Incorporation of unnatural nucleotides and methods thereof

Abstract

To provide kits and methods for synthesizing proteins containing unnatural amino acids with mutant tRNAs.SOLUTION: Provided herein is a kit for producing a protein containing an unnatural amino acid, the kit comprising: (a) a plasmid comprising a sequence encoding a mutant tRNA having a mutant anticodon sequence, where Y is an unnatural nucleotide comprising a first unnatural nucleobase of a specific structure; and (b) a cell.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 531,325, filed Jul. 11, 2017, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Oligonucleotides and their applications have revolutionized biotechnology. However, oligonucleotides containing both DNA and RNA each contain only 4 natural nucleotides, namely adenosine (A), guanosine (G), cytosine (C), thymine (T) for DNA, and adenosine (A), guanosine (G), cytosine (C), and uridine (U) for RNA, thereby greatly limiting the potential functions and uses of oligonucleotides.

[0003] For example, the ability to sequence-specifically synthesize / amplify oligonucleotides (DNA or RNA) using polymerase by a PCR or isothermal amplification system (e.g., transcription using T7 RNA polymerase) has revolutionized biotechnology. In addition to all potential applications in nanotechnology, this has enabled a wide range of new technologies such as in vitro development via SELEX (Systematic Evolution of Ligands by Exponential Enrichment) of RNA and DNA aptamers and enzymes. See, for example, Oliphant AR, Brandl CJ & Struhl K (1989), Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 proteins, Mol. Cell Biol.,9:2944-2949;Tuerk C & Gold L (1990), Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase, Science, 249:505-510;Ellington AD & Szostak JW (1990), In vitro selection of RNA molecules that bind specific ligands, Nature, 346:818-822.

[0004] In some embodiments, such applications are limited by the limited chemical / physical diversity present in the natural genetic alphabet (the four natural nucleotides A, C, G, and T in DNA, and the four natural nucleotides A, C, G, and U in RNA). Additional methods for generating nucleic acids containing an expanded genetic alphabet are disclosed herein. SUMMARY OF THE INVENTION

[0005] This specification discloses, in certain embodiments, a method for producing a protein containing a non-natural amino acid, the method comprising the steps of preparing a mutant tRNA, wherein the mutant tRNA comprises a mutant anticodon sequence selected from Table 1 or 2; preparing a mutant mRNA, wherein the mutant mRNA comprises a mutant codon sequence selected from Table 1 or 2; and synthesizing a protein containing a non-natural amino acid using the mutant tRNA and the mutant mRNA. In some examples, the protein is synthesized in a cell-free translation system. In some examples, the protein is synthesized in a cell (semi-synthetic organism or SSO). In some examples, the semi-synthetic organism includes a microorganism. In some examples, the semi-synthetic organism includes a bacterium. In some examples, the semi-synthetic organism includes Escherichia coli. In some examples, the mutant anticodon of the mutant tRNA pairs with a mutant codon selected from Table 1-3. In some examples, the non-natural amino acid contains at least one non-natural nucleotide. In some examples, the non-natural nucleotide contains a non-natural nucleobase. In some examples, the non-natural base of the non-natural nucleotide is 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl (I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, as well as 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidine, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorine-added cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine, 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl-substituted adenine and guanine; N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of double formation, universal nucleic acids, hydrophobic nucleic acids, nucleic acids with intermixed hybridization, nucleic acids with size expansion, fluorine-treated nucleic acids, 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-clamp, phenoxazine cytidine (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one, carbazole cytidine (2H-pyrimido[4,5-b]indole-2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxyhydroxymethyluracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-Dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wybutosine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyluracil, (acp3)w, and 2,6-diaminopurine, and those in which the purine base or pyrimidine base is substituted by a heterocyclic compound, are selected from the group consisting of. In some examples, the unnatural nucleotides are selected from the group consisting of the following formulas (showing only the nucleobase portion, and omitting the ribose and phosphate backbone for clarity).

[0006]

Chemical formula

[0007] In some examples, the unnatural nucleotides are selected from the group consisting of the following formulas (showing only the nucleobase portion, and omitting the ribose and phosphate backbone for clarity).

[0008]

Chemical formula

[0009] In some examples, the unnatural nucleotide further includes an unnatural sugar moiety. In some examples, the unnatural sugar moiety of the unnatural nucleotide is a modification at the 2’ position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkyl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2, CH3, 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, selected from the group consisting of, where 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 -ONH2, and -O(CH2) n ON[(CH2) nIt may also be [[CH3]]2, where n and m are from 1 to about 10; and / or, the unnatural sugar moiety is a modification at the 5' position: 5'-vinyl, 5'-methyl (R or S), a modification at the 4' position, 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage group, a reporter group, an intervening substance, 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. In some examples, the mutant anticodon or mutant codon further comprises an unnatural backbone. In some examples, both the mutant anticodon and the mutant codon further comprise an unnatural backbone. In some examples, the unnatural nucleotide is recognized by a DNA polymerase, an RNA polymerase, or a reverse transcriptase. In some examples, the unnatural nucleotide is incorporated during transcription into mRNA by an RNA polymerase to produce a mutant mRNA containing the mutant codon. In some examples, the unnatural nucleotide is incorporated during transcription into tRNA by an RNA polymerase to produce a mutant tRNA containing the mutant anticodon. In some examples, the unnatural nucleotide is incorporated during transcription into mRNA by an RNA polymerase to produce a mutant mRNA. In some examples, the unnatural nucleotide is incorporated during transcription into tRNA by an RNA polymerase to produce a mutant tRNA. In some examples, the mutant tRNA is charged with an unnatural amino acid residue. In some examples, a protein containing an unnatural amino acid is produced using the mutant tRNA and the mutant mRNA during translation. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Various aspects of the disclosure are described, among other things, in the appended claims. A better understanding of the features and advantages of the disclosure can be obtained by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the disclosure are used, and the accompanying drawings:

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[0011] Table 4 | Relative abundance of amino acids at position 151 in sfGFP for the experiments described in FIGS. 1F and 2D. sfGFP purified from cells expressing sfGFP with or without the tRNA having the codon and anticodon for position 151 shown respectively was analyzed by LC-MS / MS. Reporter peptide LEYNFNSHNVX 151 ITADK (X = PrK, or a natural amino acid identified excluding K or R) and LEYNFNSHNVX 151 (When X = K or R), the extracted MS1 ion intensity is expressed as a percentage of the sum of the ion intensities for all observable reporter peptides. The table of values corresponds to the average relative abundance and 95% CI of all amino acids detected at position 151 of sfGFP, n = 4 purified sfGFP samples, each propagated from individual colonies from the culture. Values <0.1% (average, for the codons shown in each figure) are excluded from the data presented in FIGS. 1F and 2D.<0.1%

[0012] Table 5 | Retention of UBP. Retention of UBP in plasmids having the codon for the indicated position 151 of sfGFP and the anticodon of the indicated tRNA is determined at the time point before sfGFP induction and at the end of induction, as described in the method. The reported values are the average UBP retention over the induction period (calculated from the retention at these two time points) of ±95% CI, n = 4 cultures each propagated from individual colonies, and the values indicated by asterisks are excluded, n = 3. n / a indicates not applicable (because the relevant sequences are natural or absent). All plasmids were isolated from cultures grown in the presence of 20 mM PrK or 5 mM pAzF (except for the Ser decoding experiment). SerRS indicates charging by the endogenous E. coli synthetase. The minus sign indicates the tRNA PylMeans the absence of PylRS in cells having it, or the absence of ectopically expressed tRNA. Retention in the rows indicated by § corresponds to culturing, from which sfGFP is further purified and analyzed by LC-MS / MS and / or Western blot of TAMRA-conjugated sfGFP (see Figure 1F (Ser), Figure 2D (PrK), and Figure 3B (pAzF)); the rows with asterisks correspond to the cultures analyzed in Figure 7A-D. Despite the fact that all four unnatural triphosphates enter the cell via the same transporter, thereby competitively inhibiting each other's uptake, differences in UBP retention were not observed in the presence (+) or absence (-) of NaMTP and / or TPT3TP in the medium. These data, and the requirement for both unnatural ribotriphosphates for high-level sfGFP expression with high-fidelity PrK incorporation (Figure 7A-D), collectively demonstrate that the expression level of the PtNTT2 transporter in YZ3 is required to maintain UBP replication and transcription, and to import an essential level of unnatural triphosphates.

