleotides) for use with the methods, cells, or engineered microorganisms described herein contain one or more ORFs, with or without unnatural nucleotides. ORFs can be derived from any suitable source, sometimes genomic DNA, mRNA, reverse-transcribed RNA, or complementary DNA (cDNA), or a nucleic acid library containing one or more of the foregoing, and can be derived from any organism containing a nucleic acid sequence, protein, or activity of interest. Non-limiting examples of organisms from which ORFs can be obtained include, for example, bacteria, yeast, fungi, humans, insects, nematodes, cattle, horses, dogs, cats, rats, or mice. In some embodiments, the nucleotide and / or nucleic acid reagents or other reagents described herein are isolated or purified. ORFs containing unnatural nucleotides can be produced by published in vitro methods. In some cases, the nucleotide or nucleic acid reagents contain unnatural nucleobases.

[0125] The nucleic acid reagent may include a nucleotide sequence adjacent to the ORF that is translated in conjunction with the ORF and encodes an amino acid tag. The nucleotide sequence encoding the tag is located 3' and / or 5' of the ORF in the nucleic acid reagent, thereby encoding the tag at the C-terminus or N-terminus of the protein or peptide encoded by the ORF. Any tag that does not abolish in vitro transcription and / or translation may be utilized and may be appropriately selected by a technician. The tag may facilitate isolation and / or purification of the desired ORF product from the culture or fermentation medium. In some cases, a library of nucleic acid reagents is used with the methods and compositions described herein. For example, a library of at least 100, 1000, 2000, 5000, 10,000, or more than 50,000 unique polynucleotides is present in the library, and each polynucleotide comprises at least one unnatural nucleobase.

[0126] Nucleic acids or nucleic acid reagents, which may or may not contain non-natural nucleotides, may contain certain elements, such as regulatory elements, which are often selected according to the intended use of the nucleic acid. Any of the following elements may be included or excluded from a nucleic acid reagent. For example, a nucleic acid reagent may contain one or more or all of the following nucleotide elements: one or more promoter elements, one or more 5' untranslated regions (5'UTRs), one or more regions into which a target nucleotide sequence may be inserted ("insertion elements"), one or more target nucleotide sequences, one or more 3' untranslated regions (3'UTRs), and one or more selection elements. A nucleic acid reagent may contain: One or more of such elements may be provided, and other elements may be inserted into the nucleic acid before the nucleic acid is introduced into a desired organism. In some embodiments, the provided nucleic acid reagent includes a promoter, a 5'UTR, an optional 3'UTR, and an insertion element into which a target nucleotide sequence is inserted (i.e., cloned) into the nucleic acid reagent. In certain embodiments, the provided nucleic acid reagent includes a promoter, an insertion element, and an optional 3'UTR, and the 5'UTR / target nucleotide sequence is inserted along with the optional 3'UTR. The elements may be arranged in any order suitable for expression in a selected expression system (e.g., expression in a selected organism, or, for example, expression in a cell-free system); in some embodiments, the nucleic acid reagent includes the following elements in a 5'→3' direction: (1) a promoter element, a 5'UTR, and an insertion element; (2) a promoter element, a 5'UTR, and a target nucleotide sequence; (3) a promoter element, a 5'UTR, an insertion element, and a 3'UTR; and (4) a promoter element, a 5'UTR, a target nucleotide sequence, and a 3'UTR. In some embodiments, UTRs can be optimized to alter or increase transcription or translation of ORFs that are completely natural or contain non-natural nucleotides.

[0127] Nucleic acids (e.g., mRNAs) comprising the nucleobases described herein, in some cases, comprise a 5' UTR and / or a 3' UTR that enhance mRNA stability in vivo (e.g., in eukaryotic cells or organisms). In some instances, the 5' or 3' UTR, or both, are engineered to reduce mRNA degradation or decay in vivo. Non-limiting examples of 5' and 3' UTRs that enhance mRNA stability in eukaryotic systems disclosed herein include the CS2 3' and 5' UTRs. In some embodiments, mRNAs are modified to reduce the rate of removal of the poly(A) tail of mRNA compared to mRNAs comprising otherwise unmodified nucleobases described herein. In some embodiments, cis-acting AU-rich elements (AREs) are shielded from intracellular and extracellular signaling that promotes mRNA decay. In some embodiments, premature stop codons in mRNAs are removed from the mRNA to reduce nonsense-mediated decay (NMD) of the mRNA.

[0128] In some cases, 5' and / or 3' UTR directly or indirectly increases the translation of mRNA into polypeptide.A non-limiting example of the way that 5' UTR or 3' UTR directly affects the translation of mRNA into polypeptide includes the recruitment of RNA binding proteins, which bind to 5' or 3' cis elements and result in the recruitment of ribosomes or effector proteins (e.g., mRNA deadenylase, decapping enzyme).A non-limiting example of the way that 5' UTR or 3' UTR indirectly affects the translation of mRNA into polypeptide includes the formation of 5' and 3' UTR secondary structures that block or enhance the binding of RNA binding proteins to the 5' or 3' UTR region, and mRNA subcellular localization.

[0129] In some embodiments, the 5'UTR and / or 3'UTR increase the translation efficiency of the mRNA in vitro or in vivo compared to the translation efficiency of an mRNA containing unengineered nucleobases. In some embodiments, translation efficiency is increased by engineering the mRNA to reduce skipping of selected AUG (start codon) by the ribosome during scanning. In some embodiments, the mRNA contains a sequence element that improves start codon recognition, such as a Kozak sequence or a variant thereof. In some embodiments, the 5'UTR of the mRNA is engineered to reduce the overall guanine-cytosine (GC) content.

[0130] In some embodiments, the formation of secondary structures in mRNA involving the AUG start codon in the 5'UTR (e.g., RNA G-quadruplex structures, RG4) is reduced, thereby In some embodiments, the 5'UTR is engineered to have a negative folding free energy (ΔG) compared to the unengineered mRNA. In some embodiments, ΔG is at most -40, -41, -42, -43, -44, -45, -46, -47, -48, -49, -50, -51, -52, -53, -54, -55, -56, -57, -58, -59, or -60. In some embodiments, the mRNA is chemically modified in the 5'UTR or 3'UTR to promote translation efficiency. In some embodiments, the chemical modification is N 6-methyladenosine. In in vitro systems (e.g., engineered eukaryotic cells or semi-synthetic organisms), overexpression of eIF4A, a subunit of the eIF4F complex that cooperates with eIF3B and eIF4H to promote the unwinding of RNA secondary structures, increases mRNA translation efficiency. In some embodiments, knockout or knockdown of a stabilizing protein (e.g., fragile X mental retardation protein (FMRP)) that promotes mRNA secondary structure formation reduces the formation of secondary structures, thereby increasing mRNA translation efficiency. In some embodiments, a trans-acting agent (e.g., small RNA molecule, protein) is introduced into cells (e.g., eukaryotic cells) to promote mRNA translation.

[0131] In some instances, the 5'UTR and / or 3'UTR promote intracellular localization of the mRNA, thereby promoting translation of the mRNA in vivo. In some embodiments, a 3' or 5'UTR cis-acting element, such as an mRNA zipcode, is modified to suppress or enhance binding of the mRNA zipcode by a zipcode-binding protein (e.g., Staufen), thereby increasing the efficiency of mRNA translation.

[0132] Nucleic acid reagents, such as expression cassettes and / or expression vectors (e.g., for expressing a heterologous tRNA synthetase), can contain a variety of regulatory elements, including promoters, enhancers, translation initiation sequences, transcription termination sequences, and other elements. A "promoter" is generally a sequence of DNA that functions when located in a relatively fixed position relative to the transcription start site. For example, a promoter can be upstream of a nucleoside triphosphate transporter nucleic acid segment. A "promoter" contains core elements required for basic interaction of RNA polymerase and transcription factors and may also contain upstream elements and response elements. An "enhancer" generally refers to a sequence of DNA that functions at a non-fixed distance from the transcription start site and can be either 5' or 3' relative to the transcription unit. Furthermore, enhancers can be present within introns and within the coding sequence itself. They are usually between 10 and 300 nucleotides in length and function in cis. Enhancers function to increase transcription from nearby promoters. Like promoters, enhancers often contain response elements that mediate transcriptional regulation. Enhancers often determine the regulation of expression and can be used to alter or optimize expression of ORFs, including ORFs that are entirely natural or contain non-natural nucleotides.

[0133] As described above, nucleic acid reagents may also contain one or more 5' UTRs and one or more 3' UTRs. For example, expression vectors used in eukaryotic host cells (e.g., yeast, fungi, insects, plants, animals, humans, or eukaryotic cells) and prokaryotic host cells (e.g., viruses, bacteria) may contain sequences that signal transcription termination, which can affect mRNA expression. These regions may be transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated region also includes a transcription termination site. In some preferred embodiments, the transcription unit contains a polyadenylation region. One advantage of this region is that the transcribed unit is more likely to be processed and transported like mRNA. The identification and use of polyadenylation signals in expression constructs is well established. In some preferred embodiments, a homologous polyadenylation signal is used in transgene constructs. It can be used as follows.

[0134] A 5' UTR may contain one or more elements endogenous to the nucleotide sequence from which it is derived, and sometimes contains one or more exogenous elements. A 5' UTR may be derived from any suitable nucleic acid, such as genomic DNA, plasmid DNA, RNA, or mRNA, for example, from any suitable organism (e.g., virus, bacteria, yeast, fungus, plant, insect, or mammal). A skilled artisan may select appropriate elements for a 5' UTR based on the selected expression system (e.g., expression in a selected organism, or, for example, expression in a cell-free system). A 5' UTR may contain one or more of the following elements known to skilled artisans: enhancer sequences (e.g., transcription or translation), transcription initiation sites, transcription factor binding sites, translational regulatory sites, translation initiation sites, translation factor binding sites, accessory protein binding sites, feedback regulator binding sites, Pribnow boxes, TATA boxes, -35 elements, E-boxes (helix-loop-helix binding elements), ribosome binding sites, replicons, internal ribosome entry sites (IRES), silencer elements, etc. In some embodiments, the promoter element may be separated such that all 5'UTR elements necessary for proper conditional regulation are contained within the promoter element fragment, or within a functional subsequence of the promoter element fragment.

[0135] The 5'UTR in a nucleic acid reagent may contain a translational enhancer nucleotide sequence. The translational enhancer nucleotide sequence is often located between the promoter and target nucleotide sequence of the nucleic acid reagent. The translational enhancer sequence often binds to ribosomes and may be an 18S rRNA-binding ribonucleotide sequence (i.e., a 40S ribosome-binding sequence) or an internal ribosome entry sequence (IRES). The IRES generally forms an RNA scaffold with a precisely positioned RNA tertiary structure that contacts the 40S ribosomal subunit through a number of specific intermolecular interactions. Examples of ribosomal enhancer sequences are known and can be identified by a skilled artisan (e.g., Mignone et al., Nucleic Acids Research 33:D141-D146 (2005); Paulous et al., Nucleic Acids Research 31:722-733 (2003); Akbergenov et al., Nucleic Acids Research 32:239-247 (2004); Mignone et al., Genome Biology 3(3):reviews0004.1-0001.10 (2002); Gallie, Nucleic Acids Research 30:3401-3411 (2002); Shaloiko et al., DOI:10.1002 / bit.20267; and Gallie et al., Nucleic Acids Research 15:3257-3273 (1987)).

[0136] The translational enhancer sequence may be a eukaryotic sequence such as a Kozak consensus sequence or other sequence (e.g., the Hydrozoa polyp sequence, GenBank Accession No. U07128). The translational enhancer sequence may be a prokaryotic sequence such as a Shine-Dalgarno consensus sequence. In certain embodiments, the translational enhancer sequence is a viral nucleotide sequence. The translational enhancer sequence may be derived from the 5'UTR of a plant virus, such as tobacco mosaic virus (TMV), alfalfa mosaic virus (AMV); tobacco etch virus (ETV); potato virus Y (PVY); turnip mosaic (poty) virus, and pea seed-borne mosaic virus. In certain embodiments, an approximately 67-base omega sequence from TMV is included in the nucleic acid reagent as the translational enhancer sequence (e.g., lacking guanosine nucleotides and containing a 25-nucleotide poly(CAA) central region).

[0137] The 3'UTR contains one or more elements endogenous to the nucleotide sequence from which it is derived. The 3'UTR may contain a sequence encoding a nucleotide ... The 3'UTR often or may not include a polyadenosine tail, and if a polyadenosine tail is present, one or more adenosine moieties may be added to or deleted from it (e.g., about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 adenosine moieties may be added to or deleted from it).

