Incorporation of non-natural nucleotides and methods thereof

Abstract

To provide methods, compositions and kits for synthesis of proteins containing unnatural amino acids that utilize a mutant tRNA.SOLUTION: A method of producing a protein containing an unnatural amino acid comprises: preparing a mutant tRNA, where the mutant tRNA comprises a specific mutant anticodon sequence; preparing a mutant mRNA, where the mutant mRNA comprises the specific mutant codon sequence; and synthesizing the protein containing an unnatural amino acid utilizing the mutant tRNA and the mutant mRNA.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 July 11, 2017, which is incorporated by reference in its entirety. [Background technology]

[0002] Oligonucleotides and their applications have revolutionized biotechnology, however, oligonucleotides, including both DNA and RNA, each contain only the four naturally occurring nucleotides adenosine (A), guanosine (G), cytosine (C), and thymine (T) in DNA, and the four naturally occurring nucleotides adenosine (A), guanosine (G), cytosine (C), and uridine (U) in RNA, which severely limits the potential functions and applications of oligonucleotides.

[0003] For example, the ability to sequence-specifically synthesize / amplify oligonucleotides (DNA or RNA) using polymerases (e.g., transcription using T7 RNA polymerase) via PCR or isothermal amplification systems has revolutionized biotechnology. In addition to all of the potential applications in nanotechnology, this has enabled a diverse range of new techniques such as the in vitro development of RNA and DNA aptamers and enzymes via SELEX (Systematic Evolution of Ligands by Exponential Enrichment). 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 restricted by the limited chemical / physical diversity that exists 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.) Disclosed herein are additional methods for generating nucleic acids containing an expanded genetic alphabet. Summary of the Invention

[0005] Disclosed herein, in certain embodiments, is a method for producing a protein containing an unnatural amino acid, the method comprising the steps of: preparing a mutant tRNA, the mutant tRNA comprising a mutant anticodon sequence selected from Table 1 or 2; preparing a mutant mRNA, the mutant mRNA comprising a mutant codon sequence selected from Table 1 or 2; and synthesizing a protein containing an unnatural 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 comprises a microorganism. In some examples, the semi-synthetic organism comprises a bacterium. In some examples, the semi-synthetic organism comprises E. coli. In some examples, the mutant anticodon of the mutant tRNA is paired with a mutant codon selected from Tables 1-3. In some examples, the unnatural amino acid comprises at least one unnatural nucleotide. In some examples, the unnatural nucleotide comprises an unnatural nucleobase. In some instances, the unnatural base of the unnatural nucleotide is 2-aminoadenine-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, hypoxanthine-9-yl (I), 5-amino-2'-deoxy-2'-propane, 5-amino ... -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, fluoropyrimidines, 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 ine), 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, those that increase duplex stability, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated 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]pyrro 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-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-Mannosil-Eosin, 5'-Methoxycarboxymethyluracil, 5-Methoxyuracil, 2-Methylthio-N6-Isopentenyladenine, Uracil-5-oxyacetate, Wybutosin, Pseudouracil, Queosin, 2-Thiocytosine, 5-Methyl-2-Thiouracil, 2-Thiouracil, 4-Thiocytosine, The non-natural nucleotide is selected from the group consisting of uracil, 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. In some examples, the non-natural nucleotide is selected from the group consisting of the following formulas (only the nucleobase portion is shown, with the ribose and phosphate backbones omitted for clarity):

[0006] [ka]

[0007] In some instances, the non-natural nucleotide is selected from the group consisting of the following formulas (only the nucleobase portion is shown, with the ribose and phosphate backbones omitted for clarity):

[0008] [ka]

[0009] In some instances, the unnatural nucleotide further comprises an unnatural sugar moiety. In some instances, the unnatural sugar moiety of the unnatural nucleotide is selected from the group consisting of a modification at the 2' position: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, 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, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C1-C2-C3 ... 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) nCH3]2, where 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 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 any combination thereof. In some instances, the mutant anticodon or mutant codon further comprises an unnatural backbone. In some instances, the mutant anticodon and mutant codon further comprise an unnatural backbone. In some instances, the unnatural nucleotide is recognized by a DNA polymerase, an RNA polymerase, or a reverse transcriptase. In some instances, the unnatural nucleotide is incorporated into an mRNA by an RNA polymerase during transcription to generate a mutant mRNA containing the mutant codon. In some instances, unnatural nucleotides are incorporated into tRNA by RNA polymerase during transcription to generate a mutant tRNA containing a mutant anticodon. In some instances, unnatural nucleotides are incorporated into mRNA by RNA polymerase during transcription to generate a mutant mRNA. In some instances, unnatural nucleotides are incorporated into tRNA by RNA polymerase during transcription to generate a mutant tRNA. In some instances, the mutant tRNA is charged with an unnatural amino acid residue. In some instances, a protein containing an unnatural amino acid is produced using a mutant tRNA and a mutant mRNA during translation. [Brief description of the drawings]