[0013] Table 6 | Yields of sfGFP proteins expressed in the uptake experiments of Ser, Prk, and pAzF. Yields were calculated from the total amount of purified protein and the volume of the culture used for purification (see methods). Data are mean ± s.d. (n = 4 sfGFP samples, purified from cultures propagated from individual colonies), and were determined from the same cultures analyzed in Figure 1F (for SerRS) and Figure 2D (for PylRS), similar to the cultures corresponding to the (+) pAzF sample in Figure 3A (for pAzFRS). The yields of purified sfGFP are comparable to the average total fluorescence (OD 600 not normalized to) of the cultures from which they were purified. Fluorescence values correspond to the time point when cells were collected for sfGFP purification. See Figure 1C (Ser), Figure 2B (PrK), and Figure 3A (pAzF).

Mode for Carrying Out the Invention

[0014] 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 restrictive of the claimed subject matter. In this application, the use of the singular includes the plural unless specifically stated otherwise. It should be noted that as used in the 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 specifically stated otherwise. Further, the use of the term “including” is without limitation, as are other forms such as “include,” “includes,” and “included.”

[0015] As used herein, ranges and amounts can be expressed as “about” a particular value or range. “About” also includes the exact amount. Thus, “about 5 μL” means both “about 5 μL” and “5 μL.” In general, the term “about” includes amounts that are expected to be within experimental error.

[0016] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0017] Summary The information of life is encoded by a four-letter genetic alphabet, which is made possible by the selective formation of two base pairs: (d)G-(d)C and (d)A-dT / U. The formation of a third unnatural base pair (UBP) between two synthetic nucleotides expands this system, thereby increasing the potential for information storage and having potential academic and practical implications. Among the various synthetic nucleotide analogs reported, some pair stably with each other within other native DNA double helices but are not recognized by polymerases, indicating that the forces governing stable pairs in double-stranded DNA are not the same as those governing polymerase-mediated replication. As a result, various approaches have been taken to develop replicable UBPs, such as those designed to interact via complementary hydrogen-bond (H-bond) patterns not utilized by natural nucleotides. Although natural base pairs are formed via H-bonds, there is no reason to deductively assume that H-bonds are the only forces sufficient to underlie the storage (or retrieval) of genetic information. For example, it has been demonstrated that the Klenow fragment of Escherichia coli DNA polymerase I (Kf) pairs dA with the unnatural nucleotide dF, whose difluorotoluene nucleobase has a mimetic shape of thymine where significant H-bonding is not possible. This supports the "geometric selection" mechanism of DNA replication and suggests that forces other than H-bonds also contribute to replication.

[0018] The in vitro development of UBP that is replicated, transcribed, and translated into proteins provides insights into the forces underlying the storage and retrieval of natural information and enables broad applications in chemical and synthetic biology. However, the ultimate goal of much of the effort to develop UBP is its in vivo use as the basis for semi-synthetic organisms (SSOs), organisms that stably store and retrieve increased (meaning non-natural or synthetic, created by humans) information. Furthermore, such SSOs have innovative practical applications, including those related to human health. Most notably, SSOs are revolutionizing the growth of the field of protein therapy. However, compared to conventional small molecule therapies, protein therapies have severely limited molecular properties due to the limited chemical diversity available with the 20 natural amino acids.

[0019] We recently reported the creation of an E. coli SSO that imports the necessary non-natural triphosphates from the medium by the nucleoside triphosphate transporter from the marine pennate diatom (Phaeodactylum tricornutum) (PtNTT2) and then uses them to replicate a plasmid containing UBP dNaM-dTPT3. Since then, we have shown that DNA containing UBP can be transcribed in the SSO by T7 RNA polymerase and that various tRNAs containing ncAAs charged and containing cognate non-natural nucleotides in the anticodon can efficiently and selectively decode non-natural codons when non-natural nucleotides are incorporated into the codons of mRNA. Since UBP can be combined at various positions of various codons, this suggests that it is possible to use UBP to encode proteins with many different ncAAs.

[0020] This specification discloses, in certain embodiments, methods, compositions, and kits for the synthesis of proteins containing unnatural amino acids using mutant tRNAs. In some examples, the proteins are synthesized in a cell-free translation system. In some examples, the proteins are synthesized in a cell or a semi-synthetic organism (SSO). In some examples, the semi-synthetic organism includes a microorganism. In some examples, the semi-synthetic organism includes a bacterium. In some examples, the semi-synthetic organism includes Escherichia coli. In some examples, the mutant tRNA contains a mutant anticodon sequence. In some examples, the mutant anticodon sequence is the anticodon sequence exemplified in Table 1. In some examples, the mutant anticodon sequence is the anticodon sequence exemplified in Table 2. In some examples, the mutant anticodon sequence is the anticodon sequence exemplified in Table 3.

[0021]

Table 1

[0022]

Table 2

[0023]

Table 3

[0024] In some examples, the mutant anticodon of the mutant tRNA pairs with a mutant codon. In some embodiments, the mutant codon is the mutant codon exemplified in Table 1. In some embodiments, the mutant codon is the mutant codon exemplified in Table 2. In some embodiments, the mutant codon is the mutant codon exemplified in Table 3.

[0025] In some embodiments, Y and X exemplified in Tables 1, 2, and 3 represent unnatural bases of unnatural nucleotides. In some embodiments, the unnatural bases are 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl (I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidine, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorine-added cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl derivatives of adenine and guanine, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine, 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and adenine and guanine substituted with 8-hydroxyl;N4-Ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, nucleic acids with disrupted hybridization, nucleic acids with size expansion, fluorine-treated nucleic acids, tricyclic pyrimidines, phenoxazines, cytidine ([5,4-b][1,4]benzoxazin-2(3H)-one, phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one, G-clamp, 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), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one), 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxyhydroxymethyluracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5’-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, wybutosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl ester of uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyluracil, (acp3)w, and 2,6-diaminopurine, and those in which the purine base or pyrimidine base is replaced by a heterocyclic compound, are selected from the group consisting of;

[0026] In some examples, the unnatural nucleotide is selected from the group consisting of the following formulas (only showing the nucleobase moiety, and omitting the ribose and phosphate backbones for clarity).

[0027]

Chem.

[0028] In some examples, the unnatural nucleotide is selected from the group consisting of the following formulas (only showing the nucleobase moiety, and omitting the ribose and phosphate backbones for clarity).

[0029]

Chem.

[0030] In some examples, the unnatural nucleotide further comprises an unnatural sugar moiety. In some examples, the unnatural sugar moiety is a modification at the 2'-position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkyl, 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-Oalkyl, 2'-F, 2'-OCH3, 2'-O(CH2)2OCH3, selected from the group consisting of, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkyl, C2-C 10 alkenyl, C2-C 10It may also be alkynyl, -O[(CH2)nO]mCH3, -O(CH2)nOCH3, -O(CH2)nNH2, -O(CH2)nCH3, -O(CH2)n-ONH2, and -O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10; and / or the unnatural sugar moiety is a modification at the 5' position: 5'-vinyl, 5'-methyl (R or S), a modification at the 4' position, 4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage group, a reporter group, an intervening agent, 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, selected from the group consisting of.

[0031] In some examples, the mutant anticodon or mutant codon further comprises an unnatural backbone. In some examples, the mutant anticodon further comprises an unnatural backbone. In some examples, the mutant codon further comprises an unnatural backbone. In some instances, the unnatural backbone is selected from the group consisting of phosphorothioate, chiral phosphorothioate, dithiophosphate, phosphotriesterase, aminoalkyl phosphotriesterase, C1-C 10 Phosphonate, 3'-alkylene phosphonate, chiral phosphonate, phosphinate, phosphoramidate, 3'-aminophosphoramidate, aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriesterase, and boranophosphate.