[0138] In some embodiments, modifications of the 5' UTR and / or 3' UTR are used to alter (e.g., increase, add, decrease, or substantially eliminate) promoter activity. Altering promoter activity can then alter the activity (e.g., enzymatic activity) of a peptide, polypeptide, or protein by altering transcription of a nucleotide sequence of interest from an operably linked promoter element containing the modified 5' or 3' UTR. For example, a microorganism may be genetically engineered to express a nucleic acid reagent containing a modified 5' or 3' UTR that can add a new activity (e.g., an activity not normally found in the host organism) or, in certain embodiments, increase expression of an existing activity by increasing transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest (e.g., a homologous or heterologous nucleotide sequence of interest). In some embodiments, a microorganism may be genetically engineered to express a nucleic acid reagent containing a modified 5' or 3' UTR that can, in certain embodiments, decrease expression of an activity by reducing or substantially eliminating transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest.

[0139] Expression of a heterologous polypeptide, such as a tRNA synthetase, from an expression cassette or expression vector is controlled by any promoter capable of expression in prokaryotic or eukaryotic cells. Promoter elements are typically required for DNA and / or RNA synthesis. Promoter elements often contain a region of DNA that can promote transcription of a particular gene by providing an initiation site for synthesis of the RNA corresponding to that gene. Promoters are generally located near the genes they regulate, upstream of the genes (e.g., 5' of the genes), and in some embodiments, on the same DNA strand as the sense strand of the gene. In some embodiments, promoter elements may be isolated from genes or organisms and inserted in functional association with polynucleotide sequences to allow for altered and / or regulated expression. Non-native promoters (e.g., promoters not normally associated with a given nucleic acid sequence) used to express nucleic acids are often referred to as heterologous promoters. In certain embodiments, a heterologous promoter and / or 5'UTR may be inserted in functional association with a polynucleotide encoding a polypeptide having a desired activity as described herein. As used herein with respect to promoters, the terms "operably linked" and "operably associated" refer to the relationship between the coding sequence and the promoter element. A promoter is operably linked or functionally associated with a coding sequence when expression from the coding sequence via transcription is regulated or controlled by the promoter element. The terms "operably linked" and "in functional association" are used interchangeably herein with respect to promoter elements.

[0140] Promoters often interact with RNA polymerase. Polymerases are enzymes that catalyze the synthesis of nucleic acids using existing nucleic acid reagents. When the template is a DNA template, RNA molecules are transcribed before protein synthesis. Enzymes with polymerase activity suitable for use in the methods of the present invention include any polymerase that is active in a selected system using a selected template to synthesize a protein. In some embodiments, a promoter (e.g., a heterologous promoter), also referred to herein as a promoter element, can be operably linked to a nucleotide sequence or an open reading frame (ORF). Transcription from the promoter element can catalyze the synthesis of RNA corresponding to the nucleotide sequence or ORF sequence operably linked to the promoter, which then results in the synthesis of a desired peptide, polypeptide, or protein.

[0141] Promoter elements can be responsive to regulatory control. Promoter elements can also be regulated by selective agents. That is, transcription from a promoter element can be turned on, off, up-regulated, or down-regulated in response to changes in environmental, nutrient, or internal conditions or signals (e.g., heat-inducible promoters, light-regulated promoters, feedback-regulated promoters, hormone-influenced promoters, tissue-specific promoters, oxygen- and pH-influenced promoters, promoters responsive to selective agents (e.g., kanamycin), etc.). Promoters that are influenced by environmental, nutrient, or internal signals are often influenced by signals (direct or indirect) that bind to or near the promoter and increase or decrease expression of the target sequence under certain conditions. As with all methods disclosed herein, the inclusion of native or modified promoters can be used to alter or optimize expression of fully native ORFs (e.g., aaRSs) or ORFs containing non-natural nucleotides (e.g., mRNAs or tRNAs).

[0142] Non-limiting examples of selective agents or modulators that affect transcription from promoter elements for use in the embodiments described herein include, but are not limited to, the following: (1) nucleic acid segments that encode products that confer resistance to an otherwise toxic compound (e.g., an antibiotic); (2) nucleic acid segments that encode products that are otherwise lacking in the recipient cell (e.g., essential products, tRNA genes, auxotrophic markers); (3) nucleic acid segments that encode products that repress the activity of a gene product; (4) nucleic acid segments that encode products that can be easily identified (e.g., phenotypic markers such as antibiotics (e.g., β-lactamase), β-galactosidase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and cell surface proteins); (5) nucleic acid segments that bind products that are otherwise deleterious to cell survival and / or function; and (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments listed above. (7) nucleic acid segments that bind to a substrate-modifying product (e.g., a restriction endonuclease); (8) nucleic acid segments that can be used to isolate or identify a desired molecule (e.g., a specific protein binding site); (9) nucleic acid segments that encode specific nucleotide sequences that would not otherwise function (e.g., for PCR amplification of a subpopulation of molecules); (10) nucleic acid segments that, if not present, directly or indirectly confer resistance or sensitivity to a particular compound; (11) nucleic acid segments that encode products that are toxic or that convert relatively non-toxic compounds into toxic compounds (e.g., herpes simplex thymidine kinase, cytosine deaminase) in recipient cells; (12) nucleic acid segments that inhibit the replication, partitioning, or heritability of nucleic acid molecules that contain them; (13) nucleic acid segments that encode conditional replication functions, e.g., replication in a particular host or host cell line or under particular environmental conditions (e.g., temperature, nutrient conditions, etc.); and / or (14) nucleic acid segments that encode one or more unnatural nucleotides. Nucleic acids encoding the above mRNA or tRNA. In some embodiments, a modulating or selective agent may be added to alter the existing growth conditions to which the organism is subjected (e.g., growth in liquid culture, growth in a fermenter, growth on solid nutrient plates, etc.).

[0143] In some embodiments, modulation of promoter elements can be used to alter (e.g., increase, add, decrease, or substantially eliminate) the activity of a peptide, polypeptide, or protein (e.g., enzymatic activity, etc.). For example, a microorganism may be genetically engineered to express a nucleic acid reagent that can add a new activity (e.g., an activity not normally found in the host organism) or, in certain embodiments, increase the expression of an existing activity by increasing transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest (e.g., a homologous or heterologous nucleotide sequence of interest). In some embodiments, a microorganism may be genetically engineered to express a nucleic acid reagent that can, in certain embodiments, decrease the expression of an activity by reducing or substantially eliminating transcription from a homologous or heterologous promoter operably linked to a nucleotide sequence of interest.

[0144] Nucleic acids encoding heterologous proteins, such as tRNA synthetases, may be inserted into or used in any suitable expression system. In some embodiments, nucleic acid reagents are sometimes stably integrated into the chromosome of the host organism, or nucleic acid reagents may, in certain embodiments, be deletions of portions of the host chromosome (e.g., in genetically modified organisms, modifications of the host genome confer the ability to selectively or preferentially maintain desired organisms with genetic modifications). Such nucleic acid reagents (e.g., nucleic acids or genetically modified organisms whose modified genomes confer selectable traits on organisms) can be selected for their ability to direct the production of desired proteins or nucleic acid molecules. Optionally, nucleic acid reagents may be modified so that codons (i) use different tRNAs than those specified in the native sequence to encode the same amino acids, or (ii) encode unusual amino acids, including unconventional or unnatural amino acids (including detectably labeled amino acids).

[0145] Recombinant expression is usefully achieved using an expression cassette, which may be part of a vector such as a plasmid. The vector may include a promoter operably linked to the nucleic acid. The vector may also include other elements necessary for transcription and translation, as described herein. The expression cassette, expression vector, and sequences in the cassette or vector may be heterologous to the cell in contact with the non-natural nucleotide.

[0146] A variety of prokaryotic and eukaryotic expression vectors suitable for carrying, encoding, and / or expressing heterologous proteins, such as tRNA synthetase, can be generated. Examples of such expression vectors include pET, pET3d, pCR2.1, pBAD, pUC, and yeast vectors. The vectors can be used, for example, in a variety of in vivo and in vitro contexts. Non-limiting examples of prokaryotic promoters that can be used include SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters. Non-limiting examples of eukaryotic promoters that can be used include constitutive promoters, such as viral promoters, such as CMV, SV40, and RSV promoters, and regulatable promoters, such as inducible or repressible promoters, such as tet promoters, hsp70 promoters, and synthetic promoters regulated by CRE. Examples of vectors for bacterial expression include pGEX-5X-3, and examples of vectors for eukaryotic expression include pCIneo-CMV. Viral vectors that can be used include those related to lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polio viruses, AIDS viruses, neurotrophic viruses, Sindbis and other viruses, and any virus that shares the properties of these viruses and is suitable for use as a vector. Retroviral vectors that can be used include those described in Verma, American Society for Microbiology, pp. 229-232, Washington, (1985). For example, such retroviral vectors include murine Maloney leukemia virus (MMLV), and other retroviruses that express desired characteristics. Typically, viral vectors contain nonstructural early genes, structural late genes, RNA polymerase III transcripts, inverted terminal repeats necessary for replication and encapsidation, and promoters that control the transcription and replication of the viral genome. When designed as a vector, viruses usually have one or more early genes removed, and a gene or gene / promoter cassette is inserted into the viral genome in place of the removed viral nucleic acid.

[0147] Cloning Any convenient cloning strategy known in the art may be used to incorporate elements such as ORFs into a nucleic acid reagent. Elements may be inserted into a template independently of the inserted element using known methods, such as (1) cleaving the template at one or more existing restriction enzyme sites and ligating the desired element, and (2) adding restriction enzyme sites to the template by hybridizing oligonucleotide primers containing one or more appropriate restriction enzyme sites and amplifying by polymerase chain reaction (described in more detail herein). Other cloning strategies utilize one or more insertion sites present in or inserted into the nucleic acid reagent, such as oligonucleotide primer hybridization sites for PCR and others described herein. In some embodiments, a cloning strategy may be combined with genetic manipulation, such as recombination (e.g., recombination of a nucleic acid reagent bearing a nucleic acid sequence of interest into the genome of an organism to be modified, as further described herein). In some embodiments, the cloned ORFs can be used to generate modified or wild-type polymerases (directly or indirectly) by engineering a microorganism with one or more ORFs of interest, such that the microorganism contains an altered polymerase activity.

[0148] A nucleic acid can be specifically cleaved by contacting the nucleic acid with one or more specific cleaving agents, which often specifically cleave at specific sites and according to specific nucleotide sequences. Examples of enzyme-specific cleaving agents include, but are not limited to, endonucleases (e.g., DNases (e.g., DNase I, II); RNases (e.g., RNase E, F, H, P); Cleavase™ enzyme; Taq DNA polymerase; E. coli DNA polymerase I and eukaryotic structure-specific endonucleases; mouse FEN-1 endonuclease; type I, type II, or type III restriction endonucleases, such as Acc I, Afl III, Alu I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II, Bcl I, Bgl I, Bgl II, Bln I, Bsa I, Bsm I, BsmBI, BssH II, BstE II, Cfo I, CIa I, Dde I, Dpn I, Dra I, EcIX I, EcoR I, EcoR II, EcoR V, Hae II, Hae II, Hind II, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MIuN I, Msp I, Nci I, Nco I, Nde I, Nde II, Nhe I, Not I, Nru I, Nsi I, Pst I, Pvu I, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi I, Sma I, Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho I); glycosylases (e.g., uracil-DNA glycosylase (UDG), 3-methyladenine DNA glycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNA glycosylase, FaPy-DNA glycosylase, Examples of suitable enzymes include 5-hydroxymethyluracil DNA glycosylase, thymine mismatch-DNA glycosylase, hypoxanthine-DNA glycosylase, 5-hydroxymethyluracil DNA glycosylase (HmUDG), 5-hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenine DNA glycosylase; exonucleases (e.g., exonuclease III); ribozymes; and DNAzymes. The sample nucleic acid may be treated with chemicals or synthesized using modified nucleotides, and the modified nucleic acid may be cleaved. In a non-limiting example, the sample nucleic acid can be treated with (i) an alkylating agent such as methylnitrosourea, which generates several alkylated bases, including N3-methyladenine and N3-methylguanine, which are recognized and cleaved by alkylpurine DNA glycosylase; (ii) sodium bisulfite, which deaminates cytosine residues in DNA to form uracil residues that can be cleaved by uracil N-glycosylase; and (iii) a chemical agent that converts guanine to its oxidized form, 8-hydroxyguanine, which can be cleaved by formamidopyrimidine DNA N-glycosylase. Examples of chemical cleavage processes include, but are not limited to, alkylation (e.g., alkylation of phosphorothioate-modified nucleic acids); acid-labile cleavage of P3'-N5'-phosphoramidate-containing nucleic acids; and osmium tetroxide and piperidine treatment of nucleic acids.