[0010] Various aspects of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings: [Figure 1A]1 illustrates the chemical structure of the dNaM-dTPT3 UBP and the natural dA-dT base pair. [Figure 1B] The gene cassettes used to express sfGFP(AXC)151 and tRNA(GYT)Ser are illustrated. PT7 and TT7 refer to the T7 RNAP promoter and terminator, respectively. In the control where sfGFP is expressed in the absence of serT, the sequence following the sfGFP T7 terminator is not present. [Figure 1C] Figure 1 illustrates a graph of fluorescence of cells expressing sfGFP and tRNASer, where the position 151-codon and anticodon are indicated, respectively. The minus sign denotes the absence of serT in the expression cassette. t=0 corresponds to the addition of IPTG to induce expression of T7 RNAP and tRNASer (if present); aTc was added at t=0.5 h to induce expression of sfGFP. AGT, natural Ser codon; TAG, amber stop codon; CTA amber suppressor anticodon. Data are shown as mean ± sd, n=4 cultures, each propagated from an individual colony. [Figure 1D] Illustrates a graph of the growth of cells expressing sfGFP and tRNASer, where position 151-codon and anticodon are indicated, respectively. The minus sign denotes the absence of serT in the expression cassette. t=0 corresponds to the addition of IPTG to induce expression of T7 RNAP and tRNASer (if present); aTc was added at t=0.5 h to induce expression of sfGFP. AGT, natural Ser codon; TAG, amber stop codon; CTA amber suppressor anticodon. Data are shown as mean ± sd, n=4 cultures, each propagated from an individual colony. [Figure 1E] Illustrated are Western blots (normalized by OD600) of lysates from cells collected at the last time points indicated in FIG. 1C and FIG. 1D and probed with an α-GFP antibody (N-terminal epitope). [Figure 1F]1 illustrates a graph of the relative abundance of amino acids (indicated by single letter codes in the figure legend) detected at position 151 of sfGFP purified from cells expressing sfGFP(AGT)151 or sfGFP(AXC)151 and tRNASer(GYT) as determined by LC-MS / MS and quantification based on precursor ion intensity (amino acids detected at <0.1% (on average, for both codons) are not shown; see Methods for details and Table 4 for a complete list of detected amino acids). Data are shown as the average of individual data points, from n=4 purified sfGFP samples, cultures each propagated from an individual colony, collected at the final time points shown in FIG. 1C and FIG. 1D. [Figure 2A] FIG. 2 illustrates a graph of fluorescence of cells expressing sfGFP, where the codon at position 151 is shown, in the presence (+) or absence (−) of tRNAPyl with the cognate anticodon, PylRS, or 20 mM PrK(N6-[(2-propynyloxy)carbonyl]-L-lysine in the medium. Fluorescence was assessed at the last time point indicated in FIG. 2B. Asterisks denote the absence of tRNAPyl in cells expressing sfGFP(TAC). TAC, natural Tyr codon; TAG, amber stop codon; nd, not determined. Data are shown as the average of individual data points, each propagated from an individual colony. [Figure 2B] Figure 2 illustrates a time course analysis of a subset of the data shown in Figure 2A. Plus and minus symbols denote the presence or absence of 20 mM PrK in the medium, respectively. t = 0 corresponds to the addition of IPTG to induce expression of PylRS, T7 RNAP, and tRNAPyl; aTc was added at t = 1 h to induce expression of sfGFP. Data are shown as mean ± sd, n = 4 cultures, each propagated from an individual colony. [Figure 2C]Illustrated are Western blots of sfGFP purified from cells expressing sfGFP and tRNAPyl with the indicated codon and anticodon at position 151, with or without click conjugation of TAMRA and / or addition of 20 mM PrK to the medium, respectively. tRNAPyl is absent in cells expressing sfGFP(TAC)151 (-). sfGFP was purified from cultures harvested at the last time point shown in FIG. 2B. Western blots were probed and imaged with an α-GFP antibody to detect both sfGFP and conjugated TAMRA. [Figure 2D] Illustrated are graphs of the relative abundance of amino acids (indicated by single letter codes in figure legends) at position 151 in sfGFP purified from cells expressing sfGFP(TAC)151 (collected at the last time point shown in FIG. 2B) or in sfGFP and tRNAPyl with the indicated position 151 codon and cognate anticodon, as determined by LC-MS / MS and precursor ion intensity-based quantification, respectively (amino acids detected at <0.1% (average, for all codons) not shown; see Methods for details, and Table 4 for a complete list of detected amino acids). Data are shown as the average of individual data points, from n=4 purified sfGFP samples, cultures each propagated from an individual colony. [Figure 3A]Illustrated is a graph of fluorescence of cells expressing sfGFP(TAC)151 of sfGFP and tRNApAzF with codons and cognate anticodons at the indicated positions 151 in the presence (+) or absence (-) of 5 mM pAzF in the medium, respectively. t=0 corresponds to the addition of IPTG to induce expression of pAzFRS, T7 RNAP, and tRNApAzF; aTc was added at t=0.5 h to induce expression of sfGFP. TAC, natural Tyr codon; TAG, amber stop codon; data are shown as mean ± sd, n=4 cultures, each propagated from an individual colony. Fluorescence observed with sfGFP(AXC)151 in the absence of pAzF is due to charging of tRNApAzF(GYT) with the natural amino acid (presumably Tyr). [Figure 3B] Illustrated are Western blots of sfGFP purified from cells expressing sfGFP and tRNApAzF with the indicated codon and anticodon at position 151, with or without click conjugation of TAMRA and / or addition of 5 mM pAzF to the medium, respectively. Where indicated, minus signs denote the absence of tRNApAzF in cells expressing sfGFP(TAC)151. sfGFP was purified from cultures harvested at the last time point shown in FIG. 3A. Western blots were probed and imaged with an α-GFP antibody to detect both sfGFP and conjugated TAMRA. [Figure 4] Fluorescence of cells expressing sfGFP with various codons at position 151 is illustrated. Cells carrying sfGFP plasmids with the indicated codons at position 151 were grown to an OD600 of 0.5 and induced with IPTG and aTc. Fluorescence measurements were taken 3 hours after induction. Data are shown as the average with individual data points, n=3 cultures split from a single colony and grown in parallel. [Figure 5A]Figure 1 illustrates the decoding of the natural near-cognate AXC codon with graphs of fluorescence of cells expressing sfGFP(AXC)151 with or without tRNASer with the indicated anticodon. Cells were induced as described in Figure 1C and Figure 1D, and fluorescence measurements correspond to the last time points shown in Figure 1C. Values ​​for the GYT anticodon and in the absence of tRNASer (-tRNA) correspond to the same values ​​in Figure 1C and D. Data are shown as mean ± sd, n = 4 cultures, each propagated from an individual colony. [Figure 5B] Figure 1 illustrates the decoding of the native near-cognate AXC codon with a graph of the growth of cells expressing sfGFP(AXC)151 with or without tRNASer with the indicated anticodon. Cells were induced as described in Figure 1C and Figure 1D, and fluorescence measurements correspond to the last time points shown in Figure 1C. Values ​​for the GYT anticodon and in the absence of tRNASer (-tRNA) correspond to the same values ​​in Figure 1C and D. Data are shown as mean ± sd, n = 4 cultures, each propagated from an individual colony. [Figure 6A] Illustrated are Western blots and growth of cells decoding AXC and GXC codons with tRNAPyl. Western blots of lysates (normalized by OD600) from cells carrying the indicated codon at position 151 in the presence (+) or absence (-) of tRNAPyl with the cognate anticodon, PylRS, or 20 mM PrK in the medium. Blots were probed with α-GFP antibody (N-terminal epitope). Cells were induced and harvested at equivalent time points as described in FIG. 2B. [Figure 6B]Illustrated is the growth of the culture analyzed in Figure 6A. The fold change in OD600 between induction of sfGFP (t=1 h) and the final time point is greatest when all components necessary to aminoacylate tRNAPyl are present. The variation in absolute OD600 is due to small variations in cell density at the start of induction of T7 RNAP (and tRNAPyl, if present) (t=0). Data are shown as mean ± sd, n=4 cultures, each propagated from an individual colony. [Figure 7A] Illustrates decoding of AXC and GXC codons with tRNAPyl and cell growth in response to added unnatural ribotriphosphate. Fluorescence of sfGFP (lower panel) purified from cells expressing sfGFP and tRNAPyl with the indicated codon / anticodon at position 151, with or without 20 mM PrK, in the presence (+) or absence (-) of each unnatural ribotriphosphate. Cells were induced as described in FIG. 2B, and fluorescence measurements were taken at the end of induction (~3.5 h) before harvesting cells and purifying sfGFP protein for click conjugation of TAMRA and western blots. [Figure 7B] Illustrates a gel decoding AXC and GXC codons with tRNAPyl in response to added unnatural ribotriphosphate. Western blots were probed with α-GFP antibody and imaged to detect both sfGFP and conjugated TAMRA; all lanes represent sfGFP purified from cells grown with added PrK. Data are shown as averages with individual data points, n=3 cultures, each propagated from an individual colony; nd, not determined. [Figure 7C]Illustrates graphs of fluorescence and growth of cells expressing sfGFP(TAC)151 in the presence (+) or absence (-) of both unnatural deoxyribotriphosphate and each unnatural ribotriphosphate. t=0 corresponds to the addition of IPTG to induce expression of T7 RNAP; aTc was added at t=1 h to induce expression of sfGFP. Data are shown as mean ± sd, n=3 cultures, each propagated from an individual colony. At the concentrations used (see methods), dNaMTP and dTPT3TP do not inhibit cell growth, whereas both unnatural ribotriphosphates, specifically TPT3TP, show some degree of growth inhibition. [Figure 7D] A graph of cell growth corresponding to cultures supplemented with PrK (20 mM) is illustrated, and the fluorescence is shown in FIG. 2B. Cells expressing sfGFP with natural codons were grown without any unnatural triphosphate, whereas cells expressing sfGFP with unnatural codons were grown with both unnatural deoxyribotriphosphate and ribotriphosphate. Data are shown as mean ± sd, n=4 cultures, each propagated from an individual colony. [Figure 8A] Illustrates a gel decoding AXC and GXC codons with tRNAPyl as a function of PrK concentration in the medium. Western blot of purified sfGFP from cells expressing sfGFP and tRNAPyl with the indicated codon / anticodon at position 151 with click conjugation of TAMRA and addition of PrK to the medium at the indicated concentrations. sfGFP was derived and purified from cells collected as described in FIG. 2B. Western blots were probed and imaged with α-GFP antibody to detect both sfGFP and conjugated TAMRA. [Figure 8B]Figure 2 illustrates a graph of the decoding of AXC and GXC codons with tRNAPyl as a function of PrK concentration in the medium. Fluorescence of cells expressing sfGFP and tRNAPyl (measured at the last time point shown in c) with the indicated codon and anticodon at position 151 as a function of PrK concentration in the medium, respectively. Fluorescence values ​​at 0 and 20 mM PrK are the same as the (-) and (+) PrK values ​​shown in Figure 2B, respectively. Data are shown as mean ± sd, n = 4 cultures, each propagated from an individual colony. [Figure 8C] Fluorescence time course analysis is illustrated. For clarity, only one representative culture is shown for each codon / anticodon pair and PrK concentration. Without being bound by theory, we attribute the low level of sfGFP produced in the absence of PrK to the endogenous tRNA and loss of UBP retention in sfGFP for decoding (Table 5). However, the relative amount of sfGFP with PrK (Figure 8A), and the absolute amount of sfGFP expressed (Figures 8B and 8C) increased in a dose-dependent manner with increasing PrK in the medium, eventually resulting in nearly complete PrK incorporation, suggesting that endogenous readthrough of AXC and GXC codons can be efficiently suppressed by sufficient concentrations of charged PrK-tRNAPyl(GYT) or PrK-tRNAPyl(GYC). [Figure 8D] 8C illustrates a time course analysis of cell growth at various concentrations of PrK for the experiment shown in FIG. 8C. [Figure 9] Fluorescence illustrates cell growth of the cultures shown in Figure 3 A. Data are presented as mean ± sd, n = 4 cultures, each propagated from an individual colony.

[0011] Table 4 | Relative abundance of amino acids at position 151 in sfGFP for the experiments described in Figure 1F and Figure 2D. sfGFP purified from cells expressing sfGFP with or without tRNAs with the codon and anticodon at position 151 indicated, respectively, was analyzed by LC-MS / MS. Reporter peptide LEYNFNSHNVX 151 ITADK (X=PrK or any identified natural amino acid except K or R) and LEYNFNSHNVX 151 Extracted MS1 ion intensities for (when X=K or R) are expressed as a percentage of the sum of ion intensities for all observable reporter peptides. Table of values ​​corresponds to the mean relative abundance and 95% CI of all amino acids detected at position 151 of sfGFP, n=4 purified sfGFP samples, each propagated from an individual colony from culture. Values ​​<0.1% (mean, for the codons indicated in the respective figures) are excluded from the data presented in Figure 1F and Figure 2D.