[0032] In some examples, non-natural nucleotides are recognized by a polymerase. In some examples, the polymerase is a DNA polymerase, an RNA polymerase, or a reverse transcriptase. In some examples, the polymerase is Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, ThermoSequenase®, 9°Nm™, Therminator™ DNA polymerase, Tne, Tma, TfI, Tth, TIi, Stoffel fragment, Vent® and Deep Vent® DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA polymerase, PGB-D, UlTma DNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III, archaeal DP1I / DP2 DNA polymerase II, 9°N DNA polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA polymerase, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, and SuperScript® III reverse transcriptase.

[0033] In some examples, the polymerase is DNA polymerase 1-Klenow fragment, Vent polymerase, Phusion® DNA polymerase, KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase, Therminator™ DNA polymerase, POLB polymerase, SP6 RNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, or SuperScript® III reverse transcriptase.

[0034] In some examples, unnatural nucleotides are incorporated during transcription into mRNA by a polymerase to produce mutant mRNA containing mutant codons. In some examples, unnatural nucleotides are incorporated during transcription into mRNA by a polymerase to produce mutant mRNA.

[0035] In some examples, unnatural nucleotides are incorporated during transcription into tRNA by a polymerase to produce mutant tRNA containing mutant anticodons. In some examples, unnatural nucleotides are incorporated during transcription into tRNA by a polymerase to produce mutant tRNA.

[0036] In some examples, mutant tRNA represents an unnatural amino acid residue. In some examples, the unnatural amino acid residue is an unnatural amino acid as described in Liu C.C., Schultz, P.G. Annu. Rev. Biochem. 2010, 79, 413.

[0037] In some examples, proteins containing unnatural amino acids are produced during translation using mutant tRNA and mutant mRNA. In some examples, proteins containing unnatural amino acids are produced in a cell-free translation system. In some examples, the protein is synthesized in a cell or a semi-synthetic organism (SSO). In some examples, the semi-synthetic organism includes a microorganism. In some examples, the semi-synthetic organism includes a bacterium. In some examples, the semi-synthetic organism includes Escherichia coli.

[0038] Nucleic acid A nucleic acid (e.g., also referred to herein as a target nucleic acid, target nucleotide sequence, desired nucleic acid sequence, or desired nucleic acid region) can be any source or composition, such as, for example, DNA, cDNA, gDNA (genomic DNA), RNA, siRNA (short interfering RNA), RNAi, tRNA, or mRNA, and can be in any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). A nucleic acid can contain nucleotides, nucleosides, or polynucleotides. A nucleic acid can include natural and non-natural nucleic acids. A nucleic acid can further include non-natural nucleic acids such as analogs of DNA or RNA (e.g., base analogs, sugar analogs, and / or non-natural backbones, etc.). The term "nucleic acid" is understood to refer to or mean a particular length of a polynucleotide chain, and thus polynucleotides and oligonucleotides are also included in 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. With respect to RNA, the uracil base is uridine. Nucleic acids are often vectors, plasmids, phages, autonomously replicating sequences (ARS), centromeres, artificial chromosomes, yeast artificial chromosomes (e.g., YAC), or other nucleic acids that are capable of replicating or being replicated. Non-natural nucleic acids can be nucleic acid analogs.

[0039] Non-natural nucleic acid Nucleotide analogs (i.e., unnatural nucleotides) include nucleotides containing some modification in either the base, sugar, or phosphate moiety. The modification can include chemical modification. The modification can be, for example, a modification of the 3’OΗ or 5’OΗ group, backbone, sugar component, or nucleotide base. The modification can include the addition of unnatural linker molecules and / or inter- or intra-strand crosslinks. In one aspect, the modified nucleic acid includes one or more modifications of the 3’OΗ or 5’OΗ group, backbone, sugar component, or nucleotide base, and / or the addition of an unnatural linker molecule. In one aspect, the modified backbone includes a backbone other than the phosphodiester backbone. In one aspect, the modified sugar includes a sugar other than deoxyribose (in modified DNA) or ribose (in modified RNA). In one aspect, the modified base includes 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).

[0040] The nucleic acid can include at least one modified base. Modifications of the base moiety will include natural and synthetic modifications of A, C, G, and T / U, as well as different purine or pyrimidine bases. In some embodiments, the modification is to a modified form of adenine, guanine, cytosine, or thymine (in modified DNA), or adenine, guanine, cytosine, or uracil (in modified RNA).

[0041] Modified bases of unnatural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl, cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl of adenine and guanine and other alkyl derivatives, 2-propyl of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil, 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, especially 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. Specific 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 double-strand formation, universal nucleic acids, hydrophobic nucleic acids, nucleic acids with intermixed hybridization, nucleic acids with size expansion, fluorine-treated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and purines substituted at N-2, N-6, and O-6 including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl of adenine and guanine and other alkyl derivatives, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl(-C≡C-CI 1 / 4Uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azauracil, 6-azacytosine, 6-azathymine, 5-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, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8-azadenine, 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-clamp, 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-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin-2-one), purine or pyrimidine bases 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, 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 U.S. Patent Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882;U.S. Patent Nos. 5,093,232; 5,130,302; 5,134,066; 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; 5,645,985; 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, J.I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and, Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, Crooke, S.T. and Lebleu, B., Eds., CRC Press, 1993, 273-288. Additional base modifications are found, for example, in U.S. Patent No. 3,687,808, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B. ed., CRC Press, 1993.;

[0042] Unnatural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, and such nucleic acids can contain one or more heterocyclic bases other than the five major base components of natural nucleic acids. For example, heterocyclic bases include uracil-5-yl, cytosine-5-yl, adenine-7-yl, adenine-8-yl, guanine-7-yl, guanine-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, and purines are attached to the sugar moiety of the nucleic acid via the 9-position, pyrimidines are attached to the sugar moiety of the nucleic acid via the 1-position, pyrrolopyrimidines are attached to the sugar moiety of the nucleic acid via the 7-position, and pyrazolopyrimidines are attached to the sugar moiety of the nucleic acid via the 1-position.

[0043] Nucleotide analogs can also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, phosphate moieties such that the linkage between two nucleotides is phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl, and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, and boranophosphate. These phosphate linkages or modified phosphate linkages between two nucleotides may occur via a 3'-5' linkage or a 2'-5' linkage, and it will be understood that the linkage may include reverse polarities such as from 3'-5' to 5'-3' or from 2'-5' to 5'-2'. Also included are various salts, mixed salts, and free acid forms. Many U.S. patents teach how to make and use nucleotides containing modified phosphates, including, but not limited to, U.S. Patent Nos. 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; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is incorporated herein by reference.

[0044] Non-natural nucleic acids can include 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, 60, 788-789; Wang et al, Bioorganic & Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al, Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et al, 1995, 14(3-5), 901-905; and Eppacher et al, Helvetica Chimica Acta, 2004, 87, 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 using modified bases (Wang et al, Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

[0045] Unnatural nucleic acids can include modifications at the 5'- and 2'-positions of the sugar ring (PCT / US94 / 02993), for example, 5'-CH2 substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al. Bioconjugate Chem. 1999, 10, 921-924). Unnatural nucleic acids can include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleoside (from 5' to 3') in the dimer contains 2'-OCH3 and 5'-(S)-CH3 (Mesmaeker et al, Synlett, 1997, 1287-1290). Unnatural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Unnatural nucleic acids can include 5'-methylene phosphonate DNA and RNA monomers, and dimers (Bohringer et al, Tet. Lett., 1993, 34, 2723-2726; Collingwood et al, Synlett, 1995, 7, 703-705; and, Hutter et al, Helvetica Chimica Acta, 2002, 85, 2777-2806). Unnatural nucleic acids can include 5'-phosphonate monomers having 2'-substitutions (US2006 / 0074035), and other modified 5'-phosphonate monomers (WO97 / 35869). Unnatural nucleic acids can include 5'-modified methylene phosphonate monomers (EP614907 and EP629633).Non-natural nucleic acids can include analogs of 5'- or 6'-phosphonate ribonucleosides containing a hydroxyl group at the 5'- or 6'-position (Chen et al, Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al, Bioorg. Med. Chem., 2000, 8, 2501-2509, Gallier et al, Eur. J. Org. Chem., 2007, 925-933 and Hampton et al, J. Med. Chem., 1976, 19(8), 1029-1033). Non-natural nucleic acids can include 5'-phosphonate deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et al, Oligonucleotides, 2006, 16(1), 68-82). Non-natural nucleic acids can include nucleosides having a 6'-phosphonate group, and the 5'- or / and 6'-position is unsubstituted or substituted with 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, 4, 467-472; Kappler et al, J. Med. Chem., 1986, 29, 1030-1038 and J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et al, J. Med. Chem., 1987, 30, 888-894; Hampton et al, J. Med. Chem., 1976, 19, 1371-1377; Geze et al, J. Am. Chem. Soc, 1983, 105(26), 7638-7640 and Hampton et al, J. Am. Chem. Soc, 1973, 95(13), 4404-4414).