[0149] In some embodiments, the nucleic acid reagent comprises one or more recombinase insertion sites. Recombinase insertion sites are recognition sequences on nucleic acid molecules that participate in integration / recombination reactions by recombination proteins. For example, the recombination site for Cre recombinase is loxP, a 34-base pair sequence consisting of two 13-base pair inverted repeats (which function as recombinase binding sites) flanking an 8-base pair core sequence (e.g., Sauer, Curr. Opin. Biotech. 5:521-527 (1994)). Other examples of recombination sites include the attB, attP, attL, and attR sequences, as well as mutants, fragments, variants, and derivatives thereof, recognized by the recombination protein λInt and by the auxiliary proteins integration host factor (IHF), FIS, and excision enzyme (Xis) (e.g., U.S. Pat. Nos. 5,888,732; 6,143,557; 6,171,861; 6,270,969; 6,277,608; and 6,720,140; U.S. Patent Application Publication Nos. 09 / 517,466 and 09 / 732,914; U.S. Patent Application Publication No. 2002 / 0007051; and Landy, Curr. Opin. Biotech. 3:699-707 (1993)).

[0150] An example of a recombinase cloning nucleic acid is the Gateway® system (Invitrogen, California), which contains at least one recombination site for cloning a desired nucleic acid molecule in vivo or in vitro. In some embodiments, this system utilizes a vector containing at least two different site-specific recombination sites, often based on the bacteriophage lambda system (e.g., att1 and att2), mutated from the wild-type (att0) site. Each mutated site has unique specificity for its cognate partner att site (i.e., its binding partner recombination site) of the same type (e.g., attB1 and attP1, or attL1 and attR1) and does not cross-react with other mutated recombination sites or with the wild-type att0 site. The different site specificities allow for directional cloning or ligation of the desired molecule, thus providing a desired orientation of the cloned molecule. Nucleic acid fragments flanked by recombination sites are cloned and subcloned using the Gateway® system by replacing selectable markers (e.g., ccdB) flanking att sites on a recipient plasmid molecule, sometimes called the Destination Vector. Desired clones are then isolated by transformation of a ccdB-sensitive host strain and positive selection for the marker on the recipient molecule. Similar strategies for negative selection (e.g., the use of toxic genes) can be used in other organisms, such as thymidine kinase (TK) in mammals and insects.

[0151] A nucleic acid reagent may contain one or more origin of replication (ORI) elements. In some embodiments, a template contains two or more ORIs, one that functions efficiently in one organism (e.g., bacteria) and another that functions efficiently in another organism (e.g., a eukaryote such as yeast). In some embodiments, an ORI may function efficiently in one species (e.g., S. cerevisiae, etc.) and another ORI may function efficiently in a different species (e.g., S. pombe, etc.). A nucleic acid reagent may also contain one or more transcriptional regulatory sites.

[0152] A nucleic acid reagent, such as an expression cassette or vector, can contain a nucleic acid sequence encoding a marker product. The marker product is used to determine whether a gene has been delivered to a cell and is expressed after delivery. Examples of marker genes include the E. coli lacZ gene, which encodes β-galactosidase and green fluorescent protein. In some embodiments, the marker can be a selectable marker. When such a selectable marker is successfully transferred to a host cell, the transformed host cell can survive when placed under selective pressure. There are two widely used distinct categories of selection regimes. The first category is based on cellular metabolism and the use of mutant cell lines that lack the ability to grow independently of supplemented media. The second category is dominant selection, which refers to a selection scheme used with any cell type and does not require the use of mutant cell lines. These schemes typically use drugs to inhibit host cell growth. Those cells carrying the novel gene express a protein conferring drug resistance and survive selection. Examples of such dominant selection use the drugs neomycin (Southern et al., J. Molec. Appl. Genet. 1:327 (1982)), mycophenolic acid (Mulligan et al., Science 209:1422 (1980)), or hygromycin (Sugden et al., Mol. Cell. Biol. 5:410-413 (1985)).

[0153] A nucleic acid reagent can contain one or more selection elements (e.g., an element for selecting for the presence of the nucleic acid reagent, but not for the activation of a promoter element that can be selectively regulated). Selection elements are often utilized using known processes to determine whether a nucleic acid reagent is included in a cell. In some embodiments, a nucleic acid reagent contains two or more selection elements, one element that functions efficiently in one organism and another element that functions efficiently in another organism. Examples of selection elements include, but are not limited to: (1) a nucleic acid segment that encodes a product that provides resistance to an otherwise toxic compound (e.g., an antibiotic); (2) a nucleic acid segment that encodes a product that is otherwise lacking in the recipient cell (e.g., an essential product, a tRNA gene, an auxotrophic marker); (3) a nucleic acid segment that encodes a product that suppresses the activity of a gene product; (4) a nucleic acid segment that encodes a product that can be easily identified (e.g., an antibiotic (e.g., β-lactamase), β-galactosidase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP)). (5) nucleic acid segments that bind to products that are otherwise detrimental to cell survival and / or function; (6) nucleic acid segments that otherwise inhibit the activity of any of the nucleic acid segments listed above (e.g., antisense oligonucleotides); (7) nucleic acid segments that bind to products that modify substrates (e.g., restriction endonucleases); (8) nucleic acid segments that can be used to isolate or identify desired molecules (e.g., specific protein binding sites); (9) nucleic acid segments that encode specific nucleotide sequences that may not otherwise function (e.g., molecules (10) nucleic acid segments that, when absent, directly or indirectly confer resistance or sensitivity to a particular compound; (11) nucleic acid segments that encode products that are toxic or that convert relatively non-toxic compounds into toxic compounds in recipient cells (e.g., herpes simplex thymidine kinase, cytosine deaminase); (12) nucleic acid segments that inhibit the replication, partitioning, or heritability of nucleic acid molecules that contain them; and / or (13) nucleic acid segments that encode conditional replication functions, e.g., replication in a particular host or host cell line or under particular environmental conditions (e.g., temperature, nutrient status, etc.).

[0154] Nucleic acid reagents can be in any form useful for in vivo transcription and / or translation. Nucleic acids can be plasmids, such as supercoiled plasmids, yeast artificial chromosomes (e.g., YACs), linear nucleic acids (e.g., linear nucleic acids produced by PCR or restriction digestion), single-stranded, or sometimes double-stranded. Nucleic acid reagents can also be prepared by amplification processes, such as polymerase chain reaction (PCR) or transcription-mediated amplification (TMA). In TMA, two enzymes are used in an isothermal reaction to generate amplification products that are detected by luminescence (e.g., Biochemistry 1996 Jun 25;35(25):8429-38). Standard PCR processes are known (e.g., U.S. Patent Nos. 4,683,202; 4,683,195; 4,965,188; and 5,565,493), and are generally performed in cycles. Each cycle includes heat denaturation (where hybrid nucleic acids dissociate), cooling (where primer oligonucleotides hybridize), and oligonucleotide extension by a polymerase (i.e., Taq polymerase). An example of a PCR cycling process is treating a sample at 95°C for 5 minutes; repeating 45 cycles of 95°C for 1 minute, 59°C for 1 minute and 10 seconds, and 72°C for 1 minute and 30 seconds; then treating the sample at 72°C for 5 minutes. Multiple cycles are often performed using commercially available thermal cyclers. PCR amplification products may be temporarily stored at low temperatures (e.g., 4°C) or frozen (e.g., -20°C) before analysis.

[0155] DNA containing unnatural nucleotides can be generated using cloning strategies similar to those described above. For example, an oligonucleotide containing an unnatural nucleotide at the desired position is synthesized using standard solid-phase synthesis and purified by HPLC. The oligonucleotide is then inserted into a plasmid containing the desired sequence context (i.e., UTR and coding sequence) using a cloning method (such as Golden Gate assembly) using a cloning site such as the BsaI site (although others as described above may also be used).

[0156] Kit / manufactured product In certain embodiments, disclosed herein are kits and articles of manufacture for use in one or more of the methods described herein. Such kits include a carrier, package, or container compartmentalized to receive one or more containers, such as vials, tubes, etc., each of which contains one of the distinct elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials, such as glass or plastic.

[0157] In some embodiments, the kit includes suitable packaging materials for housing the contents of the kit. In some cases, the packaging materials are constructed by well-known methods, preferably to provide a sterile and contaminant-free environment. Packaging materials as used herein include, for example, those conventionally utilized in commercially available kits sold for use with nucleic acid sequencing systems. Exemplary packaging materials include, but are not limited to, the packaging materials used in the present invention. Examples include glass, plastic, paper, foil, etc., which are capable of retaining the components described in the specification within a certain range.

[0158] The packaging material may include a label indicating the specific use of the components. The use of the kit indicated by the label may be one or more of the methods described herein that are appropriate for the particular combination of components present in the kit. For example, the label may indicate that the kit is useful for a method of synthesizing polynucleotides or for a method of sequencing nucleic acids.

[0159] Instructions for use of the packaged reagents or components may also be included in the kit, and typically include specific wording describing reaction parameters such as the relative amounts of kit components and sample to be mixed, maintenance periods for the reagent / sample mixture, temperature, buffer conditions, etc.

[0160] It is understood that not all components required for a particular reaction need be present in a particular kit. Rather, one or more additional components may be provided from other sources. Instructions accompanying the kit may identify the additional components provided and where they can be obtained.

[0161] In some embodiments, kits are provided that are useful for stably incorporating non-native nucleic acids into cellular nucleic acids, for example, using the methods provided by the present invention for producing genetically engineered mammalian cells (e.g., CHO or HEK293T cells). In one embodiment, the kits described herein include genetically engineered cells and one or more non-native nucleic acids.

[0162] In additional embodiments, the kits described herein provide cells and nucleic acid molecules comprising heterologous genes for introduction into the cells, thereby providing genetically engineered cells, such as expression vectors comprising the nucleic acids of any of the above embodiments described in this paragraph.

[0163] In some embodiments, the cells described herein are delivered to an organism, which may be a multicellular organism such as a mammal, e.g., a human. As such, eukaryotic cells containing a polypeptide with an unnatural amino acid can be introduced into the organism.

[0164] Numbered Embodiments The present disclosure includes the following non-limiting numbered embodiments: Embodiment 1. A method for producing a polypeptide comprising one or more unnatural amino acids in a eukaryotic cell, comprising: (a) (i) a transfer RNA (tRNA) having an anticodon that includes a first unnatural base; (ii) providing a eukaryotic cell comprising a messenger RNA (mRNA) having a codon that includes a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryotic cell; (b) translating a polypeptide comprising one or more unnatural amino acids from the mRNA using tRNA by a ribosome endogenous to the eukaryotic cell; A method comprising:

[0165] Embodiment 2. The method of embodiment 1, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the first position (XNN) in the codon of the mRNA.

[0166] Embodiment 3. The method of embodiment 1, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the central position (NXN) in the codon of the mRNA.

[0167] Embodiment 4. The method of embodiment 1, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the last position (NNX) in the codon of the mRNA.

[0168] Embodiment 5. The first unnatural base or the second unnatural base is: (i) 2-thiouracil, 2-thio-thymine, 2'-deoxyuridine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl(I), 5-halouracil; 5-propynyl-uracil, 6-azo-thymine, 6-azo-uracil, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, pseudouracil, uracil-5-oxaacetic acid methyl ester, uracil-5-oxaacetic acid, 5- methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil or dihydrouracil; (ii) 5-hydroxymethylcytosine, 5-trifluoromethylcytosine, 5-halocytosine, 5-propynylcytosine, 5-hydroxycytosine, cyclocytosine, cytosine arabinoside, 5,6-dihydrocytosine, 5-nitrocytosine, 6-azocytosine, azacytosine, N4-ethylcytosine, 3-methylcytosine, 5-methylcytosine, 4-acetylcytosine, 2-thiocytosine, phenoxazine cytidine ([5,4-b][1,4]benzoxazine-2 (3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one) or pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one); (iii) 2-aminoadenine, 2-propyladenine, 2-amino-adenine, 2-F-adenine, 2-amino-propyl-adenine, 2-amino-2'-deoxyadenosine, 3-deazaadenine, 7-methyladenine, 7-deaza-adenine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl and 8-hydroxyl substituted adenines, N6-isopentenyladenine, 2-methyladenine, 2,6-diaminopurine, 2-methylthio-N6-isopentenyladenine or 6-aza-adenine; (iv) 2-methylguanine, 2-propyl and alkyl derivatives of guanine, 3-deazaguanine, 6-thio-guanine, 7-methylguanine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-8-azaguanine, 8-azaguanine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl and 8-hydroxyl substituted guanines, 1-methylguanine, 2,2-dimethylguanine, 7-methylguanine or 6-aza-guanine; and (v) The method of any one of embodiments 1 to 4, wherein the compound is selected from the group consisting of hypoxanthine, xanthine, 1-methylinosine, queuosine, beta-D-galactosylqueuosine, inosine, beta-D-mannosylqueuosine, wybutoxosine, hydroxyurea, (acp3)w, 2-aminopyridine, or 2-pyridone.

[0169] Embodiment 6. The first unnatural base or the second unnatural base is: [ka] 5. The method of any one of embodiments 1 to 4, wherein the wavy line indicates the bond to the ribosyl moiety.