[0012] Table 5 | Retention of UBP. Retention of UBP in plasmids bearing the codon at the indicated position 151 of sfGFP and the anticodon of the indicated tRNA is determined at a time point prior to sfGFP induction and at the end of induction as described in Methods. Values ​​reported are the mean UBP retention over the period of induction (calculated from the retention at these two time points) with ±95% CI, n=4 cultures each propagated from an individual colony, values ​​indicated with an asterisk are excluded, n=3. n / a indicates not applicable (because the relevant sequence is natural or absent). All plasmids were isolated from cultures grown in the presence of 20 mM PrK or 5 mM pAzF (except for Ser decoding experiments). SerRS indicates charging by endogenous E. coli synthetases. The minus sign indicates charging by tRNA PylThis indicates the absence of PylRS in cells with tRNA or the absence of ectopically expressed tRNA. Retention in rows indicated by § corresponds to cultures from which sfGFP was further purified and analyzed by LC-MS / MS and / or Western blot of TAMRA-conjugated sfGFP (see Fig. 1F (Ser), Fig. 2D (PrK), and Fig. 3B (pAzF)); rows with asterisks correspond to cultures analyzed in Fig. 7A-D. Despite the fact that all four unnatural triphosphates enter cells via the same transporter and thereby competitively inhibit each other's import, no differences in UBP retention were observed in the presence (+) or absence (-) of NaMTP and / or TPT3TP in the medium. These data, and the requirement of both unnatural ribotriphosphates for high levels of sfGFP expression accompanied by high fidelity PrK import (Figure 7A-D), collectively demonstrate that expression levels of the PtNTT2 transporter in YZ3 import essential levels of unnatural triphosphates required to sustain UBP replication and transcription.

[0013] Table 6 | Yields of sfGFP protein expressed in Ser, Prk, and pAzF uptake experiments. Yields were calculated from the total amount of purified protein and the volume of culture used for purification (see Methods). Data are means ± sd (n = 4 sfGFP samples, each 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) as the culture corresponding to the (+)pAzF sample in Figure 3A (for pAzFRS). Yields of purified sfGFP are calculated as the average total fluorescence (OD 600 Fluorescence values ​​correspond to the time points at which cells were harvested for sfGFP purification. See Figure 1C (Ser), Figure 2B (PrK), and Figure 3A (pAzF). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Specific Terms Unless otherwise specified, 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 illustrative only and are not intended to limit the scope 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 content clearly dictates otherwise. In this application, the use of "or" means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" is open-ended, 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" includes the exact amount. Thus, "about 5 μL" also means "about 5 μL" and "5 μL." In general, the term "about" includes an amount that is 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] overview 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. A third unnatural base pair (UBP), formed between two synthetic nucleotides, extends this system, thereby increasing the possibility of information storage, with potential academic and practical implications. Among the wide variety of synthetic nucleotide analogs reported, some stably pair with each other in otherwise natural DNA double helices, but are not recognized by polymerases, indicating that the forces governing stable pairing in double-helical DNA are not the same as those governing polymerase-mediated replication. As a result, various approaches have been taken to develop replicable UBPs, for example UBPs designed to interact through complementary hydrogen bond (H-bond) patterns not utilized by natural nucleotides. Although natural base pairs are formed through H-bonds, there is no reason to assume a priori that H-bonds are the only force sufficient to be the basis for the storage (or retrieval) of genetic information. For example, it has been demonstrated that the Klenow fragment of E. coli DNA polymerase I (Kf) pairs with dA through the unnatural nucleotide dF, whose difluorotoluene nucleobase mimics thymine in a manner that does not allow significant H-bonding, supporting a "geometric selection" mechanism for DNA replication and suggesting that forces other than H-bonding are also responsible for replication.

[0018] The development of UBPs that can be replicated, transcribed, and translated into proteins in vitro provides insight into the forces underlying the storage and retrieval of natural information, and further allows for broad applications in chemical and synthetic biology. However, the ultimate goal of many efforts to develop UBPs is their in vivo use as a platform for semisynthetic organisms (SSOs), organisms that stably store and retrieve increased (meaning non-natural or synthetic, created by humans) information. Moreover, such SSOs have innovative practical applications, including for human health. Most notably, SSOs revolutionize the growing field of protein therapeutics. However, compared to traditional small molecule therapeutics, protein therapeutics are severely limited in their molecular properties by the finite chemical diversity available with the 20 natural amino acids.

[0019] We recently reported the construction of an E. coli SSO that imports the required unnatural triphosphates from the medium using a nucleoside triphosphate transporter from the marine pennate diatom Phaeodactylum tricornutum (PtNTT2) and then uses them to replicate a plasmid containing the UBP dNaM-dTPT3. We have since shown that DNA containing the UBP can be transcribed in the SSO by T7 RNA polymerase, and that when the unnatural nucleotide is incorporated into a codon of an mRNA, various tRNAs charged with an ncAA and containing the cognate unnatural nucleotide in the anticodon can efficiently and selectively decode the unnatural codon. Because UBPs can combine at various positions in various codons, this suggests that UBPs can be used to encode proteins with many different ncAAs.

[0020] Disclosed herein, in certain embodiments, are methods, compositions, and kits for the synthesis of proteins containing unnatural amino acids utilizing 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 semisynthetic organism (SSO). In some examples, the semisynthetic organism comprises a microorganism. In some examples, the semisynthetic organism comprises a bacterium. In some examples, the semisynthetic organism comprises E. coli. In some examples, the mutant tRNA comprises a mutant anticodon sequence. In some examples, the mutant anticodon sequence is an anticodon sequence exemplified in Table 1. In some examples, the mutant anticodon sequence is an anticodon sequence exemplified in Table 2. In some examples, the mutant anticodon sequence is an 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 is paired with a mutant codon. In some embodiments, the mutant codon is a mutant codon exemplified in Table 1. In some embodiments, the mutant codon is a mutant codon exemplified in Table 2. In some embodiments, the mutant codon is a mutant codon exemplified in Table 3.

[0025] In some embodiments, Y and X as 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, 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 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 adenines and guanines;N4-ethylcytosine, N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, those that increase duplex formation 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]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 ... Sil, 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 selected from the group consisting of adenine, uracil-5-oxyacetic acid, wybutosine, 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 with a heterocyclic compound;

[0026] In some instances, 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):

[0027] [ka]

[0028] In some instances, 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):

[0029] [ka]

[0030] In some instances, the unnatural nucleotide further comprises a unnatural sugar moiety. In some instances, the unnatural sugar moiety is a modification at the 2' position selected from the group consisting of: OH; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2F; O-alkyl, S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl; O-alkyl-Oalkyl, 2'-F, 2'-OCH3, 2'-O(CH2)2OCH3, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C1-C2 ... 10 Alkyl, C2-C 10 Alkenyl, C2-C 10alkynyl, -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 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.

[0031] In some instances, the mutant anticodon or mutant codon further comprises a non-natural backbone. In some instances, the mutant anticodon further comprises a non-natural backbone. In some instances, the mutant codon further comprises a non-natural backbone. In some instances, the non-natural backbone is a phosphorothioate, a chiral phosphorothioate, a dithiophosphate, a phosphotriesterase, an aminoalkylphosphotriesterase, a C1-C 10 The phosphonate is selected from the group consisting of phosphonates, 3' alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, 3' amino phosphoramidates, aminoalkyl phosphoramidates, thiono phosphoramidates, thiono alkyl phosphonates, thiono alkyl phosphotriesterases, and borano phosphates.

[0032] In some instances, the non-natural nucleotide is recognized by a polymerase, hi some instances, 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 instances, non-naturally occurring nucleotides are incorporated into an mRNA by a polymerase during transcription to generate a mutant mRNA that contains a mutant codon. In some instances, non-naturally occurring nucleotides are incorporated into an mRNA by a polymerase during transcription to generate a mutant mRNA.

[0035] In some instances, non-naturally occurring nucleotides are incorporated into a tRNA by a polymerase during transcription to generate a mutant tRNA that contains a mutant anticodon. In some instances, non-naturally occurring nucleotides are incorporated into a tRNA by a polymerase during transcription to generate a mutant tRNA.

[0036] In some instances, the mutant tRNA displays an unnatural amino acid residue. In some instances, the unnatural amino acid residue is an unnatural amino acid as described in Liu CC, Schultz, PG Annu. Rev. Biochem. 2010, 79, 413.

[0037] In some instances, proteins containing unnatural amino acids are produced during translation using mutant tRNAs and mutant mRNAs. In some instances, proteins containing unnatural amino acids are produced in cell-free translation systems. In some instances, proteins are synthesized in cells or semisynthetic organisms (SSOs). In some instances, semisynthetic organisms include microorganisms. In some instances, semisynthetic organisms include bacteria. In some instances, semisynthetic organisms include E. coli.