[0046] Non-natural nucleic acids can include modifications to the sugar moiety. The nucleic acids of the present invention can optionally include one or more nucleosides, and the sugars are modified. Such sugar-modified nucleosides can confer enhanced nuclease stability, increased binding affinity, or some other beneficial 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 (including 5' and / or 2' substituents); crosslinking of two ring atoms to form bicyclic nucleic acids (BNAs); replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C 12 alkyl, or a protecting group); and combinations thereof. Examples of chemically modified sugars can be found in WO 2008 / 101157, US2005 / 0130923, and WO 2007 / 134181.

[0047] Modified nucleic acids can include 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, and the sugar "analog" cyclopentyl group. The sugar can be in pyranosyl or furanosyl form. The sugar moiety can be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkyl ribose, and the sugar can be attached to each heterocyclic base in either the [α] or [β] 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 and 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" where the above sugars or analogs are attached to heterocyclic bases (nucleic acid bases) is known. Sugar modifications can be made using other modifications and can be combined with other modifications.

[0048] Modifications of the sugar moiety include natural modifications of ribose and deoxyribose, as well as non-natural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2'-position: OH; F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C 10 alkenyl, or C2-C 10 alkynyl and alkyl. The 2'-sugar modifications further 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)J2, where n and m range from 1 to about 10.

[0049] Other modifications at the 2'-position include, but are not limited to, C1-C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavage 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 other substituents having similar properties. Similar modifications can also be made at other positions on the sugar, particularly at the 3'-terminal nucleotide or at the 3'-position of the sugar in a 2'-5' linked oligonucleotide, and at the 5'-position of the 5'-terminal nucleotide. Modified sugars further include those containing modifications with crosslinking rings such as oxygen with CH2 and S. Nucleotide sugar analogs can have a sugar mimetic such as a cyclobutyl moiety in the place of the pentofuranosyl sugar. There are many U.S. patents teaching the preparation of such modified sugar structures, for example, U.S. Pat. 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; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 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 hereby incorporated by reference in its entirety and details and describes a series of base modifications.Each of these patents is incorporated herein by reference.

[0050] Examples of nucleic acids having 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 can be selected from allyl, amino, azido, thio, O-allyl, and O-C C10 alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-O-N(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ), where R m and R n are each independently H or substituted or unsubstituted C1-C 10 alkyl.

[0051] In certain embodiments, the nucleic acids of the invention include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid includes a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the nucleic acids provided herein include one or more bicyclic nucleic acids, where the bridge includes a 4'–2' bicyclic nucleic acid. Such 4'–2' bicyclic nucleic acids include, but are not limited to, one of the following formulas: 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' and 4'-CH(CH2OCH3)-0-2', and analogs thereof (U.S. Patent No. 7,399,845, issued July 15, 2008); 4'-C(CH3)(CH3)-0-2' and analogs thereof (WO2009 / 006478, WO2008 / 150729, US2004 / 0171570, U.S. Patent No. 7,427,672, Chattopadhyaya, et al, J. Org. Chem., 2009, 74, 118-134), and WO2008 / 154401, published December 8, 2008).For example, Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al, Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al, J. Am. Chem. Soc, 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al, Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8, 1-7; Oram et al, Curr. Opinion Mol Ther., 2001, 3, 239-243; U.S. Patent Nos. 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; International Applications WO2004 / 106356, WO1994 / 14226, WO2005 / 021570, and WO2007 / 134181; U.S. Patent Publications US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618; U.S. Patent Serial Nos. 12 / 129,154, 60 / 989,574, 61 / 026,995, 61 / 026,998, 61 / 056,564, 61 / 086,231, 61 / 097,787, 61 / 099,844; and PCT International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922, PCT / DK98 / 00393; and U.S. Patents 4,849,513; 5,015,733; 5,118,800; and 5,118,802. See also.

[0052] In certain embodiments, the nucleic acid can include linked nucleic acids. The nucleic acids can be joined together using any internucleic acid bond. Two main classes of internucleic acid bonding groups are defined by the presence or absence of a phosphorus atom. Representative phosphorous-containing internucleic acid bonds include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorous-containing internucleic acid bonds include, but are not limited to, methylene methylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thiocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and, N,N-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In certain embodiments, internucleic acid bonds having chiral atoms can be prepared as racemic mixtures with another enantiomer, for example, alkylphosphonate and phosphorothioate. The unnatural nucleic acid can contain a single modification. The unnatural nucleic acid can contain multiple modifications within one of the plurality of moieties or between different moieties.

[0053] Modifications of the backbone phosphates to the nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridged or unbridged), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate and can be used in any combination. Other non-phosphate bonds can also be used.

[0054] In some embodiments, backbone modifications (e.g., methyl phosphonate, phosphorothioate, phosphoramidate, and phosphorodithioate nucleotide internucleic acid bonds) can impart immunomodulatory activity to the modified nucleic acid and / or enhance its in vivo stability.

[0055] Phosphite derivatives (or modified phosphate groups) can be attached to a sugar or sugar analog moiety and can be monophosphates, diphosphates, triphosphates, alkylphosphonates, phosphorothioates, phosphorodithioates, phosphoramidates, etc. Exemplary polynucleotides containing modified phosphate linkages or non-phosphate linkages can be found 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, (DJ. Chadwick and G. Cardew, ed.) John Wiley and Sons, New York, NY; (Zon (1993) “Oligonucleoside Phosphorothioates” in Protocols for Oligonucleotides and Analogs, Synthesis and Properties (Agrawal, ed.) 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); Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179.

[0056] Skeletal modifications include replacing the phosphodiester bond with alternative moieties such as anionic groups, neutral groups, or cationic groups. Examples of such modifications include: anionic internucleoside linkages; N3’-P5’ phosphoramidate modifications; boranophosphate DNA; pro-oligonucleotides; neutral internucleoside linkages such as methyl phosphonate; amide bond DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; skeletons containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligos (Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179). Modified nucleic acids can contain chimeric or mixed skeletons that include one or more modifications, such as combinations of phosphate linkages, for example, combinations of phosphodiester and phosphorothioate linkages.

[0057] The phosphate substituents can be, for example, short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. These include 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; alkenes containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 components. Many 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; 5,470,967; 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, each of which is incorporated herein by reference. It is also understood in nucleotide substitution that both the sugar and phosphate moieties of the nucleotide can be replaced, for example, with an amide-type linkage (aminoethylglycine) (PNA). U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNA molecules, each of which is incorporated herein by reference. (Similarly, Nielsen et al., see also Science, 1991, 254, 1497-1500). The conjugate can be chemically linked to a nucleotide or nucleotide analog. Such conjugates include, but are not limited to, cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioether (e.g., hexyl-S-tritylthiol) (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), thiolcholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), aliphatic chain, e.g., dodecanediol or undecyl residue (Saison-Behmoaras et al., EM5OJ, 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycerol-S-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmitoyl moiety (Mishra et al., Biochem.(Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Many US patents teach the preparation of such conjugates, including, but not limited to: US Patent No. 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; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667; 5,514,785; 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, each of which is hereby incorporated herein by reference in its entirety.

