Compositions comprising non-standard amino acids and uses thereof

Abstract

Provided herein are stabilized DNase I polypeptides comprising non-standard amino acids, which retain enzymatic activity even under harsh conditions, such as a reducing environment. The stabilized DNase I polypeptides have enzymatic activity in a reducing environment that is higher than a corresponding DNase I polypeptide without the non-standard amino acids under the same conditions. Also provided herein are polynucleotides encoding the stabilized DNase I polypeptides, cells for expressing and / or producing the stabilized DNase I polypeptides, and methods for using the stabilized DNase I polypeptides.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 695,505, filed on July 9, 2018, which is incorporated herein by reference in its entirety. Background Art

[0003] The activity of many enzymes depends on the environment in which they catalyze their respective reactions, for example, buffers with reducing conditions. Typically, enzymes rely on certain structural features to maintain their activity, and these structural features may be impaired by certain conditions. For example, under reducing conditions, the intermolecular and / or intramolecular disulfide bonds of the enzyme can be reduced, resulting in structural changes of the enzyme, which ultimately reduce or lose activity. For example, many methods for single-cell sequencing and single-cell PCR require the use of harsh lysis buffers to lyse cells in droplets in order to extract or amplify polynucleotides (e.g., mRNA) or extract proteins from single cells. After lysis, reactions are typically used, including reverse transcription reactions, polymerase-catalyzed reactions such as replication and / or synthesis of polynucleotides, ligation reactions, amplification reactions, nuclease-catalyzed reactions, and protease-mediated reactions. However, due to the harsh nature of the lysis buffers in which the enzymes catalyzing these reactions are located, the catalysts for these reactions are typically weakened or impossible. Summary of the invention

[0004] Compositions and methods for overcoming these problems are provided herein. Stabilized enzymes are provided herein, which remain active even under harsh conditions, such as in a reducing environment. Stabilized enzymes comprising non-standard amino acids are provided herein, which have enzymatic activity under harsh conditions, such as in a reducing buffer, and whose enzymatic activity is higher than the corresponding enzyme without non-standard amino acids under the same conditions. Polynucleotides encoding these stabilized enzymes, cells for expressing and / or producing these stabilized enzymes, and methods for using these stabilized enzymes are also provided herein. In some aspects, the stabilized enzyme comprises a stabilized deoxyribonuclease I (DNase I) polypeptide.

[0005] In some aspects, provided herein is a composition comprising a stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized DNase I polypeptide comprises one or more non-standard amino acids.

[0006] In some aspects, provided herein is a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids, wherein the stabilized DNase I polypeptide has a higher endonuclease activity on a DNA substrate in an environment than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids on the DNA substrate.

[0007] In some aspects, provided herein is a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids, wherein the stabilized DNase I polypeptide is not destabilized in an environment in which a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids is destabilized.

[0008] In some aspects, provided herein is a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids, wherein the stabilized DNase I polypeptide has a melting temperature (Tm) that is lower than the Tm of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids. m At least 5°C higher.

[0009] In some embodiments, the melting temperature (Tm) of the stabilized DNase I polypeptide, its functional fragment or variant thereof is lower than the Tm of the corresponding recombinant enzyme, its functional fragment or variant thereof that does not include the one or more non-standard amino acids. m At least 5°C higher.

[0010] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or its variant has a higher endonuclease activity on a DNA substrate in the environment than the endonuclease activity of a corresponding DNase I polypeptide, its functional fragment or its variant that does not include the one or more non-standard amino acids on the DNA substrate.

[0011] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or its variant is not destabilized in an environment where the corresponding DNase I polypeptide, its functional fragment or its variant that does not include the one or more non-standard amino acids is destabilized.

[0012] In some embodiments, at least one, two, three, four or more of the one or more non-standard amino acids is selenocysteine.

[0013] In some embodiments, at least two of the one or more non-standard amino acids are directly linked by a bond.

[0014] In some embodiments, at least four of the one or more non-standard amino acids are directly linked by a bond, wherein a first pair of the at least four of the one or more non-standard amino acids are directly linked by a bond and a second pair of the at least four of the one or more non-standard amino acids are directly linked by a bond.

[0015] In some embodiments, the bond is a diselenide bond.

[0016] In some embodiments, the diselenide bond is an intermolecular bond or an intramolecular bond.

[0017] In some embodiments, the half-life of the stabilized DNase I polypeptide, its functional fragment or variant thereof is at least 1.1 times higher than the half-life of the corresponding DNase I polypeptide, its functional fragment or variant thereof that does not include the one or more non-standard amino acids.