[0170] Embodiment 7. The first unnatural base is: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 7. The method of embodiment 6, wherein the wavy line indicates the bond to the ribosyl moiety.

[0171] Embodiment 8. The first unnatural base is: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0172] Embodiment 9. The first unnatural base is: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 7. The method of embodiment 6, wherein the wavy line indicates the bond to the ribosyl moiety.

[0173] Embodiment 10. The first unnatural base is: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 7. The method of embodiment 6, wherein the wavy line indicates the bond to the ribosyl moiety.

[0174] Embodiment 11. The first unnatural base is: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 7. The method of embodiment 6, wherein the wavy line indicates the bond to the ribosyl moiety.

[0175] Embodiment 12. The first unnatural base is: [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 7. The method of embodiment 6, wherein the wavy line indicates the bond to the ribosyl moiety.

[0176] Embodiment 13. The first unnatural base or the second unnatural base is Modifications at the 2' position: OH, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2'-F, 2'-OCH, 2'-O(CH)OCH, (wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 , alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -NH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10; and / or modifications at the 5' position: 5'-vinyl, 5'-methyl (R or S); Modification at position 4: 13. The method of any one of embodiments 1-12, comprising a modified sugar moiety selected from the group consisting of 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide or a group for improving the pharmacodynamic properties of an oligonucleotide, and any combination thereof.

[0177] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the cells are human cells.

[0178] Embodiment 15. The method of embodiment 14, wherein the human cell is a HEK293T cell.

[0179] Embodiment 16. The method of any one of embodiments 1 to 13, wherein the cell is a hamster cell.

[0180] Embodiment 17. The method of embodiment 16, wherein the hamster cell is a Chinese hamster ovary (CHO) cell.

[0181] Embodiment 18. The unnatural amino acid is: It is a lysine analog; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or 18. The method of any one of embodiments 1 to 17, comprising an aldehyde or ketone group.

[0182] Embodiment 19. The unnatural amino acid is selected from the group consisting of N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fulvastatin, and the like. Fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenyl 18. The method of any one of embodiments 1 to 17, wherein the hydroxybenzoate is selected from the group consisting of N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0183] Embodiment 20. The method of embodiment 19, wherein the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK).

[0184] Embodiment 21. A method of producing a polypeptide in a eukaryotic cell, wherein the polypeptide comprises one or more unnatural amino acids, the method comprising: (a) providing a eukaryotic organism, the eukaryotic organism comprising: (i) an mRNA containing a codon containing one or more unnatural bases; (ii) a tRNA comprising an anticodon comprising one or more unnatural bases, wherein the one or more unnatural bases comprising the codon in the mRNA and the one or more unnatural bases comprising the anticodon in the tRNA form complementary base pairs; (iii) a tRNA synthetase that preferentially aminoacylates the tRNA with one or more unnatural amino acids relative to natural amino acids; and (b) providing one or more unnatural amino acids to a eukaryotic cell, wherein the eukaryotic cell produces a polypeptide comprising the one or more unnatural amino acids.

[0185] Embodiment 22. The method of embodiment 21, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the first position (XNN) in the codon of the mRNA.

[0186] Embodiment 23. The method of embodiment 21, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the central position (NXN) in the codon of the mRNA.

[0187] Embodiment 24. The method of embodiment 21, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the last position (NNX) in the codon of the mRNA.

[0188] Embodiment 25. The one or more unnatural base containing codons in an mRNA are of the formula [ka] or [ka] wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide, and the wavy line indicates the bond to the ribosyl moiety.

[0189] Embodiment 26. The first unnatural base or the second unnatural base is [ka] 25. The method of any one of embodiments 21-24, wherein the wavy line indicates the bond to the ribosyl moiety.

[0190] Embodiment 27. The first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] then the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0191] Embodiment 28. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0192] Embodiment 29. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0193] Embodiment 30. The first unnatural base is [ka] then the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0194] Embodiment 31. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0195] Embodiment 32. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0196] Embodiment 33. The first unnatural base is [ka] and the second unnatural base is [ka] 27. The method of embodiment 26, wherein the wavy line indicates the bond to the ribosyl moiety.

[0197] Embodiment 34. The non-natural nucleotide containing codon in mRNA is [ka] 25. The method of any one of embodiments 21-24, wherein the wavy line indicates the bond to the ribosyl moiety.

[0198] Embodiment 35. The non-natural nucleotide containing codon in mRNA is [ka] 35. The method of embodiment 34, wherein the wavy line indicates the bond to the ribosyl moiety.

[0199] Embodiment 36. The non-natural nucleotide containing codon in mRNA is [ka] 35. The method of embodiment 34, wherein the wavy line indicates the bond to the ribosyl moiety.

[0200] Embodiment 37. The non-natural nucleotide containing codon in mRNA is [ka] 35. The method of embodiment 34, wherein the wavy line indicates the bond to the ribosyl moiety.

[0201] Embodiment 38. The codon of the mRNA comprises three consecutive nucleobases (NNN), and an unnatural base (X) is located at the first position (XNN) in the codon of the mRNA, and the unnatural base is [ka] 22. The method of embodiment 21, wherein the wavy line indicates the bond to the ribosyl moiety.

[0202] Embodiment 39. The unnatural base is [ka] 39. The method of embodiment 38, wherein the wavy line indicates the bond to the ribosyl moiety.

[0203] Embodiment 40. The unnatural base is [ka] 39. The method of embodiment 38, wherein the wavy line indicates the bond to the ribosyl moiety.

[0204] Embodiment 41. The unnatural base is [ka] 39. The method of embodiment 38, wherein the wavy line indicates the bond to the ribosyl moiety.

[0205] Embodiment 42. The codon of the mRNA comprises three consecutive nucleobases (NNN), and the unnatural base (X) is located at the central position (NXN) in the codon of the mRNA, and the unnatural base is [ka] 22. The method of embodiment 21, wherein the wavy line indicates the bond to the ribosyl moiety.

[0206] Embodiment 43. The unnatural base is [ka] 43. The method of embodiment 42, wherein the wavy line indicates the bond to the ribosyl moiety.

[0207] Embodiment 44. The unnatural base is [ka] 43. The method of embodiment 42, wherein the wavy line indicates the bond to the ribosyl moiety.

[0208] Embodiment 45. The unnatural base is [ka] 43. The method of embodiment 42, wherein the wavy line indicates the bond to the ribosyl moiety.

[0209] Embodiment 46. The codon of the mRNA comprises three consecutive nucleobases (NNN), and the unnatural base (X) is located at the last position (NNX) in the codon of the mRNA, and the unnatural base is [ka] 22. The method of embodiment 21, wherein the wavy line indicates the bond to the ribosyl moiety.

[0210] Embodiment 47. The unnatural base is [ka] 47. The method of embodiment 46, wherein the wavy line indicates the bond to the ribosyl moiety.

[0211] Embodiment 48. The unnatural base is [ka] 47. The method of embodiment 46, wherein the wavy line indicates the bond to the ribosyl moiety.

[0212] Embodiment 49. The unnatural base is [ka] 47. The method of embodiment 46, wherein the wavy line indicates the bond to the ribosyl moiety.

[0213] Embodiment 50. The method of embodiment 21, wherein the anticodon of the tRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the first position (XNN) in the anticodon of the tRNA.

[0214] Embodiment 51. The unnatural base is [ka] 51. The method of embodiment 50, wherein the wavy line indicates the bond to the ribosyl moiety.

[0215] Embodiment 52. The unnatural base is [ka] 52. The method of embodiment 51, wherein the wavy line indicates the bond to the ribosyl moiety.

[0216] Embodiment 53. The unnatural base is [ka] 52. The method of embodiment 51, wherein the wavy line indicates the bond to the ribosyl moiety.

[0217] Embodiment 54. The unnatural base is [ka] 52. The method of embodiment 51, wherein the wavy line indicates the bond to the ribosyl moiety.

[0218] Embodiment 55. The method of embodiment 21, wherein the anticodon of the tRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the central position (NXN) in the anticodon of the tRNA.

[0219] Embodiment 56. The unnatural base is [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0220] Embodiment 57. The unnatural base is [ka] 56. The method of embodiment 55, wherein the wavy line indicates the bond to the ribosyl moiety.

[0221] Embodiment 58. The unnatural base is [ka] 56. The method of embodiment 55, wherein the wavy line indicates the bond to the ribosyl moiety.

[0222] Embodiment 59. The unnatural base is [ka] 56. The method of embodiment 55, wherein the wavy line indicates the bond to the ribosyl moiety.

[0223] Embodiment 60. The method of embodiment 21, wherein the anticodon of the tRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the last position (NNX) in the anticodon of the tRNA.

[0224] Embodiment 61. The unnatural base is [ka] 61. The method of embodiment 60, wherein the wavy line indicates the bond to the ribosyl moiety.

[0225] Embodiment 62. The unnatural base is [ka] 62. The method of embodiment 61, wherein the wavy line indicates the bond to the ribosyl moiety.

[0226] Embodiment 63. The unnatural base is [ka] 62. The method of embodiment 61, wherein the wavy line indicates the bond to the ribosyl moiety.

[0227] Embodiment 64. The unnatural base is [ka] 62. The method of embodiment 61, wherein the wavy line indicates the bond to the ribosyl moiety.

[0228] Embodiment 65. The method of embodiment 21, wherein the codon and anticodon each comprise three consecutive nucleobases (NNN), the codon in the mRNA comprises a first unnatural base (X) located at the first position of the codon (XNN), and the anticodon in the tRNA comprises a second unnatural base (Y) located at the last position of the anticodon (NNY).

[0229] Embodiment 66. The method of embodiment 65, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different.

[0230] Embodiment 67. The method of embodiment 66, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are identical.

[0231] Embodiment 68. The method of embodiment 66, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are different.

[0232] Embodiment 69. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are: [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0233] Embodiment 70. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are: [ka] 70. The method of embodiment 69, wherein the ribosyl moiety is selected from the group consisting of:

[0234] Embodiment 71. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 71. The method of embodiment 70, wherein the wavy line indicates the bond to the ribosyl moiety.

[0235] Embodiment 72. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 71. The method of embodiment 70, wherein the wavy line indicates the bond to the ribosyl moiety.

[0236] Embodiment 73. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 71. The method of embodiment 70, wherein the wavy line indicates the bond to the ribosyl moiety.

[0237] Embodiment 74. The first unnatural base (X) located in the codon of the mRNA is [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from [ka] and in each case the wavy line indicates a bond to a ribosyl moiety.

[0238] Embodiment 75. The first unnatural base (X) located in the codon of the mRNA is [ka] 75. The method of embodiment 74, wherein

[0239] Embodiment 76. The first unnatural base (X) located in the codon of the mRNA is [ka] 75. The method of embodiment 74, wherein

[0240] Embodiment 77. The method of embodiment 21, wherein the codon and anticodon each comprise three consecutive nucleobases (NNN), the codon in the mRNA comprises a first unnatural base (X) located in the middle position of the codon (NXN), and the anticodon in the tRNA comprises a second unnatural base (Y) located in the middle position of the anticodon (NYN).

[0241] Embodiment 78. The method of embodiment 77, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different.

[0242] Embodiment 79. The method of embodiment 78, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are identical.

[0243] Embodiment 80. The method of embodiment 78, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are different.

[0244] Embodiment 81. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are: [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0245] Embodiment 82. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are: [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0246] Embodiment 83. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 83. The method of embodiment 82, wherein the wavy line indicates the bond to the ribosyl moiety.

[0247] Embodiment 84. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 83. The method of embodiment 82, wherein the wavy line indicates the bond to the ribosyl moiety.

[0248] Embodiment 85. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 83. The method of embodiment 82, wherein the wavy line indicates the bond to the ribosyl moiety.

[0249] Embodiment 86. The first unnatural base (X) located in the codon of the mRNA is [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from [ka] and in each case the wavy line indicates a bond to a ribosyl moiety.

[0250] Embodiment 87. The first unnatural base (X) located in the codon of the mRNA is [ka] 87. The method of embodiment 86, wherein

[0251] Embodiment 88. The first unnatural base (X) located in the codon of the mRNA is [ka] 87. The method of embodiment 86, wherein

[0252] Embodiment 89. The method of embodiment 21, wherein the codon and anticodon each comprise three consecutive nucleobases (NNN), the codon in the mRNA comprises a first unnatural base (X) located at the last position of the codon (NNX), and the anticodon in the tRNA comprises a second unnatural base (Y) located at the first position of the anticodon (YNN).

[0253] Embodiment 90. The method of embodiment 89, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different.

[0254] Embodiment 91. The method of embodiment 89, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are identical.

[0255] Embodiment 92. The method of embodiment 89, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are different.

[0256] Embodiment 93. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are: [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0257] Embodiment 94. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are: [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0258] Embodiment 95. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 95. The method of embodiment 94, wherein the wavy line indicates the bond to the ribosyl moiety.