[0058] Polymerase One particularly useful function of polymerase is to catalyze the polymerization of nucleic acid strands using an existing nucleic acid as a template. Other useful functions are described elsewhere in this specification. Examples of useful polymerases include DNA polymerase and RNA polymerase.

[0059] The ability to improve the specificity, processing ability, or other characteristics of polymerase for non-natural nucleic acids is highly desirable in a variety of contexts where non-natural nucleic acid incorporation is desired, including, for example, amplification, sequencing, labeling, detection, cloning, and many others. The present invention provides polymerases having modified properties for non-natural nucleic acids, methods of making such polymerases, methods of using such polymerases, and many other features that will become apparent upon a complete review of the following.

[0060] In some examples, disclosed herein are, for example, polymerases that incorporate non-natural nucleic acids into growing template copies during DNA amplification. In some embodiments, the polymerase can be modified to modify the active site of the polymerase to reduce steric hindrance of non-natural nucleic acids to the active site. In some embodiments, the polymerase can be modified to provide complementarity with one or more non-natural features of the non-natural nucleic acid. Accordingly, the present invention includes compositions comprising heterologous or recombinant polymerases and methods of using them.

[0061] Polymerases can be modified using methods related to protein engineering. For example, molecular modeling can be performed based on the crystal structure to identify positions in the polymerase where mutations can be made to alter the target activity. Residues identified as targets for substitution can be exchanged with the selected residues using energy minimization modeling, homology modeling, and / or conservative amino acid substitutions as described, for example, in Bordo, et al. J Mol Biol 217: 721-729 (1991) and Hayes, et al. Proc Natl Acad Sci, USA 99: 15926-15931 (2002).

[0062] Any of a variety of polymerases can be used in the methods or compositions described herein, including, for example, protein-based enzymes isolated from biological systems and their functional variants. References to specific polymerases, such as those exemplified below, will be understood to include their functional variants unless otherwise specified. In some embodiments, the polymerase is a wild-type polymerase. In some embodiments, the polymerase is a modified polymerase or a mutant polymerase.

[0063] Polymerases having features for improving the entry of unnatural nucleic acids into the active site region and for coordinating with unnatural nucleotides in the active site region can also be used. In some embodiments, the modified polymerase has a modified nucleotide binding site.

[0064] In some embodiments, the modified polymerase has specificity for unnatural nucleic acids that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the wild-type polymerase for unnatural nucleic acids. In some embodiments, the modified polymerase or wild-type polymerase has specificity for unnatural nucleic acids containing modified sugars that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the wild-type polymerase for natural and / or unnatural nucleic acids that do not contain modified sugars. In some embodiments, the modified polymerase or wild-type polymerase has specificity for unnatural nucleic acids containing modified bases that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the wild-type polymerase for natural and / or unnatural nucleic acids that do not contain modified bases. In some embodiments, the modified polymerase or wild-type polymerase has specificity for unnatural nucleic acids containing triphosphates that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the wild-type polymerase for natural nucleic acids containing triphosphates and / or unnatural nucleic acids that do not contain triphosphates. For example, the modified wild-type polymerase or wild-type polymerase has specificity for unnatural nucleic acids containing triphosphates that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity of the wild-type polymerase for unnatural nucleic acids having diphosphates or monophosphates, unnatural nucleic acids having no phosphates, or combinations thereof.

[0065] In some embodiments, the modified polymerase or wild-type polymerase has relaxed specificity for non-natural nucleic acids. In some embodiments, the modified polymerase or wild-type polymerase has specificity for non-natural nucleic acids and at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity for natural nucleic acids of the wild-type polymerase for natural nucleic acids. In some embodiments, the modified polymerase or wild-type polymerase has specificity for non-natural nucleic acids containing modified sugars and at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity for natural nucleic acids of the wild-type polymerase for natural nucleic acids. In some embodiments, the modified polymerase or wild-type polymerase has specificity for non-natural nucleic acids containing modified bases and at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the specificity for natural nucleic acids of the wild-type polymerase for natural nucleic acids.

[0066] Absence of exonuclease activity can be a wild-type characteristic or a characteristic conferred by a mutant or recombinant polymerase. For example, exo-minus Klenow fragment is a mutant form of the Klenow fragment lacking 3’-5’ proofreading exonuclease activity.

[0067] The method of the present invention can be used to extend the substrate range of any DNA polymerase that lacks endogenous 3'-5' exonuclease proofreading activity or has, for example, 3'-5' exonuclease proofreading activity inactivated by mutation. Examples of DNA polymerases include polA, polB (see, e.g., Parrel & Loeb, Nature Struc Biol 2001), polC, polD, polY, polX, and reverse transcriptase (RT), but preferably a polymerase with high processivity and fidelity (PCT / GB2004 / 004643). In some embodiments, the modified polymerase or wild-type polymerase substantially lacks 3'-5' proofreading exonuclease activity. In some embodiments, the modified polymerase or wild-type polymerase substantially lacks 3'-5' proofreading exonuclease activity with respect to non-natural nucleic acids. In some embodiments, the modified polymerase or wild-type polymerase has 3'-5' proofreading exonuclease activity. In some embodiments, the modified polymerase or wild-type polymerase has 3'-5' proofreading exonuclease activity with respect to natural nucleic acids and substantially lacks 3'-5' proofreading exonuclease activity with respect to non-natural nucleic acids.

[0068] In some embodiments, the modified polymerase has 3’-to-5’ proofreading exonuclease activity that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the proofreading exonuclease activity of the wild-type polymerase. In some embodiments, the modified polymerase has 3’-to-5’ proofreading exonuclease activity for non-natural nucleic acids that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the proofreading exonuclease activity of the wild-type polymerase for natural nucleic acids. In some embodiments, the modified polymerase has 3’-to-5’ proofreading exonuclease activity for non-natural nucleic acids and 3’-to-5’ proofreading exonuclease activity for natural nucleic acids that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the proofreading exonuclease activity of the wild-type polymerase for natural nucleic acids. In some embodiments, the modified polymerase has 3’-to-5’ proofreading exonuclease activity for natural nucleic acids that is at least about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% of the proofreading exonuclease activity of the wild-type polymerase for natural nucleic acids.

[0069] In related aspects, the present invention provides a method for producing a modified polymerase, the method comprising structurally modeling a parent polymerase (e.g., a DNA polymerase), identifying one or more complex stabilities, or nucleotide interaction features that affect the stability of a complex or nucleotide access or binding at the active site, or complementary features to nucleotide analogs at the active site, and mutating the parent polymerase to include or remove these features, thereby providing a method for producing a modified polymerase. For example, the polymerase can be mutated to improve the steric access of non-natural nucleotides to the active site or to improve the charge-charge or hydrophobic interactions between the non-natural nucleotides and the polymerase. The method also includes determining whether the resulting modified polymerase exhibits an increase in the incorporation of nucleotides or non-natural nucleotides into growing nucleic acid copies as compared to the parent polymerase.

[0070] Polymerases can be characterized according to their rate of dissociation from nucleic acids. In some embodiments, the polymerase has a relatively low rate of dissociation from one or more natural and non-natural nucleic acids. In some embodiments, the polymerase has a relatively high rate of dissociation from one or more natural and non-natural nucleic acids. The dissociation rate is the activity of the polymerase that can be adjusted to match the reaction rate by the methods described herein.

[0071] When a polymerase is used with a specific natural nucleic acid and / or unnatural nucleic acid, or a collection of natural nucleic acids and / or unnatural nucleic acids, it can be characterized according to their fidelity. Fidelity generally refers to the accuracy with which a polymerase incorporates the correct nucleic acid into the growing nucleic acid strand when making a copy of a nucleic acid template. When natural and unnatural nucleic acids compete for strand synthesis at the same site in the polymerase-strand template nucleic acid binary complex, for example, when present at equal concentrations, the fidelity of a DNA polymerase can be measured as the ratio of correct natural and unnatural nucleic acid incorporation to incorrect natural and unnatural nucleic acid incorporation. DNA polymerase fidelity can be calculated as the ratio of (kcat / KM) of natural and unnatural nucleic acids to (k cat / K m ) of incorrect natural and unnatural nucleic acids: where k cat and k m are Michaelis-Menten parameters in steady-state enzyme kinetics (Fersht, A. R. (1985) Enzyme Structure and Mechanism, 2nd ed., p 350, W. H. Freeman & Co., New York., incorporated herein by reference). In some embodiments, the polymerase has a fidelity value of at least about 100, 1000, 10,000, 100,000, or 1x10 6 regardless of the presence or absence of proofreading activity.