[0018] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof has at least 70%, 75%, 80%, 85%, 90%, 95% sequence identity with SEQ ID NO:1.

[0019] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or its variant comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% sequence identity with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 261 consecutive amino acids of SEQ ID NO:1.

[0020] In some embodiments, the one or more non-standard amino acids are located at position 102 of SEQ ID NO:1, position 105 of SEQ ID NO:1, position 174 of SEQ ID NO:1, or position 210 of SEQ ID NO:1.

[0021] In some embodiments, the non-standard amino acid at position 102 is directly linked to the non-standard amino acid at position 105 by a bond.

[0022] In some embodiments, the non-standard amino acid at position 174 is directly linked to the non-standard amino acid at position 210 by a bond.

[0023] In some embodiments, the bond is a diselenide bond.

[0024] In some embodiments, the diselenide bond is located at the position of a disulfide bond in a corresponding recombinase that lacks the one or more non-standard amino acids.

[0025] In some embodiments, the Tm of the corresponding stabilized DNase I polypeptide, functional fragment thereof or variant thereof is less than 37°C.

[0026] In some embodiments, the Tm of the stabilized DNase I polypeptide, functional fragment thereof, or variant thereof is greater than 37°C, 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C.

[0027] In some embodiments, the Tm of the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof is at least 10°C higher than the Tm of the corresponding recombinase, a functional fragment thereof, or a variant thereof.

[0028] In some embodiments, the Tm of the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof is at least 15°C higher than the Tm of the corresponding recombinase, a functional fragment thereof, or a variant thereof.

[0029] In some embodiments, the half-life of the stabilized DNase I polypeptide, its functional fragment or its variant in the environment is at least 1.1 times higher than the half-life of the corresponding DNase I polypeptide, its functional fragment or its variant in the environment that does not include the one or more non-standard amino acids.

[0030] In some embodiments, the half-life of the DNase I polypeptide, its functional fragment or its variant in the environment is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or longer.

[0031] In some embodiments, the half-life of the DNase I polypeptide, its functional fragment or its variant in the environment is greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days or longer.

[0032] In some embodiments, the stabilized DNase I polypeptide has an endonuclease activity on a DNA substrate in an environment that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not include the one or more non-standard amino acids on the DNA substrate.

[0033] In some embodiments, the stabilized DNase I polypeptide has an endonuclease activity on a DNA substrate after being present in an environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times greater than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not include the one or more non-standard amino acids after being present in the environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes.

[0034] In some embodiments, the stabilized DNase I polypeptide has an endonuclease activity on a DNA substrate after being present in an environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not include the one or more non-standard amino acids after being present in the environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours.

[0035] In some embodiments, the DNA substrate is genomic DNA.

[0036] In some embodiments, the environment is an environment with a temperature of 4°C-98°C.

[0037] In some embodiments, the environment is an environment containing a lysis buffer.

[0038] In some embodiments, the environment is an environment with a concentration of 0.01% to 20% detergent.

[0039] In some embodiments, the detergent is a non-ionic detergent.

[0040] In some embodiments, the detergent is an ionic detergent.

[0041] In some embodiments, the environment comprises divalent cations at a concentration of 0.01 mM to 100 mM.

[0042] In some embodiments, the divalent cation is selected from the group consisting of Mg 2+ , Mn 2+ , Ca 2+ 、Co 2+ and Zn 2+ group.

[0043] In some embodiments, the environment includes a reducing agent at a concentration of 0.01 mM to 100 mM.

[0044] In some embodiments, the environment has a pH of 5-9.

[0045] In some embodiments, the environment has a pH of 6-8.

[0046] In some embodiments, the environment has a pH of 7-8.

[0047] In some embodiments, the environment has a salt concentration of 10 mM to 1M.

[0048] In some embodiments, the environment is within a droplet.

[0049] In some embodiments, the environment is the blood circulatory system.

[0050] In some embodiments, the environment has a voltage less than -150 mV, -160 mV, -170 mV, -180 mV, -190 mV, -200 mV, -210 mV, -220 mV, -230 mV, -240 mV, or -250 mV, -260 mV, -270 mV, -280 mV, -290 mV, -300 mV, -310 mV, -320 mV, -330 mV, -340 mV, or -350 mV, -360 mV, - Reduction potential of 370mV, -380mV, -390mV, -400mV, -410mV, -420mV, -430mV, -440mV, or -450mV, -460mV, -470mV, -480mV, -490mV, -500mV, -510mV, -520mV, -530mV, -540mV, or -550mV, -560mV, -570mV, -580mV, -590mV or -600mV.