[0038] nucleic acid Nucleic acids (e.g., also referred to herein as target nucleic acids, target nucleotide sequences, desired nucleic acid sequences, or desired nucleic acid regions) can be of any source or composition, e.g., DNA, cDNA, gDNA (genomic DNA), RNA, siRNA (short inhibitory RNA), RNAi, tRNA, or mRNA, and can be of any form (e.g., linear, circular, supercoiled, single-stranded, double-stranded, etc.). Nucleic acids can include nucleotides, nucleosides, or polynucleotides. Nucleic acids can include natural and non-natural nucleic acids. Nucleic acids 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 specific length of a polynucleotide chain, and thus, polynucleotides and oligonucleotides are also included in the definition. Exemplary natural nucleotides include, but are not limited to, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural deoxyribonucleotides include dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural ribonucleotides include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP. For 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., YACs), or other nucleic acids that can replicate or be replicated. The non-naturally occurring nucleic acid can be a nucleic acid analog.

[0039] unnatural nucleic acids Nucleotide analogs (i.e., non-natural nucleotides) include nucleotides that contain some modification in either the base, sugar, or phosphate moiety. Modifications can include chemical modifications. Modifications can be, for example, modifications of the 3'OH or 5'OH group, the backbone, the sugar moiety, or the nucleotide base. Modifications can include the addition of non-natural linker molecules and / or interstrand or intrastrand crosslinks. In one embodiment, the modified nucleic acid includes one or more modifications of the 3'OH or 5'OH group, the backbone, the sugar moiety, or the nucleotide base, and / or the addition of non-natural linker molecules. In one embodiment, the modified backbone includes a backbone other than a phosphodiester backbone. In one embodiment, the modified sugar includes a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In one embodiment, modified bases include bases other than adenine, guanine, cytosine, or thymine (in modified DNA) or bases other than adenine, guanine, cytosine, or uracil (in modified RNA).

[0040] The nucleic acid may contain 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 modifications are to modified forms of adenine, guanine, cytosine, or thymine (in modified DNA), or modified forms of adenine, guanine, cytosine, or uracil (in modified RNA).

[0041] Modified bases of non-natural nucleic acids include, but are not limited to, uracil-5-yl, hypoxanthine-9-yl (I), 2-aminoadenine-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl, cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, These include 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-deazadenine, and 3-deazaguanine and 3-deazaadenine. Certain non-natural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyl adenine, 5-propynyl uracil, 5-propynyl cytosine, 5-methyl cytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines, and 2-aminopropyl adenine, 5-propynyl uracil, 5-propynyl cytosine, Uracil and N-2, N-6, and O-6 substituted purines, including 5-propynylcytosine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-C≡C-CI 1 / 4) uracil, 5-propynylcytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azouracil, 6-azocytosine, 6-azothymine, 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-azaadenine, 7-deaza Guanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine 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 (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-ones), carbazole cytidines (2H-pyrimido[4,5-b]indol-2-ones), pyridoins Dolytidine (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. Pat. Nos. 3,687,808; 4,845,205; 4,910,300; and 4,948,882;No. 5,093,232; No. 5,130,302; No. 5,134,066; No. 5,175,273; No. 5,367,066; No. 5,432,272 ;No. 5,457,187;No. 5,459,255;No. 5,484,908;No. 5,502,177;No. 5,525,711;No. 5,552,540 Nos. 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; WO99 / 62923; Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813; The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, JI, Ed., John Wiley & Sons, 1990, 858- 859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, YS, Chapter 15, Antisense Research and Applications, Crooke, ST and Lebleu, B., Eds., CRC Press, 1993, 273-288. Additional base modifications can be found, for example, in U.S. Pat. No. 3,687,808, Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and Sanghvi, YS, Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B. ed., CRC Press, 1993.

[0042] Non-natural nucleic acids containing various heterocyclic bases and various sugar moieties (and sugar analogs) are available in the art, which may contain one or more heterocyclic bases other than the five main 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, in which purines are linked to the sugar moiety of the nucleic acid via the 9-position, pyrimidines via the 1-position, pyrrolopyrimidines via the 7-position, and pyrazolopyrimidines via the 1-position.

[0043] Nucleotide analogs may also be modified at the phosphate moiety. Modified phosphate moieties include, but are not limited to, phosphate moieties in which the bond between two nucleotides may be modified to include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. It will be understood that these phosphate bonds or modified phosphate bonds between two nucleotides may be via a 3'-5' or 2'-5' bond, and that the bond may include reverse polarity, such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Many U.S. patents teach how to make and use nucleotides containing modified phosphates, including, but not limited to, U.S. Patents 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; Nos. 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-naturally occurring nucleic acids 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 with modified bases (Wang et al, Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-337).

[0045] Non-natural nucleic acids can include modifications at the 5'- and 2'-positions of the sugar ring (PCT / US94 / 02993), such as 5'-CH2 substituted 2'-O-protected nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al. Bioconjugate Chem. 1999, 10, 921-924). Non-natural nucleic acids can include amide-linked nucleoside dimers prepared for incorporation into oligonucleotides, where the 3'-linked nucleoside (5' to 3') in the dimer contains a 2'-OCH3 and a 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-1290). Non-natural nucleic acids can include 2'-substituted 5'-CH2 (or O) modified nucleosides (PCT / US92 / 01020). Non-natural nucleic acids can include 5' methylene phosphonate DNA and RNA monomers and dimers (Bohringer et al, Tet. Lett., 1993, 34, 2723-2726; Collingwood et al, Synlett, 1995, 7, 703-705; and Hutter et al, Helvetica Chimica Acta, 2002, 85, 2777-2806). Non-natural nucleic acids can include 5'-phosphonate monomers with 2'-substitutions (US2006 / 0074035) and other modified 5'-phosphonate monomers (WO97 / 35869). The non-naturally occurring nucleic acid may comprise 5' modified methylene phosphonate monomers (EP614907 and EP629633).Non-naturally occurring nucleic acids may include 5'- or 6'-phosphonate ribonucleoside analogs that contain hydroxyl groups at the 5' or 6' positions (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-naturally occurring nucleic acids may include 5'-phosphonate deoxyribonucleoside monomers and dimers that have 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, where the 5' and / or 6'-positions are 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 may include modifications of the sugar moiety. Nucleic acids of the invention may optionally include one or more nucleosides, in which the sugar is modified. Such sugar-modified nucleosides may 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); bridging of two ring atoms to form bicyclic nucleic acids (BNAs); the modification of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R=H, C1-C 12 Examples of chemically modified sugars can be found in WO 2008 / 101157, US2005 / 0130923, and WO 2007 / 134181.

[0047] Modified nucleic acids may contain modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety may be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, and sugar "analog" cyclopentyl groups. The sugar may be in pyranosyl or furanosyl form. The sugar moiety may be a furanoside of ribose, deoxyribose, arabinose, or 2'-O-alkylribose, and the sugar may be linked to the respective 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 may 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" in which the sugar or analog is attached to a heterocyclic base (nucleobase) is known. Sugar modifications may be made with and combined with other modifications.

[0048] Modifications of the sugar moiety include natural modifications of the 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-C6 alkyl groups. 10 Alkenyl, or C2-C 10 2' sugar modifications can 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 are from 1 to about 10.

[0049] Other modifications at the 2' position include, but are not limited to, C1-C 10Examples of suitable modifications include lower 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, RNA cleaving groups, reporter groups, intercalators, groups for improving the pharmacokinetic or pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides, and the 5' position of 5' terminal nucleotide. Modified sugars further include those that contain modifications at the bridging ring oxygens, such as CH2 and S. Nucleotide sugar analogs can have a sugar mimetic, such as a cyclobutyl moiety in place of the pentofuranosyl sugar. There are many U.S. patents which teach the preparation of such modified sugar structures, e.g., U.S. Patents 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; 5,516,786; 5,518,787; 5,518,788; 5,518,789 ... ,519,134;No.5,567,811;No.5,576,427;No.5,591,722;No.5,597,909;No.5,610,30 No. 0; No. 5,627,053; No. 5,639,873; No. 5,646,265; No. 5,658,873; No. 5,670,633; No. 4,84 Nos. 5,205; 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 incorporated herein by reference in its entirety, detailing and describing a range of base modifications.Each of these patents is incorporated herein by reference.

[0050] Examples of nucleic acids with modified sugar moieties include, but are not limited to, nucleic acids containing 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3, and 2'-O(CH2)2OCH3 substituents. Substituents at the 2' position include allyl, amino, azido, thio, O-allyl, and OCClO alkyl, OCF3, O(CH2)2SCH3, O(CH2)2-ON(R m )(R n ), and O-CH2-C(=O)-N(R m )(R n ), where R m and R n each independently represents H or a substituted or unsubstituted C 10 It is an alkyl.