[0259] Embodiment 96. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 95. The method of embodiment 94, wherein the wavy line indicates the bond to the ribosyl moiety.

[0260] Embodiment 97. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both [ka] 95. The method of embodiment 94, wherein the wavy line indicates the bond to the ribosyl moiety.

[0261] Embodiment 98. The first unnatural base (X) located in the codon of the mRNA is [ka] and the second unnatural base (Y) located in the anticodon of the tRNA is selected from [ka] and in each case the wavy line indicates a bond to a ribosyl moiety.

[0262] Embodiment 99. The first unnatural base (X) located in the codon of the mRNA is [ka] 99. The method of embodiment 98, wherein

[0263] Embodiment 100. The first unnatural base (X) located in the codon of the mRNA is [ka] 99. The method of embodiment 98, wherein

[0264] Embodiment 101. The method of any one of embodiments 21, 23, 25-37, 42-45, 55-59, and 77-88, wherein the codon in the mRNA is selected from AXC, GXC, or GXU, and X is an unnatural base.

[0265] Embodiment 102. The method of embodiment 101, wherein the codon in the mRNA is AXC, and X is an unnatural base.

[0266] Embodiment 103. The method of embodiment 101, wherein the codon in the mRNA is GXC, and X is an unnatural base.

[0267] Embodiment 104. The method of embodiment 101, wherein the codon in the mRNA is GXU, and X is an unnatural base.

[0268] Embodiment 105. The method of any one of embodiments 21, 23, 25-37, 42-45, 55-59, and 77-88, wherein the codon in the mRNA is selected from AXC, GXC, or GXU, and the anticodon in the tRNA is selected from GYU, GYC, and AYC, wherein X is a first unnatural base and Y is a second unnatural base.

[0269] Embodiment 106. The method of embodiment 105, wherein X and Y are the same or different.

[0270] Embodiment 107. The method of embodiment 106, wherein X and Y are the same.

[0271] Embodiment 108. The method of embodiment 106, wherein X and Y are different.

[0272] Embodiment 109. The method of embodiment 105, wherein the codon in the mRNA is AXC and the anticodon in the tRNA is GYU.

[0273] Embodiment 110. The method of embodiment 109, wherein X and Y are the same or different.

[0274] Embodiment 111. The method of embodiment 109, wherein X and Y are the same.

[0275] Embodiment 112. The method of embodiment 109, wherein X and Y are different.

[0276] Embodiment 113. The method of embodiment 106, wherein the codon in the mRNA is GXC and the anticodon in the tRNA is GYC.

[0277] Embodiment 114. The method of embodiment 113, wherein X and Y are the same or different.

[0278] Embodiment 115. The method of embodiment 113, wherein X and Y are the same.

[0279] Embodiment 116. The method of embodiment 113, wherein X and Y are different.

[0280] Embodiment 117. The method of embodiment 106, wherein the codon in the mRNA is GXU and the anticodon is AYC.

[0281] Embodiment 118. The method of embodiment 117, wherein X and Y are the same or different.

[0282] Embodiment 119. The method of embodiment 117, wherein X and Y are the same.

[0283] Embodiment 120. The method of embodiment 117, wherein X and Y are different.

[0284] Embodiment 121. The method of any one of embodiments 21 to 120, wherein the tRNA is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

[0285] Embodiment 122. The method of any one of embodiments 21 to 120, wherein the tRNA synthetase is derived from Methanococcus jannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

[0286] Embodiment 123. The method of embodiment 122, wherein the tRNA and tRNA synthetase are derived from Methanococcus yannaschii.

[0287] Embodiment 124. The method of embodiment 122, wherein the tRNA and tRNA synthetase are derived from Methanosarcina barkeri.

[0288] Embodiment 125. The method of embodiment 122, wherein the tRNA and tRNA synthetase are derived from Methanosarcina mazei.

[0289] Embodiment 126. The method of embodiment 122, wherein the tRNA and tRNA synthetase are derived from Methanosarcina acetivorans.

[0290] Embodiment 127. The method of any one of embodiments 21 to 120, wherein the tRNA is derived from Methanococcus yannaschii and the tRNA synthetase is derived from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

[0291] Embodiment 128. The tRNA is derived from Methanosarcina barkeri and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina mazei or Methanosarcina The method according to any one of embodiments 21 to 120, wherein the acetylcholinesterase inhibitor is derived from acetylcholinesterase inhibitor ....

[0292] Embodiment 129. The method of any one of embodiments 21 to 120, wherein the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina acetivorans.

[0293] Embodiment 130. The method of any one of embodiments 21 to 120, wherein the tRNA is derived from Methanosarcina acetivorans and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina mazei.

[0294] Embodiment 131. The method of any one of embodiments 21 to 120, wherein the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanosarcina barkeri.

[0295] Embodiment 132. The method of any one of embodiments 21 to 120, wherein the cell is a human cell.

[0296] Embodiment 133. The method of embodiment 132, wherein the human cell is a HEK293T cell.

[0297] Embodiment 134. The method of any one of embodiments 21 to 120, wherein the cell is a hamster cell.

[0298] Embodiment 135. The method of embodiment 134, wherein the hamster cell is a Chinese hamster ovary (CHO) cell.

[0299] Embodiment 136. The unnatural amino acid is: It is a lysine analog; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or The method of any one of embodiments 21 to 135, comprising an aldehyde or ketone group.

[0300] Embodiment 137. The unnatural amino acid is selected from the group consisting of N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, Fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl)amino)ethyl)selanyl)propanoic acid, 2-amino-3-(phenyl) The method of any one of embodiments 21 to 135, wherein the hydroxybenzoate is selected from the group consisting of N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0301] Embodiment 138. The method of embodiment 137, wherein the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK).

[0302] Embodiment 139. A system for expression of a non-naturally occurring polypeptide in a eukaryotic cell, comprising: (a) at least one unnatural amino acid; (b) an mRNA encoding a non-natural polypeptide, the mRNA comprising at least one codon comprising one or more first non-natural bases; (c) a tRNA comprising at least one anticodon comprising one or more second unnatural bases, wherein the one or more first unnatural bases and the one or more second unnatural bases form one or more complementary base pairs; (d) one or more nucleic acid constructs comprising a nucleic acid sequence encoding a tRNA synthetase that preferentially aminoacylates tRNA with at least one unnatural amino acid; and (e) a eukaryotic cell that is capable of translating mRNA into a polypeptide containing an unnatural amino acid using tRNA and tRNA synthetase A system containing

[0303] Embodiment 140. The system of embodiment 139, wherein at least one codon of the mRNA comprises three consecutive nucleobases (NNN); and one or more first unnatural bases (X) are located at a first position (XNN) in at least one codon of the mRNA.

[0304] Embodiment 141. The system of embodiment 139, wherein at least one codon of the mRNA comprises three consecutive nucleobases (NNN); and one or more first unnatural bases (X) are located at central positions (NXN) in the codon of the mRNA.

[0305] Embodiment 142. The system of embodiment 139, wherein at least one codon of the mRNA comprises three consecutive nucleobases (NNN); and one or more first unnatural bases (X) are located at the last position (NNX) in at least one codon of the mRNA.

[0306] Embodiment 143. The one or more unnatural bases have the formula [ka] wherein R2 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide, and the wavy line indicates the bond to the ribosyl moiety.

[0307] Embodiment 144. The one or more first unnatural bases or the one or more second unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0308] Embodiment 145. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] then the second unnatural base is [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0309] Embodiment 146. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0310] Embodiment 147. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0311] Embodiment 148. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0312] Embodiment 149. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0313] Embodiment 150. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0314] Embodiment 151. The one or more first unnatural bases are [ka] and the one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0315] Embodiment 152. The one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0316] Embodiment 153. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0317] Embodiment 154. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0318] Embodiment 155. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0319] Embodiment 156. At least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at first positions (XNN) in the codon of the mRNA, and the one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0320] Embodiment 157. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0321] Embodiment 158. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0322] Embodiment 159. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0323] Embodiment 160. At least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at central positions (NXN) in the codon of the mRNA, and the one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0324] Embodiment 161. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0325] Embodiment 162. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0326] Embodiment 163. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0327] Embodiment 164. At least one codon of the mRNA comprises three consecutive nucleobases (NNN), and one or more first unnatural bases (X) are located at the last position (NNX) in the codon of the mRNA, and the one or more first unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0328] Embodiment 165. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0329] Embodiment 166. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0330] Embodiment 167. The one or more first unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0331] Embodiment 168. The system of embodiment 139, wherein at least one anticodon of the tRNA comprises three consecutive nucleobases (NNN); and one or more second unnatural bases (X) are located at the first position (XNN) in the anticodon of the tRNA.

[0332] Embodiment 169. The one or more second unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0333] Embodiment 170. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0334] Embodiment 171. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0335] Embodiment 172. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0336] Embodiment 173. The system of embodiment 139, wherein at least one anticodon of the tRNA comprises three consecutive nucleobases (NNN); and one or more second unnatural bases (X) are located at central positions (NXN) in the anticodon of the tRNA.

[0337] Embodiment 174. The one or more second unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0338] Embodiment 175. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0339] Embodiment 176. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0340] Embodiment 177. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0341] Embodiment 178. The system of embodiment 139, wherein at least one anticodon of the tRNA comprises three consecutive nucleobases (NNN); and one or more second unnatural bases (X) are located at the last position (NNX) in the anticodon of the tRNA.

[0342] Embodiment 179. The one or more second unnatural bases are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0343] Embodiment 180. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0344] Embodiment 181. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0345] Embodiment 182. The one or more second unnatural bases are [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0346] Embodiment 183. The system of embodiment 139, wherein at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (NNN), wherein at least one codon comprises one or more first unnatural bases (X) located at the first position (XNN) of the codon, and wherein at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located at the last position (NNY) of the anticodon.

[0347] Embodiment 184. The system of embodiment 183, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different.

[0348] Embodiment 185. The system of embodiment 184, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are identical.

[0349] Embodiment 186. The system of embodiment 184, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are different.

[0350] Embodiment 187. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] 187. The system according to any one of embodiments 183 to 186, wherein the wavy line indicates the bond to the ribosyl moiety.

[0351] Embodiment 188. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0352] Embodiment 189. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0353] Embodiment 190. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0354] Embodiment 191. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0355] Embodiment 192. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: [ka] and in each case the wavy line indicates the bond to the ribosyl moiety.

[0356] Embodiment 193. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] 193. The system of embodiment 192, wherein

[0357] Embodiment 194. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] 193. The system of embodiment 192, wherein

[0358] Embodiment 195. The system of embodiment 139, wherein at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (NNN), wherein at least one codon in the mRNA comprises one or more first unnatural bases (X) located in a central position (NXN) of the at least one codon, and wherein at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located in a central position (NYN) of the anticodon.

[0359] Embodiment 196. The system of embodiment 195, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different.

[0360] Embodiment 197. The system of embodiment 195, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are identical.

[0361] Embodiment 198. The system of embodiment 195, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are different.

[0362] Embodiment 199. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] 199. The system according to any one of embodiments 195-198, wherein the wavy line indicates the bond to the ribosyl moiety.

[0363] Embodiment 200. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0364] Embodiment 201. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] 201. The system of embodiment 200, wherein the wavy line indicates the bond to the ribosyl moiety.

[0365] Embodiment 202. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] 201. The system of embodiment 200, wherein the wavy line indicates the bond to the ribosyl moiety.

[0366] Embodiment 203. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] 201. The system of embodiment 200, wherein the wavy line indicates the bond to the ribosyl moiety.

[0367] Embodiment 204. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: [ka] and in each case the wavy line indicates the bond to the ribosyl moiety.

[0368] Embodiment 205. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] 205. The system of embodiment 204, wherein

[0369] Embodiment 206. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] 205. The system of embodiment 204, wherein

[0370] Embodiment 207. The system of embodiment 139, wherein at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (NNN), wherein at least one codon in the mRNA comprises one or more first unnatural bases (X) located at the last position (NNX) of the at least one codon, and wherein at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located at the first position (YNN) of the anticodon.

[0371] Embodiment 208. The system of embodiment 207, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different.

[0372] Embodiment 209. The system of embodiment 208, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are identical.

[0373] Embodiment 210. The system of embodiment 208, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are different.

[0374] Embodiment 211. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] 211. The system according to any one of embodiments 207 to 210, wherein the wavy line indicates the bond to the ribosyl moiety.

[0375] Embodiment 212. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are [ka] wherein the wavy line indicates the bond to the ribosyl moiety.

[0376] Embodiment 213. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0377] Embodiment 214. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0378] Embodiment 215. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are both [ka] and the wavy line indicates the bond to the ribosyl moiety.

[0379] Embodiment 216. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: [ka] and in each case the wavy line indicates the bond to the ribosyl moiety.