[0051] In some embodiments, the stabilized DNase I polypeptide, functional fragment thereof, or variant thereof is recombinant.

[0052] In some embodiments, the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof is bovine DNase I.

[0053] In some embodiments, a composition includes a polynucleotide encoding a composition disclosed herein.

[0054] In some embodiments, the polynucleotide is a vector.

[0055] In some embodiments, the bond directly linking two of the one or more non-standard amino acids of the stabilized DNase I polypeptide is not cleaved in an environment, wherein the bond directly linking two of the one or more standard amino acids of the corresponding DNase I polypeptide is cleaved in the same environment.

[0056] In some embodiments, the method of preparing the compositions disclosed herein comprises expressing the amino acid sequence of the stabilized DNase I polypeptide.

[0057] In some embodiments, expression includes expression in a cell or in vitro.

[0058] In some embodiments, the cell is a bacterial cell.

[0059] In some embodiments, the cell is a genomically recoded cell.

[0060] In some embodiments, the cell comprises a reassigned codon recognized by a stabilized non-standard amino acid tRNA comprising an anti-codon corresponding to the reassigned codon.

[0061] In some embodiments, the amino acid sequence of the stabilized DNase I polypeptide is encoded by a polynucleotide sequence that includes at least one codon for a native amino acid that has been replaced by the reassigned codon.

[0062] In some embodiments, the stabilized non-standard amino acid tRNA is a selenocysteine ​​tRNA.

[0063] In some embodiments, the method comprises culturing the cell under conditions whereby the amino acid sequence of the stabilized DNase I polypeptide is expressed.

[0064] In some embodiments, the reassigned codon is UAG, UAA, UGA, or a combination thereof.

[0065] In some aspects, provided herein is a method comprising contacting a DNA substrate in a buffer, in a reaction environment, or on a solid surface with a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof comprising one or more non-standard amino acids; wherein the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof catalyzes the cleavage or fragmentation of the DNA substrate at a higher rate than a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not include the one or more non-standard amino acids.

[0066] In some embodiments, the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof is a stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof disclosed elsewhere herein.

[0067] In some embodiments, the DNA substrate is genomic DNA.

[0068] In some embodiments, the DNA substrate is from a single cell.

[0069] In some embodiments, the method comprises forming a plurality of vessels, each vessel comprising a single cell from the plurality of cells; the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof; and a lysis buffer.

[0070] In some embodiments, the method further comprises lysing the single cell, thereby releasing the DNA substrate from the single cell.

[0071] In some embodiments, the method further comprises barcoding the DNA substrate or fragment thereof.

[0072] In some embodiments, the method further comprises amplifying the DNA substrate or a fragment thereof.

[0073] In some embodiments, the expanding comprises clonal expansion.

[0074] In some embodiments, the method further comprises sequencing the DNA substrate or a fragment thereof.

[0075] In some embodiments, the sequencing comprises whole genome sequencing.

[0076] In some embodiments, the sequencing comprises high-throughput sequencing, massively parallel sequencing, Sanger sequencing, or next-generation sequencing.

[0077] In some embodiments, the plurality of containers comprises a solid support.

[0078] In some embodiments, the DNA substrate is not attached to a solid support in the container.

[0079] In some embodiments, the buffer, the reaction environment, or the solid surface includes primers specific for the sequence of the DNA substrate or a fragment thereof.

[0080] In some embodiments, the plurality of cells comprises at least 2, 3, 4, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, , 9000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80, 000, 90,000, 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x106 ,9x10 6 ,1x10 7 ,2x10 7 、3x10 7 ,4x10 7 ,5x10 7 ,6x10 7 ,7x10 7 、8x10 7 ,9x10 7 ,1x10 8 ,2x10 8 、3x10 8 ,4x10 8 ,5x10 8 ,6x10 8 ,7x10 8 、8x10 8 ,9x10 8 ,1x10 9 ,2x10 9 、3x10 9 ,4x10 9 ,5x10 9 ,6x10 9 ,7x10 9 、8x10 9 ,9x10 9 ,1x10 10 ,2x10 10 、3x10 10 ,4x10 10 ,5x10 10 ,6x10 10 ,7x10 10 、8x10 10 ,9x10 10 ,1x10 11 ,2x10 11 、3x10 11 ,4x10 11 ,5x10 11 ,6x10 11 ,7x10 11 、8x10 11 ,9x10 11 ,1x10 12 ,2x10 12 、3x10 12 ,4x10 12 ,5x10 12 ,6x10 12 ,7x10 12 、8x10 12 Or 9x10 12cells.