[0051] In certain embodiments, the nucleic acids of the invention comprise one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acids comprise a bridge between the 4' and 2' ribosyl ring atoms. In some embodiments, the nucleic acids provided herein comprise one or more bicyclic nucleic acids, where the bridge comprises a 4' to 2' bicyclic nucleic acid. Such 4' to 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(CHOCH3)-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. USA,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. 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 Publication Nos. US2004 / 0171570, US2007 / 0287831, and US2008 / 0039618; U.S. Patent Serial Nos. See PCT International Application Nos. PCT / US2008 / 064591, PCT / US2008 / 066154, and PCT / US2008 / 068922, PCT / DK98 / 00393; and U.S. Patent Nos. 4,849,513; 5,015,733; 5,118,800; and 5,118,802.

[0052] In certain embodiments, the nucleic acid may include linked nucleic acids. Nucleic acids may be linked together using any internucleic acid linkage. Two main classes of internucleic acid linkage groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates (P=S). Representative non-phosphorus containing internucleic acid linkages include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiesters (-OC(O)-S-), thionocarbamates (-OC(O)(NH)-S-); siloxanes (-O-Si(H)2-O-); and N,N-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). In some embodiments, nucleic acid linkages with chiral atoms can be prepared as separate enantiomers, e.g., alkyl phosphonates and phosphorothioates, as racemic mixtures. Non-natural nucleic acids can contain a single modification. Non-natural nucleic acids can contain multiple modifications within one of the moieties or between different moieties.

[0053] Backbone phosphate modifications to nucleic acids include, but are not limited to, methyl phosphonates, phosphorothioates, phosphoramidates (bridged or non-bridged), phosphotriesters, phosphorodithioates, phosphodithioates, and boranophosphates, which may be used in any combination. Other non-phosphate linkages may also be used.

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

[0055] The phosphorous acid derivative (or modified phosphate group) can be attached to a sugar or sugar analog moiety and can be a monophosphate, diphosphate, triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate, and the like. Exemplary polynucleotides containing modified phosphate or non-phosphate linkages are described in Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al. (1996) Nucleic Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids Res. 24:2966-2973; Matteucci (1997) "Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic Agents, (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) (US Patent No. 5,453,496); Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179.

[0056] Backbone modifications include replacing phosphodiester linkages with alternative moieties such as anionic, neutral, or cationic groups. Examples of such modifications include: anionic internucleoside linkages; N3'-P5' phosphoramidate modifications; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methyl phosphonate; amide-linked DNA; methylene (methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNAs); and positively charged deoxyribonucleic acid guanidine (DNG) oligos (Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179). Modified nucleic acids can include chimeric or mixed backbones containing one or more modifications, e.g., a combination of phosphate linkages, such as a combination of phosphodiester and phosphorothioate linkages.

[0057] The phosphate substituents can be, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others with mixed N, O, S, and CH2 moieties. Many U.S. patents disclose how to make and use these types of phosphate exchangers, including, but not limited to, U.S. Patents 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; 5,471,968; 5,472,102; 5,473,102; 5,474,102; 5,475,102; 5,476,102; 5,477,102; 5,478,102; 5,479,102; 5,480,102; 5,481,102; 5,482,102; 5,483,102; 5,484,102; 5,485,102; 5,486,102; 5,487,102; 5,488,102; 5,489,102; 5,490,102; 5,50 ... Nos. 89,677; 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 by nucleotide substitution that both the sugar and phosphate moieties of the nucleotide can be replaced, for example, with an amide 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. (See also Nielsen et al.(See also, Science, 1991, 254, 1497-1500.) The conjugates can be chemically bound to the nucleotide or nucleotide analogue. Such conjugates include, but are not limited to, cholesterol moieties (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), thioethers (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), thiol cholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 2765-2770), and the like. 533-538), aliphatic chains such as dodecanediol or undecyl residues (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), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl-rac-glycero-SH-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moieties (Mishra et al., Biochem.Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesteryl moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Many United States patents teach the preparation of such conjugates, including, but not limited to, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,8 No. 02; No. 5,138,045; No. 5,414,077; No. 5,486,603; No. 5,512,439; No. 5,578,718; No. 5,608,046; No. 4,587,044; No. 4,605,735 No.4,667,025;No.4,762,779;No.4,789,737;No.4,824,941;No.4,835,263;No.4,876,335;No.4,904,582;No.4,958,013;No. No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022; No. 5,254,469; No. 5, No. 258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873; No. 5,317,098; No. 5,371,241; No. 5,391,723; No. 5,416,203; No. 5,451 Nos. 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 incorporated herein by reference in its entirety.

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

[0059] The ability to improve the specificity, processivity, or other characteristics of polymerases for non-naturally occurring nucleic acids is highly desirable in a variety of contexts in which non-naturally occurring nucleic acid incorporation is desired, including, for example, amplification, sequencing, labeling, detection, cloning, and many others. The present invention provides polymerases with modified properties for non-naturally occurring nucleic acids, methods of making such polymerases, methods of using such polymerases, and many other features that will become apparent after a full discussion below.

[0060] In some examples, disclosed herein include polymerases that incorporate non-naturally occurring nucleic acids into a growing template copy, for example, during DNA amplification. In some embodiments, the polymerase can be modified to modify the active site of the polymerase to reduce steric inhibition of entry of the non-naturally occurring nucleic acid into the active site. In some embodiments, the polymerase can be modified to provide complementarity with one or more non-natural features of the non-naturally occurring nucleic acid. Thus, the invention includes compositions comprising heterologous or recombinant polymerases and methods of their use.

[0061] Polymerases can be modified using methods related to protein engineering. For example, molecular modeling can be performed based on crystal structures to identify positions in the polymerase that can be mutated to change the target activity. Residues identified as targets for substitution can be replaced with selected residues using energy minimization modeling, homology modeling, and / or conservative amino acid substitution, such as those described 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 functional variants thereof. Reference to a particular polymerase, such as those exemplified below, will be understood to include functional variants thereof unless otherwise specified. In some embodiments, the polymerase is a wild-type polymerase. In some embodiments, the polymerase is a modified or mutant polymerase.

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

[0064] In some embodiments, the modified polymerase has a specificity for non-naturally occurring 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 the non-naturally occurring nucleic acids. In some embodiments, the modified polymerase or the wild-type polymerase has a specificity for natural nucleic acids without modified sugars and / or non-naturally occurring 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 the non-naturally occurring nucleic acids. In some embodiments, the modified or wild-type polymerase has a specificity for non-naturally occurring 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 non-naturally occurring nucleic acids that do not contain modified bases. In some embodiments, the modified or wild-type polymerase has a specificity for non-naturally occurring 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 and / or non-naturally occurring nucleic acids that do not contain triphosphates. For example, the modified wild-type polymerase or wild-type polymerase has a specificity for non-naturally occurring nucleic acids that contain 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 non-naturally occurring nucleic acids that have diphosphates or monophosphates, non-naturally occurring nucleic acids that have no phosphates, or combinations thereof.

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

[0066] The lack of exonuclease activity can be a wild-type characteristic or a characteristic conferred by a mutant or recombinant polymerase, for example, the exo-minus Klenow fragment is a mutant form of the Klenow fragment that lacks 3' to 5' proofreading exonuclease activity.

[0067] The methods of the invention can be used to extend the substrate range of any DNA polymerase that lacks endogenous 3-5' exonuclease proofreading activity or that has had its 3-5' exonuclease proofreading activity abolished, for example, 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), preferably high processive fidelity polymerases (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 on non-naturally occurring nucleic acids. In some embodiments, the modified polymerase or the wild-type polymerase has 3' to 5' proofreading exonuclease activity. In some embodiments, the modified polymerase or the wild-type polymerase has 3' to 5' proofreading exonuclease activity towards naturally occurring nucleic acids and substantially lacks 3' to 5' proofreading exonuclease activity towards non-naturally occurring nucleic acids.

[0068] In some embodiments, the modified polymerase has a 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 a 3' to 5' proofreading exonuclease activity towards non-naturally occurring 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 towards naturally occurring nucleic acids. In some embodiments, the modified polymerase has a 3' to 5' proofreading exonuclease activity towards non-naturally occurring nucleic acids and a 3' to 5' proofreading exonuclease activity towards naturally occurring 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 towards naturally occurring nucleic acids. In some embodiments, the modified polymerase has a 3' to 5' proofreading exonuclease activity towards naturally occurring 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 towards naturally occurring nucleic acids.

[0069] In a related aspect, the invention provides a method of producing a modified polymerase, the method comprising the steps of structurally modeling a parent polymerase (e.g., a DNA polymerase), identifying nucleotide interaction features that affect one or more complex stability, or complex stability, 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. For example, the polymerase can be mutated to improve steric access of non-natural nucleotides to the active site, or to improve charge-charge or hydrophobic interactions between the non-natural nucleotide and the polymerase. The method also includes determining whether the resulting modified polymerase exhibits increased incorporation of nucleotides or non-natural nucleotides into growing copies of a nucleic acid, 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 dissociation rate from one or more natural and non-natural nucleic acids. In some embodiments, the polymerase has a relatively high dissociation rate from one or more natural and non-natural nucleic acids. The dissociation rate is an activity of the polymerase that can be adjusted to match reaction rates in the methods described herein.