[0072] Polymerases from natural sources or variants thereof can be screened using an assay that detects the incorporation of unnatural nucleic acids having a specific structure. In one example, the polymerase can be screened for its ability to incorporate an unnatural nucleic acid or UBP; for example, d5SICSTP, dNaMTP, or d5SICSTP-dNaMTP UBP. Polymerases that exhibit modified properties of unnatural nucleic acids compared to wild-type polymerases, such as heterologous polymerases, can be used. For example, the modified properties can be, for example, K m , k cat , V max, polymerase processing ability in the presence of non-natural nucleic acids (or naturally occurring nucleotides), average template read length by polymerase in the presence of non-natural nucleic acids, polymerase specificity for non-natural nucleic acids, binding rate of non-natural nucleic acids, release rate of products (such as pyrophosphate, triphosphate, etc.), branching rate, or any combination thereof. In one embodiment, the modified property is a decrease in the k m of non-natural nucleic acids and / or an increase in the k cat / K m or V max / K m of non-natural nucleic acids. Similarly, the polymerase optionally has an increase in the binding rate of non-natural nucleic acids, an increase in the product release rate, and / or a decrease in the branching rate compared to the wild-type polymerase.

[0073] At the same time, the polymerase can incorporate natural nucleic acids, such as A, C, G, T, into the growing nucleic acid copy. For example, the polymerase has a specific activity of natural nucleic acids that is at least about 5% higher (e.g., 5%, 10%, 25%, 50%, 75%, 100% or more) than the corresponding wild-type polymerase and a processing ability of natural nucleic acids in the presence of a template that is at least 5% higher (e.g., 5%, 10%, 25%, 50%, 75%, 100% or more) than the wild-type polymerase in the presence of natural nucleic acids. Optionally, the polymerase has a k cat / K m or V max / K m for naturally occurring nucleotides that is at least about 5% higher (e.g., about 5%, 10%, 25%, 50%, 75%, or 100% or more) than the wild-type polymerase.

[0074] The polymerases used herein that can have the ability to incorporate non-natural nucleic acids of a particular structure can also be generated using directed evolution approaches. Nucleic acid synthesis assays can be used to screen for polymerase mutants that are specific for any of a variety of non-natural nucleic acids. For example, polymerase mutants can be screened for their ability to incorporate into nucleic acids a non-natural nucleic acid or UBP; for example, dTPT3, a dNaM analog, or a dTPT3-dNaM UBP. In some embodiments, such assays are in vitro assays that use, for example, recombinant polymerase mutants. Such directed evolution techniques can be used to screen for mutants of polymerases that are active against any of the non-natural nucleic acids described herein.

[0075] The modified polymerase of the described composition can optionally be a modified and / or recombinant Φ29-type DNA polymerase. Optionally, the polymerase can be a modified and / or recombinant Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

[0076] Generally useful nucleic acid polymerases in the present invention include DNA polymerases, RNA polymerases, reverse transcriptases, and mutants or variant forms thereof. DNA polymerases and their properties are described in detail, inter alia, in DNA Replication 2nd edition, Kornberg and Baker, W. H. Freeman, New York, N. Y. (1991). Known conventional DNA polymerases useful in the present invention include, but are not limited to, Pyrococcus furiosus (Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1, Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaels et al., 1996, Biotechniques, 20:186-8, Boehringer Mannheim), Thermus thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991, Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase (Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32), Thermococcus litoralis (Thermococcus litoralis) (Tli) DNA polymerase (also called Vent™ DNA polymerase, Cariello et al, 1991, Polynucleotides Res, 19: 4193, New England Biolabs), 9°N m™ DNA polymerase (New England Biolabs), Stoffel fragment, Thermo Sequenase® (Amersham Pharmacia Biotech UK), Therminator™ (New England Biolabs), Thermotoga maritima (Tma) DNA polymerase (Diaz and Sabino, 1998 Braz J Med.Res, 31:1239), Thermus aquaticus (Taq) DNA polymerase (Chien et al, 1976, J. Bacteriol, 127: 1550), DNA polymerase, Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (Thermococcus sp. JDF-3, from International Patent Application Publication No. 0132887), Pyrococcus GB-D (PGB-D) DNA polymerase (also called Deep Vent™ DNA polymerase, Juncosa-Ginesta et al., 1994, Biotechniques, 16:820, New England Biolabs), Ultma DNA polymerase (from the thermophilic Thermotoga maritima; Diaz and Sabino, 1998 Braz J. Med. Res, 31:1239; PE Applied Biosystems), Tgo DNA polymerase (from Thermococcus gorgonarius, Roche Molecular Biochemicals), Escherichia coli DNA polymerase I (Lecomte and Doubleday, 1983, Polynucleotides Res. 11:7505), T7 DNA polymerase (Nordstrom et al, 1981, J Biol. Chem. 256:3112), and archaeal DP1I / DP2 DNA polymerase II (Cann et al, 1998, Proc. Natl. Acad. Sci.(such as US Patent No. 9,514,250). Both mesophilic and thermophilic polymerases are contemplated. Thermophilic DNA polymerases include, but are not limited to, ThermoSequenase®, 9°N™, Therminator™, Taq, Tne, Tma, Pfu, TfI, Tth, Tli, Stoffel fragment, Vent® and Deep Vent® DNA polymerases, KOD DNA polymerase, Tgo, JDF-3, and mutants, variants, and derivatives thereof. Polymerases that are mutants lacking 3′ exonuclease activity are contemplated. Reverse transcriptases useful in the present invention include, but are not limited to, reverse transcriptases from HIV, HTLV-I, HTLV-II, FeLV, FIV, SIV, AMV, MMTV, MoMuLV, and other retroviruses (see Levin, Cell 88:5-8 (1997); Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et al, CRC Crit Rev Biochem. 3:289-347 (1975)). Further, examples of polymerases include, but are not limited to, 9°N DNA polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase, and VentR® DNA polymerase (Gardner et al. (2004) “Comparative Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase (J. Biol. Chem.,279(12), 11834-11842; Gardner and Jack “Determinants of nucleotide sugar recognition in an archaeon DNA polymerase” Nucleic Acids Research, 27(12) 2545-2553.) including. Polymerases isolated from non-thermophilic organisms can be heat-inactivated. An example is the DNA polymerase from phage. It will be understood that polymerases from any of various sources can be modified to increase or decrease their resistance to high-temperature conditions. In some embodiments, the polymerase can be thermophilic. In some embodiments, the thermophilic polymerase can be heat-inactivated. Thermophilic polymerases are typically useful for high-temperature conditions or thermal cycling conditions such as those utilized in polymerase chain reaction (PCR) techniques.

[0077] In some embodiments, the polymerase includes Φ29, B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, ThermoSequenase®, 9°Nm™, Therminator™ DNA polymerase, Tne, Tma, TfI, Tth, TIi, Stoffel fragment, Vent® and Deep Vent® DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA polymerase, PGB-D, UlTma DNA polymerase, Escherichia coli DNA polymerase I, Escherichia coli DNA polymerase III, archaeal DP1I / DP2 DNA polymerase II, 9°N DNA polymerase, Taq DNA polymerase, Phusion® DNA polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA polymerase, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (MMLV) reverse transcriptase, SuperScript® II reverse transcriptase, and SuperScript® III reverse transcriptase.

[0078] In some embodiments, the polymerase is DNA polymerase 1 - Klenow fragment, Vent polymerase, Phusion™ DNA polymerase, KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase, Therminator™ DNA polymerase, POLB polymerase, SP6 RNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (MMLV) reverse transcriptase, SuperScript™ II reverse transcriptase, or SuperScript™ III reverse transcriptase.