[0081] In some embodiments, the plurality of cells are from one or more biological samples.

[0082] In some embodiments, the one or more biological samples include at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more samples.

[0083] In some embodiments, the one or more biological samples are from a subject suffering from a disease.

[0084] In some embodiments, the plurality of cells comprises a plurality of bacterial cells or a plurality of fungal cells.

[0085] In some embodiments, the plurality of cells comprises a plurality of immune cells.

[0086] In some embodiments, the plurality of cells comprises a plurality of diseased cells.

[0087] In some embodiments, the plurality of cells comprises a plurality of cancer cells.

[0088] Join by reference

[0089] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] The features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and the accompanying drawings, which set forth exemplary embodiments utilizing the principles of the present disclosure, wherein:

[0091] Figure 1Depicted is an agarose gel after 50ng double-stranded DNA added to a mixture of 0 mm DTT (lane 6), 50 mm DTT (lane 7), 30 ng seleno-DNase (GST–seleno-DNase I fusion) or equimolar disulfide DNase (wild-type DNase I purified from bovine pancreas, Sigma DN25), incubated with DNase for 2 hours at 37°C in reaction buffer (NEB B0303S) supplemented with 0 mM DTT (-DTT, lanes 2 and 3) or 50 mM DTT (+DTT, lanes 4 and 5). Double-stranded DNA was added to each mixture and digested at 37°C for 30 min, followed by heat inactivation at 75°C for 10 min. Both enzymes digested the DNA in 0 mM DTT, but only seleno-DNase digested the DNA in 50 mM DTT. DETAILED DESCRIPTION

[0092] definition

[0093] The term "coding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (e.g., a gene, cDNA, or mRNA) as a template for synthesizing other polymers and macromolecules in a biological process, which have a determined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a determined amino acid sequence and the biological properties obtained therefrom. Therefore, if in a cell or other biological system, the transcription and translation of the mRNA corresponding to a gene produce a protein, the gene, cDNA, or RNA encodes the protein. A coding strand (whose nucleotide sequence is consistent with the mRNA sequence and is generally provided in the sequence) and a non-coding strand (used as a template for gene or cDNA transcription) can both be referred to as encoding a protein or other product of the gene or cDNA.

[0094] The term "endogenous" refers to any material derived from or produced within an organism, cell, tissue or system.

[0095] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0096] The term "expression" refers to the transcription and / or translation of a specific nucleotide sequence driven by a promoter.

[0097] The term "homology" or "identity" refers to the subunit sequence identity between two polymer molecules, such as two nucleic acid molecules, such as between two nucleic acid molecules (DNA molecules or two RNA molecules) or between two polypeptide molecules. When a subunit position in the two molecules is occupied by the same monomer subunit; for example, if a position in each of the two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; for example, if half of the positions in the two sequences (e.g., five positions in a polymer of ten subunits in length) are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 / 10) are matched or homologous, the two sequences are 90% homologous.

[0098] The term "isolated" means altered or removed from the natural state. For example, a naturally occurring nucleic acid or peptide in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment such as a host cell.

[0099] In the context of the present invention, the following abbreviations are used to represent commonly occurring nucleic acid bases: "A" represents adenosine, "C" represents cytosine, "G" represents guanosine, "T" represents thymidine, and "U" represents uridine.

[0100] The term "operably linked" or "transcriptional control" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that results in the expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be adjacent to each other and in the same reading frame (e.g., if two protein coding regions need to be linked).

[0101] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known natural nucleotide analogs that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologous genes, SNPs, and complementary sequences, as well as sequences explicitly shown. Specifically, degenerate codon replacement can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0102] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that are similar in function to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. As used herein, the term "amino acid analog" refers to a compound having the same basic chemical structure as naturally occurring amino acids, i.e., an α carbon bound to hydrogen, carboxyl, amino, and R groups, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. As used herein, the term "amino acid mimetics" refers to a chemical compound that has a structure different from the general chemical structure of an amino acid, but functions similarly to naturally occurring amino acids.

[0103] The term "non-standard amino acid" refers to any amino acid other than the 20 standard amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine). Selenocysteine ​​is a non-standard amino acid (NSAA).