[0071] Polymerases can be characterized according to their fidelity when used with a particular natural and / or non-natural nucleic acid, or with a collection of natural and / or non-natural nucleic acids. Fidelity generally refers to the accuracy with which a polymerase incorporates a correct nucleic acid into a growing nucleic acid strand when making a copy of the nucleic acid template. When natural and non-natural nucleic acids are present, for example, at equal concentrations to compete for strand synthesis at the same site in the polymerase-strand-template-nucleic acid binary complex, the fidelity of a DNA polymerase can be measured as the ratio of incorporation of correct natural and non-natural nucleic acids to incorporation of incorrect natural and non-natural nucleic acids. DNA polymerase fidelity is calculated by dividing the (kcat / KM) of natural and non-natural nucleic acids by the (kcat / KM) of incorrect natural and non-natural nucleic acids. cat / K m ), where k cat and k m is the Michaelis-Menten parameter in steady-state enzyme kinetics (Fersht, AR (1985) Enzyme Structure and Mechanism, 2nd ed., p 350, WH Freeman & Co., New York., incorporated herein by reference). In some embodiments, the polymerase has a kinetic constant of at least about 100, 1000, 10,000, 100,000, or 1x10, with or without proofreading activity. 6 has a fidelity value of

[0072] Polymerases from natural sources or variants thereof can be screened using assays that detect the incorporation of non-natural nucleic acids having specific structures. In one example, polymerases can be screened for the ability to incorporate non-natural nucleic acids or UBPs; e.g., d5SICSTP, dNaMTP, or d5SICSTP-dNaMTP UBPs. Polymerases, e.g., heterologous polymerases, that exhibit modified properties of non-natural nucleic acids compared to wild-type polymerases can be used. For example, modified properties can be determined using, e.g., K m , k cat , V max, polymerase processivity in the presence of non-naturally occurring nucleic acids (or naturally occurring nucleotides), average template read length by the polymerase in the presence of non-naturally occurring nucleic acids, specificity of the polymerase for non-naturally occurring nucleic acids, binding rate of non-naturally occurring nucleic acids, release rate of products (pyrophosphate, triphosphate, etc.), branching rate, or any combination thereof. In one embodiment, the modified property may be the k m Reduction of k and / or k of non-natural nucleic acids cat / K m Or V max / K m Similarly, the polymerase optionally has an increased rate of binding of non-natural nucleic acids, an increased rate of product release, and / or a decreased rate of branching as compared to a wild-type polymerase.

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

[0074] Polymerases used herein that may 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 polymerase mutants with specificity for any of a variety of non-natural nucleic acids. For example, polymerase mutants can be screened for the ability to incorporate non-natural nucleic acids or UBPs; e.g., dTPT3, dNaM analogs, or dTPT3-dNaM UBPs into nucleic acids. In some embodiments, such assays are in vitro assays, e.g., using recombinant polymerase mutants. Such directed evolution techniques can be used to screen polymerase mutants for activity against any of the non-natural nucleic acids described herein.

[0075] The modified polymerase of the described compositions 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] Nucleic acid polymerases generally useful in the present invention include DNA polymerases, RNA polymerases, reverse transcriptases, and mutant or modified versions thereof. DNA polymerases and their properties are described in detail, inter alia, in DNA Replication 2nd edition, Kornberg and Baker, WH Freeman, New York, NY (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), Bacterioccus vessei (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 (TIi) DNA polymerase (also known as Vent™ DNA polymerase, Cariello et al, 1991, Polynucleotides Res, 19: 4193, New England Biolabs), 9°Nm™ 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. Bacteoriol, 127: 1550), DNA polymerases, Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et al.,1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase (thermococcus sp. JDF-3, from patent application WO 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), E. 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.USA 95:14250). Both mesophilic and thermophilic polymerases are contemplated. Thermophilic DNA polymerases include, but are not limited to, ThermoSequenase®, 9°Nm™, Therminator™, Taq, Tne, Tma, Pfu, TfI, Tth, TIi, Stoffel fragment, Vent™ and Deep Vent™ DNA polymerases, KOD DNA polymerase, Tgo, JDF-3, and mutants, variants, and derivatives thereof. Polymerases that are mutants that are 3' exonuclease deficient 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 Vent® 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.). Polymerases isolated from non-thermophilic organisms may be heat inactivated. An example is DNA polymerase from phage. It will be appreciated that polymerases from any of a variety of sources may be modified to increase or decrease their resistance to high temperature conditions. In some embodiments, the polymerase may be thermophilic. In some embodiments, thermophilic polymerases may 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 is selected from the group consisting of 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 polymerases include 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 the incorporation of non-naturally occurring nucleic acid residues into DNA by a polymerase. In other embodiments, the incorporated non-naturally occurring nucleic acid can be the same as the natural residue, e.g., where a label or other moiety of the non-naturally occurring nucleic acid is removed by the action of the polymerase during incorporation, or the non-naturally occurring nucleic acid can have one or more characteristics that distinguish it from natural nucleic acids.

[0080] Since at least the last 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 life forms and functions, and the most common route to this goal is to create semisynthetic organisms (SSOs) whose DNA harbors two additional letters forming a third unnatural base pair (UBP). Previously, our efforts to generate such SSOs have resulted in the creation of strains of Escherichia coli that, thanks to a nucleoside triphosphate transporter from Phaeodactylum tricornutum (PtNTT2), can import the necessary unnatural triphosphates from the medium and then use them to replicate a plasmid containing the UBP dNaM-dTPT3 (Figure 1A). SSO stores but does not retrieve increased information, which requires the in vivo transcription of UBP into mRNA and tRNA, aminoacylation of tRNA with unnatural amino acids, and finally, efficient participation of UBP in decoding at the ribosome. Here, we report the in vivo transcription of DNA containing dNaM and dTPT3 into mRNA with two different unnatural codons and tRNA with the cognate unnatural anticodon, and their efficient decoding at the ribosome to direct the site-specific incorporation of natural or non-canonical amino acids (ncAA) into superfolder green fluorescent protein (sfGFP). The results demonstrate that interactions apart from hydrogen bonding can contribute to all steps of information storage and retrieval. The resulting SSO should both encode and retrieve increased information and serve as a platform for the creation of new life forms and functions.

[0081] Green fluorescent protein and mutants such as sfGFP served as a model system for studies on ncAA incorporation using an amber suppression system containing position Y151, which has been shown to tolerate a variety of natural and ncAAs (Figure 4). To investigate the decoding of unnatural codons, we first focused on the incorporation of Ser at position 151 in sfGFP because E. coli serine aminoacyl-tRNA synthetase (SerRS) does not rely on anticodon recognition for tRNA aminoacylation, thus eliminating the potential complication of inefficient charging. SSO strain YZ3 was engineered to express sfGFP and E. coli tRNA 151 in a 3′-terminal cytosine-1′-terminal codon. Ser The cells were transformed with a plasmid encoding the gene (serT), in which the sfGFP codon 151 (TAC) was replaced by 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 was replaced (Figure 1B). Transformants were grown in medium supplemented with dNaMTP and dTPT3TP, and then further supplemented with NaMTP and TPT3TP, and isopropyl-β-D-thiogalactoside (IPTG) to induce the expression of T7 RNA polymerase (T7 RNAP) and tRNA. Ser After a short period of tRNA induction, anhydrotetracycline (aTc) was added to induce the expression of sfGFP (AXC). 151 induced the expression of

[0082] After induction, sfGFP(AXC) 151 It encodes tRNA Ser Cells transformed with a control plasmid lacking (GYT) expressed sfGFP with a natural Ser codon at position 151 (sfGFP(AGT) 151 The cells showed dramatically reduced fluorescence compared to cells transformed with a plasmid encoding sfGFP(AXC) (Figure 1C). Furthermore, cell proliferation was enhanced by the sfGFP(AXC) gene, presumably due to ribosome stalling and sequestering. 151Upon induction of sfGFP (Figure 1D), the expression of sfGFP began to plateau. Lysates of these cells were subjected to Western blot analysis using an anti-GFP antibody, which revealed a significant decrease in sfGFP expression and the presence of sfGFP truncated at the unnatural codon (Figure 1E). In contrast, sfGFP(AXC) 151 and tRNA Ser Cells transformed with plasmids encoding both (GYT) and sfGFP(AGT) 151 (Figure 1C) showed nearly equal fluorescence to control cells expressing sfGFP(AXC) 151 (Fig. 1D) without reaching a plateau upon induction, and Western blots of lysates from these cells revealed only full-length sfGFP protein (Fig. 1E). Furthermore, we found that all four native near-cognate tRNAs (tRNA sfGFP) expressed in an identical manner to decode the AXC codon. Ser The ability of PtNTT2 to transfer both deoxytriphosphates and ribotriphosphates of unnatural nucleotides (GNT; N=G, C, A, or T) to unnatural nucleotides was evaluated. In all cases, little fluorescence was observed and growth defects persisted (Figures 5A and 5B). These data demonstrate that PtNTT2 can import both deoxytriphosphates and ribotriphosphates of unnatural nucleotides, that T7 RNA polymerase can transcribe mRNAs and tRNAs containing unnatural nucleotides in vivo, and that ribosomes efficiently decode unnatural codons using unnatural anticodons.