[0079] In addition, such polymerases can be used in DNA amplification and / or sequencing applications, including, for example, real-time applications in the context of amplification or sequencing that involve incorporation of non-natural nucleic acid residues into DNA by the polymerase. In other embodiments, the incorporated non-natural nucleic acid can be the same as the natural residue, for example, in which case the label or other moiety of the non-natural nucleic acid is removed by the action of the polymerase during incorporation, or the non-natural nucleic acid can have one or more features that distinguish it from the natural nucleic acid.

[0080] Since at least the last universal common ancestor of all life on Earth, genetic information has been stored in a four-letter alphabet that is propagated and retrieved by the formation of two base pairs. A central goal of synthetic biology is to create new living organisms and functions, and the most common route to this goal is to create semi-synthetic organisms (SSOs) whose DNA bears two additional letters that form a third unnatural base pair (UBP). Previously, our efforts to generate such SSOs resulted in the creation of strains of Escherichia coli, thanks to a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2), which imports the necessary unnatural triphosphates from the medium and then uses them to replicate plasmids containing the UBP dNaM-dTPT3 (Figure 1A). SSOs store increased information but do not retrieve it, which requires in vivo transcription of the UBP into mRNA and tRNA, aminoacylation of tRNA with unnatural amino acids, and finally, efficient participation of the UBP in decoding by ribosomes. Here, we report in vivo transcription of DNA containing dNaM and dTPT3 into mRNA with two different unnatural codons and tRNA with cognate unnatural anticodons, and their efficient decoding by ribosomes to direct site-specific incorporation of natural or non-canonical amino acids (ncAAs) into superfolder green fluorescent protein (sfGFP). The results demonstrate that interactions other than hydrogen bonding can contribute to all steps of information storage and retrieval. The resulting SSOs should encode and retrieve increased information and function as a platform for the creation of new living organisms and functions.

[0081] Green fluorescent protein and variants such as sfGFP have served as model systems for studies on ncAA incorporation using an amber suppression system that includes position Y151, which has been shown to tolerate various natural and ncAAs (Figure 4). To investigate the decoding of non-natural codons, we first focused on the incorporation of Ser at position 151 of sfGFP. This is because Escherichia coli serine aminoacyl-tRNA synthetase (SerRS) does not rely on anticodon recognition for tRNA aminoacylation and thus eliminates potential complications of inefficient charging. The SSO strain YZ3 was transformed with a plasmid encoding the sfGFP and E. coli tRNA Ser gene (serT), and the sfGFP codon 151 (TAC) was replaced with the non-natural codon AXC (sfGFP(AXC) 151 ; X = NaM), and the anticodon of serT was replaced with the non-natural anticodon GYT (tRNA Ser (GYT); Y = TPT3 (Figure 1B). The transformants were grown in medium supplemented with dNaMTP and dTPT3TP and then further supplemented with NaMTP and TPT3TP, as well as isopropyl-β-D-thiogalactoside (IPTG) to induce the expression of T7 RNA polymerase (T7 RNAP) and tRNA Ser (GYT). After a short period of tRNA induction, anhydrotetracycline (aTc) was added to induce the expression of sfGFP(AXC) 151 .

[0082] After induction, cells transformed with a control plasmid encoding sfGFP(AXC) 151 but lacking tRNA Ser (GYT) showed dramatically reduced fluorescence compared to cells transformed with a plasmid encoding sfGFP with a natural Ser codon at position 151 (sfGFP(AGT) 151 ; Figure 1C). Furthermore, cell growth was likely due to ribosome stalling and sequestering, sfGFP(AXC) 151Reached a plateau upon induction of (Figure 1D). Lysates of these cells were subjected to Western blot using an anti-GFP antibody, which revealed a significant decrease in sfGFP expression and the presence of sfGFP cleaved at the position of the unnatural codon (Figure 1E). In contrast, cells transformed with plasmids encoding both sfGFP(AXC) 151 and tRNA Ser (GYT) showed fluorescence almost equal to that of control cells expressing sfGFP(AGT) 151 (Figure 1C), and cell growth did not reach a plateau upon induction of sfGFP(AXC) 151 (Figure 1D), and Western blot of lysates from these cells revealed only the full-length sfGFP protein (Figure 1E). Furthermore, we evaluated the ability of all four natural near-cognate tRNAs (tRNA Ser (GNT); N = G, C, A, or T) expressed in the same manner to decode the AXC codon. In all cases, little fluorescence was observed and growth abnormalities remained (Figures 5A and 5B). These data demonstrate that PtNTT2 can incorporate both deoxyribophosphates and ribophosphates of both unnatural nucleotides, that T7 RNA polymerase can transcribe mRNAs and tRNAs containing unnatural nucleotides in vivo, and that ribosomes can efficiently decode unnatural codons using unnatural anticodons.

[0083] To evaluate decoding fidelity, we analyzed proteins purified from cells expressing both sfGFP(AXC) 151 and tRNA Ser (GYT) by LC / MS-MS and performed relative quantification by peak intensity. This revealed incorporation of 98.5 ± 0.7% (95% CI, n = 4) of Ser at position 151, with Ile / Leu being the major impurities (Figure 1F and Table 4). sfGFP(AXC) 151The retention of UBP in the gene was 98 ± 2% (95% CI, n = 4) (Table 5), and considering that X→T is typically the major mutation during replication (resulting in the Ile codon ATC in the case of AXC), we conclude that the majority of the protein lacking Ser at position 151 is due to the loss of UBP during replication and that the fidelity of translation using the unnatural codon is high.

[0084] [Table 4]

[0085] [Table 5]

[0086] To demonstrate the encoding of ncAAs with UBP, we constructed plasmids similar to those used above but with the gene replaced by the Methanosarcina mazei tRNA Ser (GYT). The tRNA Pyl can be selectively loaded by the Methanosarcina barkeri pyrrolysine aminoacyl-tRNA synthetase (PylRS) with the ncAA N Pyl -[(2-propynyloxy)carbonyl]-L-lysine (PrK). In addition to the codon AXC, we also analyzed the codon GXC and the corresponding tRNA 6 (GYC). SSOs carrying a separate plasmid encoding IPTG-inducible PylRS were transformed with the required plasmids and grew regardless of the addition of PrK. sfGFP(AXC) Pyl or sfGFP(GXC) Pyl in the absence of either PylRS, the cognate unnatural tRNA 151 or PrK 151In control experiments using cells expressing any of these, we observed only plateaus in low cellular fluorescence (Figure 2A), truncation of sfGFP (Figures 6A and 6B), and cell proliferation (Figure 6B). In contrast, for any unnatural mRNA with its cognate unnatural tRNA, when PylRS was present and PrK was added, we observed high fluorescence (64% and 69% for AXC and GXC of sfGFP(TAC) 151 respectively) (Figures 2A and 2B), robust production of full-length sfGFP (Figure 6A), and normal growth (Figure 6B).

[0087] To verify the incorporation of PrK, sfGFP was affinity purified from cell lysates using a C-terminal Strep-tag II and exposed to copper-catalyzed click chemistry to attach a carboxytetramethylrhodamine (TAMRA) dye (TAMRA-PEG4-N3). Since it was found that the electrophoretic mobility of sfGFP shifted during SDS-PAGE, the fidelity of PrK incorporation could be evaluated by Western blot (Figure 2C). We observed a strong TAMRA signal, and when cultured in medium supplemented with PrK, sfGFP(AXC) 151 and tRNA Pyl (GYT) or sfGFP(GXC) 151 and tRNA Pyl (GYC), we observed that substantially all of the sfGFP shifted (Figure 2C). In contrast, when NaMTP, TPT3TP, or both were absent, little or no TAMRA signal or shifted sfGFP was observed (Figures 7A and 7B). Finally, no TAMRA signal or shifted sfGFP was observed in proteins purified from cells expressing sfGFP(TAC) 151 with any unnatural tRNA (Figure 2C). This data demonstrates that PrK is specifically incorporated into sfGFP by decoding an unnatural codon with a tRNA having an unnatural anticodon.