[0104] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds comprising amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein sequence or peptide sequence can contain. Polypeptides include any peptide or protein that comprises two or more amino acids linked to each other by peptide bonds. The term used herein refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) and long chains (which are generally referred to in the art as proteins, of which there are many types). "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0105] The term "promoter" refers to a DNA sequence recognized by the cell's transcription machinery or introduced by synthetic machinery and required to initiate specific transcription of a polynucleotide sequence.

[0106] The term "constitutive" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0107] The term "inducible" promoter refers to a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, produces a gene product in a cell essentially only when an inducer corresponding to the promoter is present in the cell.

[0108] The term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0109] Range: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the range format is only for convenience and brevity, and should not be interpreted as an unchangeable limitation on the scope of the invention. Therefore, the range statement should be regarded as having explicitly disclosed all possible sub-ranges, as well as individual numerical values ​​within the range. For example, the statement of a range such as 1 to 6 should be regarded as having explicitly disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within the range, for example, 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95%-99% consistency includes something with 95%, 96%, 97%, 98% or 99% consistency, and includes sub-ranges such as 96%-99%, 96%-98%, 96%-97%.

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the claimed subject matter belongs. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not limitations of any subject matter claimed. In this application, unless otherwise expressly stated, the use of the singular includes the plural.

[0111] Compositions including non-standard amino acids

[0112] The use of non-standard amino acids in proteins provides the possibility of polypeptides with greatly expanded functionality, which can be used in a wide range of applications. For example, by incorporating selenocysteine ​​in a polypeptide, an enzyme with enhanced stability or activity level may be developed, and a highly active therapeutic polypeptide may be produced. However, these methods have been hampered so far by the inability to produce an organism that retains a stability translation pathway that predictably and reliably incorporates selenocysteine ​​in the encoded polypeptide. The research described in detail herein illustrates a stable system for selecting tRNA molecules that can incorporate selenocysteine ​​and for producing polypeptides that incorporate selenocysteine ​​positions. Importantly, this system can be easily transferred from one organism to another without the need for recombinant engineering.

[0113] Utilize progressively developed orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pairs, more than 100 kinds of NSAA with diverse chemical properties have been synthesized and co-translationally incorporated into proteins. Non-standard amino acids have been designed based on tyrosine or pyrrolysine. aaRS / tRNA can be provided on plasmids or in the genome of organisms recoded by the genome. Orthogonal aaRS / tRNA pairs can be used to incorporate NSAA bio-orthogonally into proteins. Overexpression systems based on carriers can be used to exceed natural codon functions by their redistributed functions. If natural UAG translation function is completely eliminated, far lower aaRS / tRNA functions can be sufficient to achieve effective NSAA incorporation. NSAA incorporation based on organisms recoded by the genome (Genomically recoded organism, GRO) can use carrier-based and / or genome-based aaRS / tRNA pairs. In the absence of available NSAA, genome-based aaRS / tRNA pairs have been used to reduce the misincorporation of standard amino acids. Because the UAG codon function has been completely redistributed in the organism recoded by the genome, NSAA, for example selenocysteine, can be incorporated into the organism recoded by the genome without any phenotypic results. The NSAA in the organism recoded by the genome is incorporated and can be related to the inducer of supplementing non-standard amino acids (such as selenocysteine) and aaRS to the growth medium. Or, aaRS can be constitutively expressed. Or, as in the present disclosure, endogenous seryl-tRNA synthetase can be used for serylation (serylate) selenocysteine ​​tRNA, which is subjected to the enzyme action comprising SelA and produces tRNasec (tRNA loaded with selenocysteine). Culture medium can supplement selenium source (such as sodium selenite) to improve the generation of tRNasec. The desired protein can utilize any desired protein overexpression system (such as T7-RNAP, constitutive incorporation, or inducible expression based on IPTG / allolactose, anhydrotetracycline, arabinose, rhamnose or other inducible systems) to overexpress. Protein crosslinks (diselenide bonds) can be formed spontaneously during protein folding catalytically based on proximity geometry, and the protein can be treated like any other overexpressed product.