[0083] To assess the fidelity of decoding, we analyzed sfGFP(AXC) by LC / MS-MS. 151 and tRNA Ser Proteins purified from cells expressing both sfGFP(AXC) and sfGFP(GYT) were analyzed for relative quantification by peak intensity, which revealed 98.5 ± 0.7% (95% CI, n = 4) incorporation of Ser at position 151, with Ile / Leu being the major impurity (Figure 1F and Table 4). 151Considering that the retention of the UBP in genes is 98 ± 2% (95% CI, n = 4) (Table 5) and that X → T is typically the major mutation during duplication (resulting in the Ile codon ATC in the case of AXC), we conclude that the majority of proteins that do not contain Ser at position 151 are due to loss of the UBP during duplication and that the fidelity of translation using unnatural codons is high.

[0084] [Table 4]

[0085] [Table 5]

[0086] To demonstrate the encoding of ncAAs with UBPs, we used a tRNA sequence similar to that used above, but Ser The gene is Methanosarcina mazei tRNA Pyl A plasmid was constructed in which the (GYT) gene was replaced with tRNA Pyl is ncAA N 6 10-[(2-propynyloxy)carbonyl]-L-lysine (PrK) can be selectively charged by Methanosarcina barkeri pyrrolysine aminoacyl-tRNA synthetase (PylRS). In addition to the codon AXC, we also found that codon GXC and the corresponding tRNA Pyl (GYC) was also analyzed. SSO carrying a separate plasmid encoding IPTG-inducible PylRS was transformed with the required plasmid and grown with or without the addition of PrK. PylRS, the cognate unnatural tRNA Pyl , or sfGFP(AXC) in the absence of either PrK. 151 or sfGFP(GXC) 151In control experiments with cells expressing either AXC or GXC, we observed low cellular fluorescence (Figure 2A), truncation of sfGFP (Figures 6A and 6B), and only a plateau in cell growth (Figure 6B). In contrast, for either non-natural mRNA with its cognate non-natural tRNA, when PylRS was present and PrK was added, we observed high fluorescence (sfGFP(TAC) for AXC and GXC, respectively) and no significant changes in cell growth (Figure 6C). 151 We observed 64% and 69% (Figures 2A and 2B) of the cells expressing sfGFP, robust production of full-length sfGFP (Figure 6A), and normal growth (Figure 6B).

[0087] To verify PrK uptake, sfGFP was affinity purified from cell lysates using a C-terminal Strep-tag II and subjected to copper-catalyzed click chemistry to attach a carboxytetramethylrhodamine (TAMRA) dye (TAMRA-PEG4-N3), which was found to shift the electrophoretic mobility of sfGFP during SDS-PAGE, allowing us to assess the fidelity of PrK uptake by Western blot (Figure 2C). We observed a strong TAMRA signal and that in cells cultured in medium supplemented with PrK and expressing sfGFP(AXC), 151 and tRNA Pyl (GYT) or sfGFP(GXC) 151 and tRNA Pyl We observed that virtually all of the sfGFP was shifted when purified from cells expressing (GYC) (Figure 2C). In contrast, little or no TAMRA signal or shifted sfGFP was observed in the absence of NaMTP, TPT3TP, or both (Figures 7A and 7B). Finally, no TAMRA signal or shifted sfGFP was observed in sfGFP with any of the non-natural tRNAs (TAC). 151 This was not observed in protein purified from cells expressing PrK (Figure 2C). This data demonstrates that PrK is specifically incorporated into sfGFP by decoding the unnatural codon with a tRNA bearing the unnatural anticodon.

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

[0089] To investigate the coding of other ncAAs with UBPs, we used p-azido-phenylalanine (pAzF) with AXC codon and the evolved Methanocaldococcus yannaschii TyrRS / tRNA Tyr Pair (pAzFRS / tRNA pAzF By inducing the synthetase and adding pAzF to the growth medium, we were able to express the native sfGFP(TAC) 151 The fluorescence was as strong as that of cells expressing sfGFP(AXC) 151 and tRNA pAzF We observed normal growth of GYT (Figure 3A, Figure 9). We purified full-length sfGFP (86 ± 6 μg / mL, sd, n = 4; sfGFP(TAC) 151 68% of the control (Table 6), copper-free click chemistry was performed using dibenzocyclooctyl (DBCO) groups to conjugate TAMRA (TAMRA-PEG4-DBCO). 151 and tRNA pAzFWe observed robust TAMRA binding to sfGFP isolated from cells expressing (GYT) and cultured in the presence of pAzF (Fig. 3B). Although we were unable to precisely assess the fidelity of pAzF incorporation due to degradation of the azide moiety, ∼93% of the sfGFP protein was shifted, which compares favorably with ∼95% shifted sfGFP generated by amber suppression (Fig. 3B).

[0090] [Table 6] Since at least the last common ancestor of all life on Earth, proteins have been generated by decoding codons written exclusively in a four-nucleotide genetic alphabet. We demonstrate here the decoding of two new codons written in an expanded genetic alphabet and use them to site-specifically incorporate ncAAs into proteins. We find that at every step of information storage and retrieval, the hydrogen bonds that are so obviously central to natural base pairs are at least partially replaced by complementary packing and hydrophobic forces. Despite the novel mechanism of decoding, unnatural codons can be decoded as efficiently as their fully natural counterparts. Although we only examined the decoding of two unnatural codons, the UBP is unlikely to be limited to these, and when combined with the recently reported Cas9 editing system that reinforces the retention of the UBP, it will likely make available many more codons than have been used so far. The reported SSO may therefore be just the first of a new form of semisynthetic life that can exploit a variety of forms and functions unavailable to natural organisms.

[0091] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. 1. A kit for producing a protein that contains an unnatural amino acid, the kit comprising: a. Nucleoside triphosphate transporters from diatoms; b. A mutant tRNA, wherein the mutant tRNA is GGY, GYG, YGG, GAY, GYA, YGA, GCY, GYC, YGC, GUY, GY. U, YGU, CAY, CYA, YCA, CGY, CYG, YCG, CUY, CYU, YCU, CCY, CYC, YCC, AAY, AYA, YAA, AGY , AYG, YAG, ACY, AYC, YAC, AUY, AYU, YAU, UUY, UYU, YUU, UAY, UYA, YUA, UGY, UYG, YUG, UCY, UYC, YUC, GYY, YGY, YYG, CYY, YCY, YYC, AYY, YAY, YYA, UYY, YUY, YYU, YYY, GGX, GX G, XGG, GAX, GXA, XGA, GCX, GXC, XGC, GUX, GXU, XGU, CAX, CXA, XCA, CGX, CXG, XCG, CUX , CXU, XCU, CCX, CXC, XCC, AAX, AXA, XAA, AGX, AXG, XAG, ACX, AXC, XAC, AUX, AXU, XAU, and a mutant anticodon sequence selected from UUX, UXU, XUU, UAX, UXA, XUA, UGX, UXG, XUG, UCX, UXC, XUC, GXX, XGX, XXG, CXX, XCX, XXC, AXX, XAX, XXA, UXX, XUX, XXU, or XXX, where X and Y are each 【Chemistry 1】 A mutant tRNA, which is a non-natural nucleotide comprising a non-natural nucleobase independently selected from c. aminoacyl-tRNA synthetase, d. cells, and e. A mutant mRNA or an oligonucleotide encoding said mutant mRNA, said mutant mRNA being selected from the group consisting of GGY, GYG, YGG, GAY, GYA, YGA, GCY, GYC, YGC, GUY, GYU, YGU, CAY, CYA, YCA, CGY, CYG, YCG, CUY, CYU, YCU, CCY, C YC, YCC, AAY, AYA, YAA, AGY, AYG, YAG, ACY, AYC, YAC, AUY, AYU, YAU, UUY, UYU, YUU, UAY, U YA, YUA, UGY, UYG, YUG, UCY, UYC, YUC, GYY, YGY, YYG, CYY, YCY, YYC, AYY, YAY, YYA, UYY, YU Y, YYU, YYY, GGX, GXG, XGG, GAX, GXA, XGA, GCX, GXC, XGC, GUX, GXU, XGU, CAX, CXA, XCA, CG X, CXG, XCG, CUX, CXU, XCU, CCX, CXC, XCC, AAX, AXA, XAA, AGX, AXG, XAG, ACX, AXC, XAC, AUX , AXU, XAU, UUX, UXU, XUU, UAX, UXA, XUA, UGX, UXG, XUG, UCX, UXC, XUC, GXX, XGX, XXG, CXX, XCX, XXC, AXX, XAX, XXA, UXX, XUX, XXU, or XXX, where X and Y are each 【Chemistry 2】 a mutant mRNA or an oligonucleotide encoding said mutant mRNA, Including the kit.