[0088] We purified 54 ± 4 and 55 ± 6 μg / mL of sfGFP (s.d., n = 4, sfGFP(TAC) with AXC and GXC codons, respectively) with optimal PrK enrichment (Figures 8A - 8D). 151 ~40% of the control (Table 6). Furthermore, based on mass spectrometry, the purity of sfGFP with PrK was 96.2 ± 0.3% (95% CI, n = 4) with the AXC codon and 97.5 ± 0.7% (95% CI, n = 4) with the GXC codon (Figure 2D). The yield of the purified sfGFP protein was slightly lower than that of amber suppression (87 ± 6 μg / mL, s.d., n = 4 (Table 6)) due to a moderate decrease in growth by the addition of unnatural ribotriphosphates (Figures 7C and 7D). Decoding of both unnatural codons resulted in higher fluorescence than amber suppression when normalized to cell density (Figures 2A and 2B), suggesting that decoding with unnatural codons is more efficient than amber suppression.

[0089] To examine the coding of other ncAAs with UBP, we investigated the coding of p - azido - phenylalanine (pAzF) with the AXC codon and an evolved Methanocaldococcus jannaschii TyrRS / tRNA Tyr pair (pAzFRS / tRNA pAzF ). By induction of the synthetase and addition of pAzF to the growth medium, we observed robust fluorescence equivalent to that of cells expressing native sfGFP(TAC) 151 and normal growth of sfGFP(AXC) 151 with tRNA pAzF (GYT) (Figure 3A, Figure 9). We purified full - length sfGFP (86 ± 6 μg / mL, s.d., n = 4; 68% of the sfGFP(TAC) control, Table 6) and performed copper - free click chemistry using the dibenzocyclooctyl (DBCO) group to conjugate TAMRA (TAMRA - PEG4 - DBCO). We used sfGFP(AXC) 151 and tRNA 151 and tRNA pAzFStrong binding of TAMRA to sfGFP was observed when isolated from cells expressing (GYT) and cultured in the presence of pAzF (Figure 3B). We were unable to accurately assess the fidelity of pAzF incorporation due to degradation of the azide moiety, but ~93% of the sfGFP protein was shifted, which is superior compared to ~95% of the shifted sfGFP generated by amber suppression (Figure 3B).

[0090]

Table 6

[0091] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions are presently contemplated by those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. The following claims define the scope of the present invention, and methods and structures within the scope of these claims and their equivalents are intended to be embraced thereby.

Claims

1. 1. A method for producing a protein that contains an unnatural amino acid, the method comprising: preparing a mutant tRNA, said mutant tRNA comprising a mutant anticodon sequence selected from Table 1 or 2; - preparing a mutant mRNA, said mutant mRNA comprising a mutant codon sequence selected from Table 1 or 2; and - synthesizing proteins containing unnatural amino acids using mutant tRNAs and mutant mRNAs The method according to claim 1, further comprising:

2. The method of claim 1, wherein the protein is synthesized in a cell-free translation system.

3. 2. The method of claim 1, wherein the protein is synthesized in a cell (semisynthetic organism or SSO).

4. 4. The method of claim 3, wherein the semisynthetic organism comprises a microorganism.

5. 5. The method of claim 3 or 4, wherein the semisynthetic organism comprises a bacterium.

6. 6. The method of any one of claims 3 to 5, wherein the semisynthetic organism comprises Escherichia coli.

7. 7. The method of any one of claims 1 to 6, wherein the mutant anticodon of the mutant tRNA is paired with a mutant codon selected from Tables 1-3.

8. 8. The method of any one of claims 1 to 7, wherein the unnatural amino acid comprises at least one unnatural nucleotide.

9. 9. The method of any one of claims 1 to 8, wherein the non-natural nucleotide is a non-natural nucleobase.

10. The non-natural bases of the non-natural nucleotides include 2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, 3-deazaguanine, 3-deazaadenos ... uracil, 4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthine-9-yl (I), 5-methyl-cytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 5-bromo, and 5-trifluoromethyluracil and cytosine; 5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynylcytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted pyrimidines, 5-hydroxycytosine. , 5-bromocytosine, 5-bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-iodouracil, 6-alkyl groups of adenine and guanine. Derivatives, 6-azapyrimidine, 6-azo-uracil, 6-azo-cytosine, azacytosine, 6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine, 7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine, 7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and guanines;N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, increasing duplex stability, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines, phenoxazines, cytidines ([5,4-b][1,4]benzoxazin-2(3H)-ones, phenothiazine cytidines (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-ones, G-clamps, phenoxazine cytidines (9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-ones, benzoxazin-2(3H)-one, carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindol cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-carboxyhydroxymethyluracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β- D-galactosyl eosin, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosyl eosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyl adenine, uracil-5-oxyacetic acid, Wibe 10. The method according to claim 1, wherein the uridine derivative is selected from the group consisting of tosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyluracil, (acp3)w, and 2,6-diaminopurine, and those in which the purine or pyrimidine base is replaced by a heterocyclic compound;

11. 10. The method of any one of claims 1 to 9, characterized in that the non-natural nucleotide is selected from the group consisting of the following formulas (only the nucleobase portion is shown, the ribose and phosphate backbones are omitted for clarity): 【Chemistry 1】

12. 10. The method of any one of claims 1 to 9, characterized in that the non-natural nucleotide is selected from the group consisting of the following formulas (only the nucleobase portion is shown, the ribose and phosphate backbones are omitted for clarity): 【Chemistry 2】

13. 13. The method of any one of claims 1 to 12, wherein the unnatural nucleotide further comprises an unnatural sugar moiety.

14. The unnatural sugar moiety of the unnatural nucleotide may be modified at the 2' position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH ３ , OCN, Cl, Br, CN, CF ３ , O.C.F. ３ , SOCH ３ , S.O. ２ , C.H. ３ , O.N.O. ２ , NO ２ , N ３ , N.H. ２ F; 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 ３ , 2′-O(CH ２ ) ２ OCH ３ wherein alkyl, alkenyl, and alkynyl are selected from the group consisting of 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 N.H. ２ , -O(CH ２ ) ｎ CH ３ , -O(CH ２ ) ｎ -ONH ２ , and -O(CH ２ ) ｎ ON [(CH ２ ) ｎ CH ３ ] ２ and n and m are from 1 to about 10; and / or the unnatural sugar moiety is selected from the group consisting of a modification at the 5' position: 5'-vinyl, 5'-methyl (R or S), a modification at the 4' position: 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.

15. 15. The method of any one of claims 1 to 14, wherein the mutant anticodon or the mutant codon further comprises a non-natural backbone.

16. 15. The method of any one of claims 1 to 14, wherein the mutant anticodon and the mutant codon further comprise a non-natural backbone.

17. 17. The method of any one of claims 1 to 16, wherein the non-natural nucleotide is recognized by a DNA polymerase, an RNA polymerase, or a reverse transcriptase.

18. 18. The method of any one of claims 1 to 17, wherein the non-natural nucleotide is incorporated into mRNA by RNA polymerase during transcription to generate a mutant mRNA containing a mutant codon.

19. 19. The method of any one of claims 1 to 18, wherein the non-natural nucleotide is incorporated into the tRNA by RNA polymerase during transcription to generate a mutant tRNA containing a mutant anticodon.

20. 20. The method of any one of claims 1 to 19, wherein the non-natural nucleotides are incorporated into mRNA by RNA polymerase during transcription to generate a mutant mRNA.

21. 21. The method of any one of claims 1 to 20, wherein the non-natural nucleotide is incorporated into the tRNA by RNA polymerase during transcription to generate a mutant tRNA.

22. 22. The method of any one of claims 1 to 21, wherein the mutant tRNA is charged with an unnatural amino acid residue.

23. 23. The method of any one of claims 1-22, wherein the moiety containing an unnatural amino acid is generated using the mutant tRNA and the mutant mRNA during transcription.

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