[0114] The present inventors have developed polypeptides and methods to produce polypeptides in genome recoded organisms (GROs), which are folded into biological products, which are stabilized, for example, by diselenide bonds between selenocysteine ​​amino acids. However, the disulfide bonds between cysteine ​​amino acids have an oxidation-reduction potential of about -220mV, and the diselenide bonds have an oxidation-reduction potential of about -380mV. Since bacterial cytosols generally have an oxidation-reduction potential of about -280 to -300mV, diselenide bonds, rather than disulfide bonds, avoid reduction, so that they form and exist in cytosols. Because diselenide bonds have the same geometric bond angles and torsion as disulfide bonds, and very similar bond lengths, they can be substituted into polypeptides without destroying the three-dimensional structure of polypeptides. In addition, because the expected in vivo environment (e.g., blood) contains reducing agents, such as glutathione, albumin, and thioredoxin, the disulfide bonds in the polypeptides can be reduced, causing the polypeptides to unfold, and in the case of multiple disulfide bonds, "scramble" disulfide bonds, so that incorrect cysteines are combined with each other. Both of these lead to the loss of the intended biological activity of the polypeptide.The lower redox potential of diselenide bonds makes them resistant to reduction when exposed to serum or purified reducing components of serum, giving them a longer serum half-life than their disulfide-containing counterparts.

[0115] Although peptides of selenocysteine ​​with diselenide bonds can be produced in vitro by solid phase peptide synthesis, the process cannot easily reach the required yield for therapeutic applications (particularly for proteins). However, the in vivo production of recombinant seleno-proteins is limited by the strict sequence requirements that selenocysteine ​​may appear in proteins. In particular, selenocysteine ​​insertion sequence (SECIS) elements must appear in the coding DNA sequence at the selenocysteine ​​incorporation site to recruit endogenous selenocysteine ​​translation mechanisms, including specific elongation factors (SelB). On the contrary, the recoded strains of Escherichia coli (which have unassigned codons such as amber stop codons) can be used together with engineered selenocysteine ​​tRNAs with anti-amber anticodons, which allow selenocysteine ​​targeting to polypeptides by introducing amber stop codons into corresponding DNA coding sequences. Modified tRNA interacts with endogenous elongation factor EF-Tu. As known in the art, other codons can be recoded, typically rare codons. The codons on mRNA and the anticodons on tRNA are usually triplets of complementary base sequences.

[0116] The recoded protein can be synthesized in a bacterial (e.g., E. coli) cell or in vitro, in a translation or linked transcription-translation system. The gene or mRNA encoding the recoded protein is non-naturally occurring and is a variant of a naturally occurring coding sequence. Although many proteins shown in the associated sequence table have all cysteine ​​residues that participate in the substitution of disulfide bonds by selenocysteine ​​residues, it is not necessary to replace all cysteine ​​residues to obtain the benefits of substitution. Even a diselenide bond can improve the stability of the protein. Any number of diselenide bonds (selenocysteine ​​pairs) can replace disulfide bonds in proteins. If a protein has N disulfide bonds, the protein can have any number of such bonds reduced to 1 from N, N minus 1, N minus 2, N minus 3, N minus 4.... A bond called selenylsulfide may also be formed between cysteine ​​and selenocysteine ​​residues. The redox potential (~-270mv) of this bond is lower than that of disulfide bonds (-220mv) but not lower than that of bacterial cytoplasm (-280mv). The selenosulfide bond can be used to increase resistance to reduction in certain redox environments. Using the methods described herein, selenosulfide can be used to replace diselenide by replacing a single disulfide-bonded cysteine ​​with selenocysteine, or by replacing a single diselenide-bonded selenocysteine ​​with cysteine.

[0117] Using the described method, disulfide-stabilized biologic sequences with substituted selenocysteine ​​can be produced in the cytosol of E. coli at mg / L scale in standard laboratory shake flasks and scaled up to g / L production in microbial fermenters.

[0118] Enzymes having different combinations of diselenide bonds and disulfide bonds include, but are not limited to, nucleases (eg, DNases and RNases), polymerases, ligases, reverse transcriptases, proteases, restriction endonucleases, and carbon fixation enzymes (eg, carbon capture enzymes).

[0119] Any cysteine ​​in the enzyme disclosed herein can remain as selenocysteine, as long as the presence of the selenocysteine ​​does not interfere with the expression, folding or expected function of the polypeptide. A method is provided herein for producing and verifying the presence of selenocysteine ​​in the expected diselenide bond involved in various enzymes, including but not limited to nucleases (e.g., DNase and RNase), polymerases, ligases, reverse transcriptases, proteases, restriction endonucleases, and carbon fixation enzymes (e.g., carbon capture enzymes).