[0380] Embodiment 217. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] 217. The system of embodiment 216, wherein

[0381] Embodiment 218. The one or more first unnatural bases (X) located in a codon of an mRNA are [ka] 217. The system of embodiment 216, wherein

[0382] Embodiment 219. The system of any one of embodiments 139 to 218, wherein at least one codon in the mRNA is selected from AXC, GXC, or GXU, wherein X is an unnatural base.

[0383] Embodiment 220. The system of embodiment 219, wherein at least one codon in the mRNA is AXC, and X is an unnatural base.

[0384] Embodiment 221. The system of embodiment 219, wherein at least one codon in the mRNA is GXC, and X is an unnatural base.

[0385] Embodiment 222. The system of embodiment 219, wherein at least one codon in the mRNA is GXU, wherein X is an unnatural base.

[0386] Embodiment 223. The method of any one of embodiments 139 to 218, wherein at least one codon in the mRNA is selected from AXC, GXC, or GXU, and at least one anticodon in the tRNA is selected from GYU, GYC, and AYC, wherein X is one or more first unnatural bases, and Y is one or more second unnatural bases. The system described above.

[0387] Embodiment 224. The system of embodiment 223, wherein X and Y are the same or different.

[0388] Embodiment 225. The system of embodiment 224, wherein X and Y are the same.

[0389] Embodiment 226. The system of embodiment 224, wherein X and Y are different.

[0390] Embodiment 227. The system of embodiment 223, wherein at least one codon in the mRNA is AXC and at least one anticodon in the tRNA is GYU.

[0391] Embodiment 228. The system of embodiment 227, wherein X and Y are the same or different.

[0392] Embodiment 229. The system of embodiment 228, wherein X and Y are the same.

[0393] Embodiment 230. The system of embodiment 228, wherein X and Y are different.

[0394] Embodiment 231. The system of embodiment 223, wherein at least one codon in the mRNA is GXC and at least one anticodon in the tRNA is GYC.

[0395] Embodiment 232. The system of embodiment 231, wherein X and Y are the same or different.

[0396] Embodiment 233. The system of embodiment 232, wherein X and Y are identical.

[0397] Embodiment 234. The system of embodiment 232, wherein X and Y are different.

[0398] Embodiment 235. The system of embodiment 223, wherein at least one codon in the mRNA is GXU and at least one anticodon is AYC.

[0399] Embodiment 236. The system of embodiment 235, wherein X and Y are the same or different.

[0400] Embodiment 237. The system of embodiment 236, wherein X and Y are the same.

[0401] Embodiment 238. The system of embodiment 236, wherein X and Y are different.

[0402] Embodiment 239. The system of any one of embodiments 139 to 238, wherein the tRNA is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

[0403] Embodiment 240. The system of any one of embodiments 139 to 238, wherein the tRNA synthetase is derived from Methanococcus jannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

[0404] Embodiment 241. The system of embodiment 240, wherein the tRNA and tRNA synthetase are derived from Methanococcus yannaschii.

[0405] Embodiment 242. The system of embodiment 240, wherein the tRNA and tRNA synthetase are derived from Methanosarcina barkeri.

[0406] Embodiment 243. The system of embodiment 240, wherein the tRNA and tRNA synthetase are derived from Methanosarcina mazei.

[0407] Embodiment 244. The system of embodiment 240, wherein the tRNA and tRNA synthetase are derived from Methanosarcina acetivorans.

[0408] Embodiment 245. The system of any one of embodiments 139 to 239, wherein the tRNA is derived from Methanococcus yannaschii and the tRNA synthetase is derived from Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

[0409] Embodiment 246. The system of any one of embodiments 139 to 239, wherein the tRNA is derived from Methanosarcina barkeri and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina mazei, or Methanosarcina acetivorans.

[0410] Embodiment 247. The system of any one of embodiments 139 to 239, wherein the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina acetivorans.

[0411] Embodiment 248. The system of any one of embodiments 139 to 239, wherein the tRNA is derived from Methanosarcina acetivorans and the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, or Methanosarcina mazei.

[0412] Embodiment 249. The system of any one of embodiments 139 to 239, wherein the tRNA is derived from Methanosarcina mazei and the tRNA synthetase is derived from Methanosarcina barkeri.

[0413] Embodiment 250. The system of any one of embodiments 139 to 249, wherein the cells are human cells.

[0414] Embodiment 251. The system of embodiment 250, wherein the human cells are HEK293T cells.

[0415] Embodiment 252. The system of any one of embodiments 139 to 239, wherein the cells are hamster cells.

[0416] Embodiment 253. The system of embodiment 252, wherein the hamster cells are Chinese hamster ovary (CHO) cells.

[0417] Embodiment 254. The unnatural amino acid is: It is a lysine analog; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or 254. The system of any one of embodiments 139 to 253, comprising an aldehyde or ketone group.

[0418] Embodiment 255. The unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L -lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-bromophenylalanine p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)-3-oxopropyl 254. The system according to any one of embodiments 139 to 253, wherein the hydroxyl group is selected from the group consisting of N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine or N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

[0419] Embodiment 256. The system of embodiment 255, wherein the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK).

[0420] Embodiment 257. The method of any one of embodiments 21-138, wherein the mRNA and tRNA are stabilized for degradation in eukaryotic cells.

[0421] Embodiment 258. The method of any one of embodiments 21-138 and 257, wherein the polypeptide is produced by translation of mRNA using tRNA by a ribosome that is endogenous to the eukaryotic cell.

[0422] Embodiment 259. The system of any one of embodiments 139 to 256, wherein the mRNA and tRNA are stabilized for degradation in eukaryotic cells.

[0423] Embodiment 260. The system of any one of claims 139 to 256 and 259, wherein the polypeptide is produced by translation of mRNA using tRNA by a ribosome that is endogenous to the eukaryotic cell.

[0424] Embodiment 261. (a) a messenger RNA (mRNA) having a codon comprising a first unnatural base; (b) a transfer RNA (tRNA) having an anticodon that includes a second unnatural base, wherein the first and second unnatural bases form an unnatural base pair (UBP) in the eukaryote, and wherein the mRNA can be translated in the cell to produce a polypeptide that includes at least one unnatural amino acid.

[0425] Embodiment 262. The eukaryote of embodiment 261, wherein the tRNA is charged with an unnatural amino acid.

[0426] Embodiment 263. The eukaryote of any one of embodiments 261-262, further comprising a polypeptide translated from the mRNA, wherein the polypeptide comprises an unnatural amino acid, and optionally, the polypeptide comprises a eukaryotic glycosylation pattern.

[0427] Embodiment 264. The eukaryotic cell of any one of embodiments 261-263, further comprising a tRNA synthetase, wherein the tRNA synthetase preferentially aminoacylates the tRNA with an unnatural amino acid.

[0428] Embodiment 265. The eukaryotic cell of any one of embodiments 261 to 264, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the first position (XNN) in the codon of the mRNA.

[0429] Embodiment 266. The eukaryotic cell of any one of embodiments 261 to 265, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the central position (NXN) in the codon of the mRNA.

[0430] Embodiment 267. The eukaryotic cell of any one of embodiments 261 to 266, wherein the codon of the mRNA comprises three consecutive nucleobases (NNN); and the first unnatural base (X) is located at the last position (NNX) in the codon of the mRNA.

[0431] Embodiment 268. The first unnatural base or the second unnatural base is (i) 2-thiouracil, 2-thio-thymine, 2'-deoxyuridine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl(I), 5-halouracil; 5-propynyl-uracil, 6-azo-thymine, 6-azo-uracil, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, pseudouracil, uracil-5-oxaacetic acid methyl ester, uracil-5-oxaacetic acid, 5- methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil or dihydrouracil; (ii) 5-hydroxymethylcytosine, 5-trifluoromethylcytosine, 5-halocytosine, 5-propynylcytosine, 5-hydroxycytosine, cyclocytosine, cytosine arabinoside, 5,6-dihydrocytosine, 5-nitrocytosine, 6-azocytosine, azacytosine, N4-ethylcytosine, 3-methylcytosine, 5-methylcytosine, 4-acetylcytosine, 2-thiocytosine, phenoxazine cytidine ([5,4-b][1,4]benzoxazine-2 (3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one) or pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one); (iii) 2-aminoadenine, 2-propyladenine, 2-amino-adenine, 2-F-adenine, 2-amino-propyl-adenine, 2-amino-2'-deoxyadenosine, 3-deazaadenine, 7-methyladenine, 7-deaza-adenine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl and 8-hydroxyl substituted adenines, N6-isopentenyladenine, 2-methyladenine, 2,6-diaminopurine, 2-methylthio-N6-isopentenyladenine or 6-aza-adenine; (iv) 2-methylguanine, 2-propyl and alkyl derivatives of guanine, 3-deazaguanine, 6-thio-guanine, 7-methylguanine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-8-azaguanine, 8-azaguanine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl and 8-hydroxyl substituted guanines, 1-methyl guanine, 2,2-dimethylguanine, 7-methylguanine or 6-azaguanine; and (v) hypoxanthine, xanthine, 1-methylinosine, queuosine, beta-D-galactosylqueuosine, inosine, beta-D-mannosylqueuosine, wybutoxosine, hydroxyurea, (acp3)w, 2-aminopyridine or 2-pyridone 268. A eukaryotic cell according to any one of embodiments 261 to 267, selected from the group consisting of:

[0432] Embodiment 269. The first unnatural base and the second unnatural base are each independently: [ka] 268. The eukaryotic cell of any one of embodiments 261-267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0433] Embodiment 270. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 268. The eukaryotic cell of any one of embodiments 261 to 267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0434] Embodiment 271. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 268. The eukaryotic cell of any one of embodiments 261 to 267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0435] Embodiment 272. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 268. The eukaryotic cell of any one of embodiments 261 to 267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0436] Embodiment 273. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 268. The eukaryotic cell of any one of embodiments 261 to 267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0437] Embodiment 274. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 268. The eukaryotic cell of any one of embodiments 261 to 267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0438] Embodiment 275. The first unnatural base is [ka] and the second unnatural base is [ka] and the first unnatural base is [ka] and the second unnatural base is [ka] 268. The eukaryotic cell of any one of embodiments 261 to 267, wherein the wavy line indicates the bond to the ribosyl moiety.

[0439] Embodiment 276. The first unnatural base or the second unnatural base is Modifications at the 2' position: OH, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-Alkyl-O-Alkyl, 2'-F, 2'-OCH3, 2'-O(CH2)2OCH3 (wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C1-C 10 , alkyl, C2-C 10 Alkenyl, C2-C 10 Alkynyl, -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n NH2, -O(CH2) n CH3, -O(CH2) n -NH2 and -O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10; and / or modifications at the 5' position: 5'-vinyl, 5'-methyl (R or S); Modification at position 4: 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of an oligonucleotide, or group for improving the pharmacodynamic properties of an oligonucleotide, and any combination thereof. 276. The eukaryotic cell of any one of embodiments 261-275, comprising a modified sugar moiety selected from the group consisting of:

[0440] Embodiment 277. The at least one unnatural amino acid is: It is a lysine analog; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or 277. The eukaryotic cell of any one of embodiments 263 to 276, comprising an aldehyde or ketone group.

[0441] Embodiment 278. The at least one unnatural amino acid is selected from the group consisting of N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((pro...

Claims

1. (a) a messenger RNA (mRNA) having a codon that includes a first unnatural base; (b) a transfer RNA (tRNA) having an anticodon containing a second unnatural base; A eukaryotic cell comprising: The eukaryotic cell, wherein the first and second unnatural bases are capable of forming an unnatural base pair (UBP) in a eukaryotic cell, and wherein the mRNA is capable of being translated in the cell to produce a polypeptide comprising at least one unnatural amino acid.

2. The eukaryotic cell of claim 1 , wherein the tRNA is charged with an unnatural amino acid.

3. 3. The eukaryotic cell of claim 1, further comprising a polypeptide translated from the mRNA, wherein the polypeptide comprises an unnatural amino acid, and optionally, the polypeptide comprises a eukaryotic glycosylation pattern.

4. 4. The eukaryotic cell of claim 1, further comprising a tRNA synthetase, wherein the tRNA synthetase preferentially aminoacylates the tRNA with an unnatural amino acid.

5. 5. The eukaryotic cell of any one of claims 1 to 4, wherein the codon of the mRNA comprises three consecutive nucleic acid bases (N-N-N); and the first unnatural base (X) is located at the first position (X-N-N) in the codon of the mRNA.

6. 6. The eukaryotic cell of any one of claims 1 to 5, wherein the codon of the mRNA comprises three consecutive nucleobases (N-N-N); and the first unnatural base (X) is located at the central position (N-X-N) in the codon of the mRNA.

7. 7. The eukaryotic cell of any one of claims 1 to 6, wherein the codon of the mRNA comprises three consecutive nucleic acid bases (N-N-N); and the first unnatural base (X) is located at the last position (N-N-X) in the codon of the mRNA.