2. The kit of claim 1 , wherein the kit comprises the mutant mRNA.

3. The kit of claim 1 or 2, wherein the nucleoside triphosphate transporter, the mutant tRNA, the aminoacyl-tRNA synthetase, and / or the mutant mRNA or an oligonucleotide encoding the mutant mRNA is present in a cell.

4. The kit of claim 1 or 2, wherein the nucleoside triphosphate transporter, the mutant tRNA, and the aminoacyl-tRNA synthetase are present in a cell.

5. The kit according to any one of claims 1 to 4, further comprising pol B polymerase.

6. The kit described in claim 5, characterized in that the pol B polymerase is present within a cell.

7. The kit of any one of claims 1 to 6, further comprising an oligonucleotide encoding the mutant tRNA.

8. The kit of any one of claims 1 to 7, further comprising an oligonucleotide encoding the aminoacyl-tRNA synthetase.

9. The kit according to any one of claims 1 to 8, characterized in that the kit comprises an oligonucleotide encoding the mutant mRNA.

10. 10. The kit of claim 1, wherein the mutant tRNA comprises the mutant anticodon GYU, where Y is a non-natural nucleotide comprising a selected non-natural nucleobase.

11. 10. The kit of claim 1, wherein the mutant tRNA comprises the mutant anticodon GYC, where Y is a non-natural nucleotide comprising a selected non-natural nucleobase.

12. 12. The kit of any one of claims 1 to 11, wherein the mutant mRNA comprises the mutant codon AXC, where X is a non-natural nucleotide comprising a selected non-natural nucleobase.

13. 12. The kit of any one of claims 1 to 11, wherein the mutant mRNA comprises the mutant codon GXC, where X is a non-natural nucleotide comprising a selected non-natural nucleobase.

14. The unnatural nucleobase of the unnatural nucleotide Y is 【Chemistry 3】 14. The kit according to claim 10 or 11, or claim 12 or 13 when dependent on claim 10 or 11, wherein the cation exchanger is (NaM).

15. The unnatural nucleobase of the unnatural nucleotide Y is 【Chemistry 4】 (TPT3).

16. The non-natural nucleobase of the non-natural nucleotide X is 【Chemistry 5】 16. The kit of claim 12, 13, or 15, wherein the cation exchanger is (NaM).

17. The non-natural nucleobase of the non-natural nucleotide X is 【Chemistry 6】 15. The kit according to claim 12, characterized in that the target polypeptide is TPT3.

18. X is a non-natural nucleobase 【Chemistry 7】 (NaM), and Y is an unnatural nucleobase 【Chemistry 8】 17. The kit of any one of claims 1 to 13, 15 and 16, wherein the non-natural nucleotide comprises TPT3.

19. The unnatural nucleobases of the unnatural nucleotides in the mutant codon sequence and / or the mutant anticodon sequence are each independently 【Chemistry 9】 10. The kit according to claim 1, wherein the anti-inflammatory agent is selected from the group consisting of NaM and TPT3.

20. The unnatural nucleobase of the unnatural nucleotide in the mutant codon sequence is 【Chemistry 10】 20. The kit of claim 19, wherein the cation is (NaM).

21. The non-natural nucleobase of the non-natural nucleotide in the mutant anticodon sequence is 【Chemistry 11】 21. The kit according to claim 19 or 20, characterized in that the antibody is (TPT3).

22. 22. The kit according to any one of claims 1 to 21, wherein the aminoacyl-tRNA synthetase is derived from Methanosarcina barkeri.

23. 22. The kit according to any one of claims 1 to 21, wherein the aminoacyl-tRNA synthetase is derived from Methanocaldococcus jannaschii.

24. 24. The kit according to any one of claims 1 to 23, wherein the aminoacyl-tRNA synthetase is a pyrrolysyl-tRNA synthetase.

25. 24. The kit of claim 1, wherein the aminoacyl-tRNA synthetase is a tyrosyl-tRNA synthetase.

26. 26. The kit of any one of claims 1 to 25, wherein the mutant tRNA is a mutant of a pyrrolysyl tRNA from Methanosarcina mazei.

27. 27. The kit according to any one of claims 1 to 26, wherein the cell is a microorganism.

28. 27. The kit of claim 1, wherein the cell is a bacterium.

29. 27. The kit of any one of claims 1 to 26, wherein the cell is Escherichia coli.

30. 30. The kit of any one of claims 1-29, further comprising an unnatural amino acid.

31. 31. The kit of any one of claims 1 to 30, wherein the unnatural amino acid is p-azido-phenylalanine (pAzF).

32. The unnatural amino acid is N ６ 32. The kit according to claim 1, wherein the cytosine is -[(2-propynyloxy)carbonyl]-L-lysine (PrK).

33. A method for producing a protein in a cell, the protein comprising an unnatural amino acid, the method comprising contacting a cell with a first unnatural nucleotide and a second unnatural nucleotide, the cell comprising: a. Nucleoside triphosphate transporters from diatoms; b. A mutant tRNA, wherein the mutant tRNA is GGY, GYG, YGG, GAY, GYA, YGA, GCY, GYC, YGC, GUY, GY. U, YGU, CAY, CYA, YCA, CGY, CYG, YCG, CUY, CYU, YCU, CCY, CYC, YCC, AAY, AYA, YAA, AGY , AYG, YAG, ACY, AYC, YAC, AUY, AYU, YAU, UUY, UYU, YUU, UAY, UYA, YUA, UGY, UYG, YUG, UCY, UYC, YUC, GYY, YGY, YYG, CYY, YCY, YYC, AYY, YAY, YYA, UYY, YUY, YYU, YYY, GGX, GX G, XGG, GAX, GXA, XGA, GCX, GXC, XGC, GUX, GXU, XGU, CAX, CXA, XCA, CGX, CXG, XCG, CUX , CXU, XCU, CCX, CXC, XCC, AAX, AXA, XAA, AGX, AXG, XAG, ACX, AXC, XAC, AUX, AXU, XAU, and a mutant anticodon sequence selected from UUX, UXU, XUU, UAX, UXA, XUA, UGX, UXG, XUG, UCX, UXC, XUC, GXX, XGX, XXG, CXX, XCX, XXC, AXX, XAX, XXA, UXX, XUX, XXU, or XXX, where X and Y are each 【Chemistry 12】 A mutant tRNA, which is a non-natural nucleotide comprising a non-natural nucleobase independently selected from c. aminoacyl-tRNA synthetase, and d. The mutant mRNA is GGY, GYG, YGG, GAY, GYA, YGA, GCY, GYC, YGC, GUY, G YU, YGU, CAY, CYA, YCA, CGY, CYG, YCG, CUY, CYU, YCU, CCY, CYC, YCC, AAY, AYA, YAA, AG Y, AYG, YAG, ACY, AYC, YAC, AUY, AYU, YAU, UUY, UYU, YUU, UAY, UYA, YUA, UGY, UYG, YUG , UCY, UYC, YUC, GYY, YGY, YYG, CYY, YCY, YYC, AYY, YAY, YYA, UYY, YUY, YYU, YYY, GGX, GXG, XGG, GAX, GXA, XGA, GCX, GXC, XGC, GUX, GXU, XGU, CAX, CXA, XCA, CGX, CXG, XCG, C UX, CXU, XCU, CCX, CXC, XCC, AAX, AXA, XAA, AGX, AXG, XAG, ACX, AXC, XAC, AUX, AXU, XA and a mutant codon sequence selected from U, UUX, UXU, XUU, UAX, UXA, XUA, UGX, UXG, XUG, UCX, UXC, XUC, GXX, XGX, XXG, CXX, XCX, XXC, AXX, XAX, XXA, UXX, XUX, XXU, or XXX, where X and Y are each 【Chemistry 13】 A mutant mRNA comprising a non-natural nucleotide comprising a non-natural nucleobase independently selected from wherein the mutant anticodon of the mutant tRNA pairs with the mutant codon of the mutant mRNA, and the cell is a semi-synthetic organism (SSO); the first and second unnatural nucleotides are transported into the cell via the nucleoside triphosphate transporter, the transported unnatural nucleotides are incorporated by an RNA polymerase during transcription of a double-stranded oligonucleotide comprising the unnatural oligonucleotide to generate the mutant mRNA and the mutant tRNA; and The method includes contacting the cell with an unnatural amino acid and a mutant mRNA in the cell is translated within the cell, thereby producing a protein that includes the unnatural amino acid.

34. The method of claim 33, wherein the first non-natural nucleotide and the second non-natural nucleotide each comprise a non-natural nucleobase.

35. The unnatural nucleobase in the first unnatural nucleotide 【Chemistry 14】 and the unnatural nucleobase in the second unnatural nucleotide is 【Chemistry 15】 35. The method of claim 34, wherein:

36. The method of claim 33, wherein the unnatural amino acid is p-azido-phenylalanine (pAzF).

37. The method according to any one of claims 33 to 35, wherein the unnatural amino acid is N 6 -[(2-propynyloxy)carbonyl]-L-lysine (PrK).

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