[0120] According to the present invention, stabilized enzymes can be prepared and used with the diselenide bond between two selenocysteine ​​residues. Even if this technology and modification can be used to produce an enzyme that also keeps activity under harsh conditions (for example, in a reducing environment). Provided herein is a stabilized enzyme that comprises non-standard amino acids, the enzymatic activity that it has under harsh conditions (for example, in a reducing buffer or a lysis buffer) is higher than the corresponding enzyme that does not have the non-standard amino acids under the same conditions. The stabilized enzyme can include a stabilized DNase I polypeptide. Also provided herein are polynucleotides encoding these stabilized enzymes, cells for expressing and / or producing these stabilized enzymes, and methods using these stabilized enzymes.

[0121] Enzymes having different combinations of diselenide bonds and disulfide bonds include, but are not limited to, nucleases (eg, DNases and RNases), polymerases, ligases, reverse transcriptases, proteases, restriction endonucleases, and carbon fixation enzymes (eg, carbon capture enzymes).

[0122] In some embodiments, according to the present invention, nucleases can be prepared and used with a diselenide bond between two selenocysteine ​​residues. Exemplary nucleases include, but are not limited to, DNase (e.g., DNase I of cattle), RNase, etc. For example, DNase I has two disulfide bonds. For example, RNase A has 4 disulfide bonds. In some embodiments, RNase A enzyme comprises 2, 4, 6, or 8 selenocysteine ​​residues. In some embodiments, RNase A enzyme comprises at least 1, 2, 3, or 4 diselenide bonds. For example, RNase 3 has 4 disulfide bonds. In some embodiments, RNase 3 enzyme comprises at least 2, 4, 6, or 8 selenocysteine ​​residues. In some embodiments, RNase 3 enzyme comprises at least 1, 2, 3, or 4 diselenide bonds. For example, benzonase (e.g., Serratia marcescens nuclease) comprises at least two necessary disulfide bonds and is a 30kDa homodimer. In some embodiments, benzonase comprises at least 2 or 4 selenocysteine ​​residues. In some embodiments, the benzonase comprises at least 1 or 2 diselenide bonds.

[0123] In some embodiments, nuclease can comprise one or more non-standard amino acids. In some embodiments, nuclease can comprise one or more selenocysteine ​​residues. In some embodiments, nuclease can comprise diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, nuclease can comprise one or more diselenide bonds. In some embodiments, the enzymatic activity that nuclease comprising one or more non-standard amino acids has under harsh conditions (such as reducing buffer or lysis buffer) is higher than the corresponding nuclease without the non-standard amino acids under the same conditions.

[0124] For example, the nucleases provided herein that comprise one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of polynucleotide substrates with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5.6, 5.7, 5.8, 6.9, 7.10, 7.11, 7.12, 7.13, 7.14, 7.15, 7.16, 7.17, 7.18, 7.19, 8.10, 8.11, 8.19, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 9.10, 9.11, 9.12, 9.13, 9.14, 9.15 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0125] For example, the nucleases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of polynucleotide substrates in a buffer with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0126] For example, the nucleases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of polynucleotide substrates in a buffer comprising a detergent, a reducing agent, and / or a reductase (e.g., a reductase) with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 6.21, 6.22, 6.23, 6.24, 6.25, 6.26, 6.27, 6.28, 6.29, 7.30, 7.31, 7.32, 7.33, 7.34, 7.35, 7.36, 7.37, 7.38, 7.39, 8.40, 8.41, 8.42, 8.43, 8.44, 8.45, 8.46, 8.47, 8.48, 8.49, 9.50, 9.51, 9.52, 9.53, 9.6 .1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0127] For example, the nucleases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of polynucleotide substrates in a buffer having a redox potential of less than about -150 mV with an activity at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5 4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the nucleases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of polynucleotide substrates in a buffer having an oxidation-reduction potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV, less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV, less than about -600 mV, less 0 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV or less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV or less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV 80 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV or less than about -600 mV, and the activity of the nuclease is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.65, 2.70, 2.80 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV or less than about -600 mV 7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0128] In some embodiments, according to the present invention, polymerase can be prepared and used with the diselenide bond between two selenocysteine ​​residues. In some embodiments, polymerase can include one or more non-standard amino acids. In some embodiments, polymerase can include one or more selenocysteine ​​residues. In some embodiments, polymerase can include the diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, polymerase can include one or more diselenide bonds. In some embodiments, the enzymatic activity that the polymerase comprising one or more non-standard amino acids has under harsh conditions (for example, in reducing buffer or lysis buffer) is higher than the corresponding polymerase without the non-standard amino acids under the same conditions.