8. the first unnatural base and the second unnatural base are each independently 【Chemical 1】 and the wavy line indicates the bond to the ribosyl moiety.

9. the first unnatural base is 【Chemistry 2】 and the second unnatural base is 【Chemistry 3】 and the first unnatural base is 【Chemistry 4】 and the second unnatural base is 【Chemistry 5】 and the wavy line indicates the bond to the ribosyl moiety.

10. the first unnatural base is 【Chemistry 6】 and the second unnatural base is 【Chemistry 7】 and the first unnatural base is 【Chemistry 8】 and the second unnatural base is 【Chemistry 9】 and the wavy line indicates the bond to the ribosyl moiety.

11. the first unnatural base is 【Chemistry 10】 and the second unnatural base is 【Chemistry 11】 and the first unnatural base is 【Chemistry 12】 and the second unnatural base is 【Chemistry 13】 and the wavy line indicates the bond to the ribosyl moiety.

12. the first unnatural base is 【Chemistry 14】 and the second unnatural base is 【Chemistry 15】 and the first unnatural base is 【Chemistry 16】 and the second unnatural base is 【Chemistry 17】 and the wavy line indicates the bond to the ribosyl moiety.

13. the first unnatural base is 【Chemistry 18】 and the second unnatural base is 【Chemistry 19】 and the first unnatural base is 【Chemistry 20】 and the second unnatural base is 【Chemical 21】 and the wavy line indicates the bond to the ribosyl moiety.

14. the first unnatural base is 【Chemical 22】 and the second unnatural base is 【Chemical 23】 and the first unnatural base is 【Chemistry 24】 and the second unnatural base is 【Chemistry 25】 and the wavy line indicates the bond to the ribosyl moiety.

15. The at least one unnatural amino acid is: It is a lysine analogue; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or A eukaryotic cell according to any one of claims 3 to 14, which contains an aldehyde or ketone group.

16. The at least one unnatural amino acid may be selected from the group consisting of N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidome lysine ... p-Acyl-L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl allyl-L-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyl) 16. The eukaryotic cell of claim 15, wherein the amino acid sequence is selected from the group consisting of N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

17. 17. The eukaryotic cell of claim 16, wherein the at least one unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK).

18. A eukaryotic cell according to any one of claims 1 to 17, which is a human cell.

19. The eukaryotic cell of claim 18, wherein the human cell is a HEK293T cell.

20. A eukaryotic cell according to any one of claims 1 to 18, which is a mammalian cell, optionally a hamster cell.

21. 21. The eukaryotic cell of claim 20, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell.

22. 22. The eukaryotic cell of any one of claims 18 to 21, further comprising a polypeptide translated from the mRNA, said polypeptide comprising an unnatural amino acid and a mammalian glycosylation pattern.

23. 23. The eukaryotic cell according to any one of claims 1 to 22, which is isolated.

24. A semisynthetic organism comprising a eukaryotic cell according to any one of claims 1 to 23.

25. A eukaryotic cell culture comprising a plurality of eukaryotic cells according to any one of claims 1 to 24.

26. A method of delivering a cell to an organism, said method comprising the step of contacting said organism with a cell according to any one of claims 1 to 23.

27. 27. The method of claim 26, wherein the organism is a mammal, optionally wherein the mammal is a human.

28. 1. A method for producing a polypeptide comprising at least one unnatural amino acid in a eukaryotic cell, comprising: (a) (i) a messenger RNA (mRNA) having a codon that includes a first unnatural base; and (ii) a transfer RNA (tRNA) having an anticodon containing a second unnatural base in a eukaryotic cell; into the cell, wherein the first and second unnatural bases are capable of forming an unnatural base pair (UBP) in a eukaryotic cell; (b) translating the mRNA using the tRNA to form a polypeptide comprising at least one unnatural amino acid; The method comprising:

29. 29. The method of claim 28, wherein the tRNA is charged with an unnatural amino acid.

30. 1. A method for producing a polypeptide comprising at least one unnatural amino acid in a eukaryotic cell, comprising: (a) (i) a messenger RNA (mRNA) having a codon that includes a first unnatural base; (ii) a transfer RNA (tRNA) having an anticodon containing a second unnatural base; and wherein the first and second unnatural bases are capable of forming an unnatural base pair (UBP) in the eukaryotic cell; and (b) translating a polypeptide comprising at least one unnatural amino acid from the mRNA using a tRNA by a ribosome endogenous to the eukaryotic cell. The method comprising:

31. A method for producing a polypeptide in a eukaryotic cell, wherein the polypeptide comprises at least one unnatural amino acid, the method comprising: (a) providing a eukaryotic cell, said eukaryotic cell comprising: (i) an mRNA comprising a codon comprising a first unnatural base; (ii) a tRNA comprising an anticodon comprising a second unnatural base, and the second unnatural base is capable of forming a complementary base pair; (iii) a tRNA synthetase that preferentially aminoacylates the tRNA with at least one unnatural amino acid relative to natural amino acids; The process comprising: (b) providing one or more unnatural amino acids to a eukaryotic cell, wherein the eukaryotic cell produces a polypeptide comprising at least one unnatural amino acid.

32. 32. The method of any one of claims 26 to 31, wherein the codon of the mRNA comprises three consecutive nucleobases (N-N-N); and the first unnatural base (X) is located at the first position (X-N-N) in the codon of the mRNA.

33. 32. The method of any one of claims 26 to 31, wherein the mRNA codon comprises three consecutive nucleobases (N-N-N); and the first unnatural base (X) is located at the central position (N-X-N) in the mRNA codon.

34. 32. The method of any one of claims 26 to 31, wherein the codon of the mRNA comprises three consecutive nucleobases (N-N-N); and the first unnatural base (X) is located at the last position (N-N-X) in the codon of the mRNA.

35. The one or more unnatural bases comprising the codon of the mRNA are represented by the formula 【Chemical 26】 or 【Chemical 27】 wherein R ２ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide, and the wavy line indicates the bond to the ribosyl moiety.

36. The first unnatural base or the second unnatural base is 【Chemical 28】 and the wavy line indicates the bond to the ribosyl moiety.

37. The first unnatural base is 【Chemical 29】 and the second unnatural base is 【Chemistry 30】 or the first unnatural base is 【Chemical 31】 and the second unnatural base is 【Chemical 32】 37. The method of claim 36, wherein the wavy line indicates the bond to the ribosyl moiety.

38. The first unnatural base is 【Chemical 33】 and the second unnatural base is 【Chemical Formula 34】 or the first unnatural base is 【Chemical 35】 and the second unnatural base is 【Chemical 36】 37. The method of claim 36, wherein the wavy line indicates the bond to the ribosyl moiety.

39. The first unnatural base is 【Chemical 37】 and the second unnatural base is 【Chemical 38】 or the first unnatural base is 【Chemical Formula 39】 and the second unnatural base is 【Chemistry 40】 37. The method of claim 36, wherein the wavy line indicates the bond to the ribosyl moiety.

40. The first unnatural base is 【Chemistry 41】 and the second unnatural base is 【Chemistry 42】 or the first unnatural base is 【Chemistry 43】 and the second unnatural base is 【Chemical 44】 37. The method of claim 36, wherein the wavy line indicates the bond to the ribosyl moiety.

41. The first unnatural base is 【Chemistry 45】 and the second unnatural base is 【Chemistry 46】 or the first unnatural base is 【Chemistry 47】 and the second unnatural base is 【Chemistry 48】 37. The method of claim 36, wherein the wavy line indicates the bond to the ribosyl moiety.

42. The mRNA codon comprises three consecutive nucleobases (N-N-N), and a first unnatural base (X) is located at a first position (X-N-N) in the mRNA codon, and the first unnatural base is 【Chemistry 49】 The method of any one of claims 26 to 36, wherein the wavy line indicates the bond to the ribosyl moiety.

43. An mRNA codon comprises three consecutive nucleobases (N-N-N), and a first unnatural base (X) is located at a central position (N-X-N) in the mRNA codon, and the first unnatural base is 【Chemistry 50】 The method of any one of claims 26 to 36, wherein the wavy line indicates the bond to the ribosyl moiety.

44. The mRNA codon comprises three consecutive nucleobases (N-N-N), and a first unnatural base (X) is located at the last position (N-N-X) in the mRNA codon, and the unnatural base is 【Chemistry 51】 The method of any one of claims 26 to 36, wherein the wavy line indicates the bond to the ribosyl moiety.

45. The anticodon of the tRNA comprises three consecutive nucleobases (N-N-N); a second unnatural base (X) is located at a first position (X-N-N) in the anticodon of the tRNA, said second unnatural base being 【Chemistry 52】 The method of any one of claims 26 to 36, wherein the wavy line indicates the bond to the ribosyl moiety.

46. The anticodon of the tRNA comprises three consecutive nucleobases (N-N-N); a second unnatural base (X) is located at a central position (N-X-N) in the anticodon of the tRNA, said second unnatural base being 【Chemistry 53】 The method of any one of claims 26 to 36, wherein the wavy line indicates the bond to the ribosyl moiety.

47. The anticodon of the tRNA comprises three consecutive nucleobases (N-N-N); a second unnatural base (X) is located at the last position (N-N-X) in the anticodon of the tRNA, and the second unnatural base is 【Chemical 54】 The method of any one of claims 26 to 36, wherein the wavy line indicates the bond to the ribosyl moiety.

48. 37. The method of any one of claims 26 to 36, wherein the codon and anticodon each comprise three consecutive nucleic acid bases (N-N-N), a first unnatural base (X) of the codon in the mRNA is located at a first position (X-N-N) of the codon, and a second unnatural base (Y) of the anticodon of the tRNA is located at a last position (N-N-Y) of the anticodon.

49. 37. The method of any one of claims 26 to 36, wherein the codon and anticodon each comprise three consecutive nucleic acid bases (N-N-N), the codon in the mRNA comprises a first unnatural base (X) located at a central position (N-X-N) of the codon, and the anticodon in the tRNA comprises a second unnatural base (Y) located at a central position (N-Y-N) of the anticodon.

50. 37. The method of any one of claims 26 to 36, wherein the codon and anticodon each comprise three consecutive nucleic acid bases (N-N-N), the codon in the mRNA comprises a first unnatural base (X) located at the last position of the codon (N-N-X), and the anticodon in the tRNA comprises a second unnatural base (Y) located at the first position of the anticodon (Y-N-N).

51. 51. The method of any one of claims 48 to 50, wherein the first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are the same or different.

52. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are 【Chemistry 55】 and the wavy line indicates the bond to the ribosyl moiety.

53. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are 【Chemical 56】 and the wavy line indicates the bond to the ribosyl moiety.

54. The first unnatural base (X) located in the codon of the mRNA and the second unnatural base (Y) located in the anticodon of the tRNA are both 【Chemical 57】 54. The method of claim 53, wherein the wavy line indicates the bond to the ribosyl moiety.

55. The first unnatural base (X) located in the codon of the mRNA is 【Chemistry 58】 and the second unnatural base (Y) located in the anticodon of the tRNA is selected from 【Chemical Formula 59】 and in each case the wavy line indicates a bond to a ribosyl moiety.

56. 50. The method of any one of claims 26-29, 31, 33, 35-41, 43, 46 and 49, wherein the codon in the mRNA is selected from AXC, GXC or GXU, and X is a first unnatural base.

57. 57. The method of claim 56, wherein the anticodon in the tRNA is selected from GYU, GYC, and AYC, and Y is a second unnatural base.

58. 58. The method of claim 57, wherein the codon in the mRNA is AXC and the anticodon in the tRNA is GYU.

59. 58. The method of claim 57, wherein the codon in the mRNA is GXC and the anticodon in the tRNA is GYC.

60. 58. The method of claim 57, wherein the codon in the mRNA is GXU and the anticodon is AYC.

61. The first unnatural base or the second unnatural base may have a modification at the 2′ position: OH, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH ３ , OCN, Cl, Br, CN, CF ３ , OCF ３ , SOCH ３ , S.O. ２ CH ３ , ONO ２ , NO ２ , N ３ , N.H. ２ F or a combination thereof; O-alkyl, S-alkyl, N-alkyl or combinations thereof; O-alkenyl, S-alkenyl, N-alkenyl or a combination thereof; O-alkynyl, S-alkynyl, N-alkynyl or a combination thereof; O-alkyl-O-alkyl, 2'-F, 2'-OCH ３ , 2'-O(CH ２ ) ２ OCH ３ or a combination thereof (wherein alkyl, alkenyl and alkynyl are substituted or unsubstituted C １ ~C １０ , alkyl, C ２ ~C １０ Alkenyl, C ２ ~C １０ Alkynyl, —O[(CH ２ ) ｎ O] ｍ CH ３ , -O(CH ２ ) ｎ OCH ３ , -O(CH ２ ) ｎ NH ２ , -O(CH ２ ) ｎ CH ３ , -O(CH ２ ) ｎ -NH ２ and —O(CH ２ ) ｎ ON [(CH ２ ) ｎ CH ３ )] ２ where n and m are from 1 to about 10; 5'-vinyl, 5'-methyl (R or S) or a combination thereof Modifications at the 5' position including: 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides or group for improving the pharmacodynamic properties of oligonucleotides or combinations thereof Modifications at the 4' position including: or a combination thereof 61. The method of any one of claims 26 to 60, comprising a modified sugar moiety selected from the group consisting of:

62. The at least one unnatural amino acid is: It is a lysine analogue; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or 62. The method of any one of claims 26 to 61, comprising an aldehyde or ketone group.

63. The at least one unnatural amino acid is selected from the group consisting of N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl -L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine p-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy 62. The method of any one of claims 26 to 61, wherein the amino acid residue is selected from the group consisting of N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

64. 64. The method of claim 63, wherein the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK).

65. The method of any one of claims 26 to 64, wherein the cells are human cells.

66. 66. The method of claim 65, wherein the human cell is a HEK293T cell.

67. The method of any one of claims 26 to 64, wherein the cell is a hamster cell.

68. 68. The method of claim 67, wherein the hamster cell is a Chinese hamster ovary (CHO) cell.

69. The method of any one of claims 26 to 68, wherein the tRNA is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

70. 70. The method of any one of claims 26 to 69, wherein the cell comprises a tRNA synthetase derived from Methanococcus jannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

71. 1. A system for expression of a non-naturally occurring polypeptide comprising: (a) at least one unnatural amino acid; (b) an mRNA encoding a non-natural polypeptide, the mRNA comprising at least one codon comprising one or more first non-natural bases; (c) a tRNA comprising at least one anticodon comprising one or more second unnatural bases, wherein the one or more first unnatural bases and the one or more second unnatural bases are capable of forming one or more complementary base pairs; and (d) a eukaryotic ribosome capable of translating mRNA into a polypeptide containing an unnatural amino acid using tRNA and tRNA synthetase; Including, The tRNA is charged with the unnatural amino acid, or the system further comprises a tRNA synthetase or one or more nucleic acid constructs comprising a nucleic acid sequence encoding a tRNA synthetase, wherein the tRNA synthetase preferentially aminoacylates the tRNA with at least one unnatural amino acid.

72. 72. The system of claim 71, wherein at least one codon of the mRNA comprises three consecutive nucleobases (N-N-N); and one or more first unnatural bases (X) are located at a first position (X-N-N) in at least one codon of the mRNA.

73. 72. The system of claim 71, wherein at least one codon of the mRNA comprises three consecutive nucleobases (N-N-N); and one or more first unnatural bases (X) are located at a central position (N-X-N) in the codon of the mRNA.

74. 72. The system of claim 71, wherein at least one codon of the mRNA comprises three consecutive nucleobases (N-N-N); and one or more first unnatural bases (X) are located at the last position (N-N-X) in at least one codon of the mRNA.

75. The one or more unnatural bases may be of the formula 【Chemistry 60】 wherein R ２ is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, and azide, and the wavy line indicates the bond to the ribosyl moiety.

76. The one or more first unnatural bases or the one or more second unnatural bases may be 【Hua 61】 and the wavy line indicates the bond to the ribosyl moiety.

77. The one or more first unnatural bases are 【Hua 62】 then the one or more second unnatural bases are 【Chemistry 63】 and the one or more first unnatural bases are 【Hua 64】 then the second unnatural base is 【Chemistry 65】 and the wavy line indicates the bond to the ribosyl moiety.

78. The one or more first unnatural bases are 【Hua 66】 then the one or more second unnatural bases are 【Hua 67】 and the one or more first unnatural bases are 【Chemistry 68】 then the one or more second unnatural bases are 【Chemical Formula 69】 and the wavy line indicates the bond to the ribosyl moiety.

79. The one or more first unnatural bases are 【Chemistry 70】 then the one or more second unnatural bases are 【Chemical 71】 and the one or more first unnatural bases are 【Chemical Formula 72】 then the one or more second unnatural bases are 【Chemical Formula 73】 and the wavy line indicates the bond to the ribosyl moiety.

80. The one or more first unnatural bases are 【Chemical 74】 then the one or more second unnatural bases are 【Chemistry 75】 and the one or more first unnatural bases are 【Chemical Formula 76】 then the one or more second unnatural bases are 【Chemical 77】 and the wavy line indicates the bond to the ribosyl moiety.

81. The one or more first unnatural bases are 【Chemical 78】 then the one or more second unnatural bases are 【Chemical 79】 and the one or more first unnatural bases are 【Chemistry 80】 then the one or more second unnatural bases are 【Chemistry 81】 and the wavy line indicates the bond to the ribosyl moiety.

82. The one or more first unnatural bases are 【Chemistry 82】 then the one or more second unnatural bases are 【Chemistry 83】 and the one or more first unnatural bases are 【Chemistry 84】 then the one or more second unnatural bases are 【Chemistry 85】 and the wavy line indicates the bond to the ribosyl moiety.

83. The one or more first unnatural bases are 【Chemistry 86】 then the one or more second unnatural bases are 【Hua 87】 and the wavy line indicates the bond to the ribosyl moiety.

84. The one or more first unnatural bases are 【Hua 88】 and the wavy line indicates the bond to the ribosyl moiety.

85. At least one codon of the mRNA comprises three consecutive nucleobases (N-N-N), and one or more first unnatural bases (X) are located at a first position (X-N-N) in the codon of the mRNA, and the one or more first unnatural bases are 【Chemistry 89】 and the wavy line indicates the bond to the ribosyl moiety.

86. At least one codon of the mRNA comprises three consecutive nucleobases (N-N-N), and one or more first unnatural bases (X) are located at a central position (N-X-N) in the codon of the mRNA, and the one or more first unnatural bases are 【Chemistry 90】 and the wavy line indicates the bond to the ribosyl moiety.

87. At least one codon of the mRNA comprises three consecutive nucleobases (N-N-N), and one or more first unnatural bases (X) are located at the last position (N-N-X) in the codon of the mRNA, and the one or more first unnatural bases are 【Chemistry 91】 and the wavy line indicates the bond to the ribosyl moiety.

88. At least one anticodon of the tRNA comprises three consecutive nucleobases (N-N-N); one or more second unnatural bases (X) are located at a first position (X-N-N) in the anticodon of the tRNA, and the one or more second unnatural bases are 【Chemistry 92】 and the wavy line indicates the bond to the ribosyl moiety.

89. At least one anticodon of the tRNA comprises three consecutive nucleobases (N-N-N); one or more second unnatural bases (X) are located at a central position (N-X-N) in the anticodon of the tRNA, and the one or more second unnatural bases are 【Chemistry 93】 and the wavy line indicates the bond to the ribosyl moiety.

90. At least one anticodon of a tRNA consists of three consecutive nucleobases (N-N-N) wherein one or more second unnatural bases (X) are located at the last position (N-N-X) in the anticodon of the tRNA, and the one or more second unnatural bases are 【Chemistry 94】 and the wavy line indicates the bond to the ribosyl moiety.

91. 72. The system of claim 71, wherein at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (N-N-N), wherein the at least one codon comprises one or more first unnatural bases (X) located at a first position of the codon (X-N-N), and the at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located at a last position of the anticodon (N-N-Y).

92. 92. The system of claim 91, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different.

93. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are 【Chemistry 95】 93. The system of claim 91 or 92, wherein the wavy line indicates the bond to the ribosyl moiety.

94. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are 【Chemistry 96】 and the wavy line indicates the bond to the ribosyl moiety.

95. The one or more first unnatural bases (X) located in the codon of the mRNA are 【Chemistry 97】 and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: 【Chemistry 98】 and in each case the wavy line indicates a bond to a ribosyl moiety.

96. 72. The system of claim 71, wherein at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (N-N-N), wherein at least one codon in the mRNA comprises one or more first unnatural bases (X) located in a central position of the at least one codon (N-X-N), and wherein at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located in a central position of the anticodon (N-Y-N).

97. 97. The system of claim 96, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different.

98. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are 【Hua99】 and the wavy line indicates the bond to the ribosyl moiety.

99. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are 【Chemistry 100】 and the wavy line indicates the bond to the ribosyl moiety.

100. The one or more first unnatural bases (X) located in the codon of the mRNA are 【Chemistry 101】 and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: 【Chemistry 102】 and in each case the wavy line indicates a bond to a ribosyl moiety.

101. 72. The system of claim 71, wherein at least one codon and at least one anticodon each independently comprise three consecutive nucleobases (N-N-N), wherein the at least one codon in the mRNA comprises one or more first unnatural bases (X) located at the last position of the at least one codon (N-N-X), and the at least one anticodon in the tRNA comprises one or more second unnatural bases (Y) located at the first position of the anticodon (Y-N-N).

102. 102. The system of claim 101, wherein the one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are the same or different.

103. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are 【Chemistry 103】 and the wavy line indicates the bond to the ribosyl moiety.

104. The one or more first unnatural bases (X) located in the codon of the mRNA and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are 【Chemistry 104】 and the wavy line indicates a bond to the ribosyl moiety.

105. The one or more first unnatural bases (X) located in the codon of the mRNA are 【Chemistry 105】 and the one or more second unnatural bases (Y) located in the anticodon of the tRNA are selected from: 【Chemistry 106】 and in each case the wavy line indicates a bond to a ribosyl moiety.

106. 106. The system of any one of claims 71 to 105, wherein at least one codon in the mRNA is selected from AXC, GXC, or GXU, wherein X is one or more first unnatural bases.

107. 107. The system of claim 106, wherein at least one anticodon in the tRNA is selected from GYU, GYC, and AYC, and wherein Y is one or more second unnatural bases.

108. 108. The system of claim 107, wherein at least one codon in the mRNA is AXC and at least one anticodon in the tRNA is GYU.

109. 108. The system of claim 107, wherein at least one codon in the mRNA is GXC and at least one anticodon in the tRNA is GYC.

110. 108. The system of claim 107, wherein at least one codon in the mRNA is GXU and at least one anticodon is AYC.

111. 111. The system of any one of claims 71 to 110, wherein the tRNA is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

112. 112. The system of any one of claims 71 to 111, wherein the tRNA synthetase is derived from Methanococcus yannaschii, Methanosarcina barkeri, Methanosarcina mazei, or Methanosarcina acetivorans.

113. A system according to any one of claims 71 to 112, which is in vitro or cell-free.

114. 114. The system of any one of claims 71 to 113, comprising a cell lysate.

115. A system according to any one of claims 71 to 113, which is a reconstituted system of purified components.

116. A system according to any one of claims 71 to 112 in a eukaryotic cell.

117. The system of claim 116, wherein the eukaryotic cell is a human cell.

118. The system of claim 116, wherein the eukaryotic cell is a HEK293T cell.

119. 117. The system of claim 116, wherein the eukaryotic cell is a hamster cell.

120. 120. The system of claim 119, wherein the hamster cells are Chinese hamster ovary (CHO) cells.

121. Unnatural amino acids are: It is a lysine analogue; Contains aromatic side chains; Contains an azide group; containing an alkyne group; or 121. A system according to any one of claims 71 to 120, comprising an aldehyde or ketone group.

122. Unnatural amino acids include N6-((azidoethoxy)-carbonyl)-L-lysine (AzK), N6-((propargylethoxy)-carbonyl)-L-lysine (PraK), BCN-L-lysine, norbornene lysine, TCO-lysine, methyltetrazine lysine, allyloxycarbonyl lysine, 2-amino-8-oxononanoic acid, 2-amino-8-oxooctanoic acid, p-acetyl-L-phenylalanine, p-azidomethyl-L-phenylalanine, and p-azidomethyl-L-phenylalanine. L-phenylalanine (pAMF), p-iodo-L-phenylalanine, m-acetylphenylalanine, 2-amino-8-oxononanoic acid, p-propargyloxyphenylalanine, p-propargyl-phenylalanine, 3-methyl-phenylalanine, L-dopa, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenyl Alanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, O-allyl tyrosine, O-methyl-L-tyrosine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, phosphonotyrosine, tri-O-acetyl-GlcNAcp-serine, L-phosphoserine, phosphonoserine, L-3-(2-naphthyl)alanine, 2-amino-3-((2-((3-(benzyloxy)- 122. The system of any one of claims 71 to 121, wherein the system is selected from the group consisting of N6-(((2-azidobenzyl)oxy)carbonyl)-L-lysine, N6-(((3-azidobenzyl)oxy)carbonyl)-L-lysine, and N6-(((4-azidobenzyl)oxy)carbonyl)-L-lysine.

123. 123. The system of any one of claims 71 to 122, wherein the unnatural amino acid is N6-((azidoethoxy)-carbonyl)-L-lysine (AzK).

124. 124. The system of any one of claims 71 to 123, wherein the tRNA is charged with an unnatural amino acid.

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