[0129] For example, a polymerase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can catalyze a polymerase reaction with an activity that is at least 1.1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.1, 9.2, 9.3, 9.4, 9.5, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 10.1, 10.2 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0130] For example, a polymerase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can catalyze a polymerase reaction in a buffer with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0131] For example, a polymerase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can catalyze a polymerase reaction in a buffer comprising a detergent, a reducing agent, and / or a reductase (e.g., a reductase) with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 6.30, 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 70, 71, 72, 73, 74, 75 .1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0132] For example, a polymerase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can catalyze a polymerase reaction in a buffer having a redox potential of less than about -150 mV with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 7.9, 8.1, 8. 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the polymerases provided herein that contain one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can catalyze a polymerase reaction in a buffer having an oxidation-reduction potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, or less than about -290 mV. V, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV or less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV or less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -48 0 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV or less than about -600 mV, and the activity of the polymerase is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.65, 2.70, 2.80, 2.90, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.70, 2.80, 2.90, 2.17, 2.18, 2.19, 2.20, 2.21 7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0133] In some embodiments, according to the present invention, a ligase can be prepared and used with a diselenide bond between two selenocysteine ​​residues. In some embodiments, a ligase can include one or more non-standard amino acids. In some embodiments, a ligase can include one or more selenocysteine ​​residues. In some embodiments, a ligase can include a diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, a ligase can include one or more diselenide bonds. In some embodiments, the ligase including one or more non-standard amino acids has an enzymatic activity under harsh conditions (e.g., in a reducing buffer or a lysis buffer) that is higher than the corresponding ligase without the non-standard amino acids under the same conditions.

[0134] For example, the ligases provided herein that comprise one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can ligate two or more nucleic acids together with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0135] For example, a ligase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can ligate two or more nucleic acids together in a buffer with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0136] For example, the ligases provided herein that contain one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can ligate two or more nucleic acids together in a buffer containing a detergent, a reducing agent, and / or a reductase (e.g., a reductase) with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.110, 3.120, 3.130, 3.140, 3.150, 3.160, 3.170, 3.180, 3.190, 4.210, 4.220, 4.230, 4.240, 4.250, 4.260, 4.270, 4.280, 4.290, 5.300, 5.310, 5.320, 5.330, 5.340, 5.350, 5.360, 5.370, 5.380, 5.390, 6.40 ... 70, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0137] For example, the ligases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can ligate two or more nucleic acids together in a buffer having a redox potential of less than about -150 mV with an activity at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5 .4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the ligases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can ligate two or more nucleic acids together in a buffer having a redox potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV, less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV or less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV or less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about The ligase has an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.65, 2.70, 2.80, 2.90, 3.00, 3.01, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16 ...1, 3.02, 3.03, 3.04, 3.05, 3.06, 3.07, 3.08, 3.09, 3.11, 3.12, 3.13, 3.14, 3.15 .7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0138] In some embodiments, according to the present invention, restriction endonucleases can be prepared and used with a diselenide bond between two selenocysteine ​​residues. In some embodiments, restriction endonucleases can include one or more non-standard amino acids. In some embodiments, restriction endonucleases can include one or more selenocysteine ​​residues. In some embodiments, restriction endonucleases can include a diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, restriction endonucleases can include one or more diselenide bonds. In some embodiments, the restriction endonucleases comprising one or more non-standard amino acids have an enzymatic activity that is higher than the corresponding restriction endonucleases without the non-standard amino acids under harsh conditions (e.g., in a reducing buffer or a lysis buffer) under the same conditions.

[0139] For example, a restriction endonuclease provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave one or more bonds of a polynucleotide substrate with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0140] For example, a restriction endonuclease provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave one or more bonds of a polynucleotide substrate in a buffer with an activity at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3.5, 4.5, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0 , 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0141] For example, a restriction endonuclease provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave one or more bonds of a polynucleotide substrate in a buffer comprising a detergent, a reducing agent, and / or a reductase (e.g., a reductase) with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 4.20, 4.21, 4.22, 4.23, 4.24, 4.25, 4.26, 4.27, 4.28, 4.29, 5.30, 5.31, 5.32, 5.33, 5.34, 5.35, 5.36, 5.37, 5.38, 5.39, 6.40, 6.41, 6.42, 6.43, 6.44, 6.45, 6.46, 6.47, 6.48, 6.50, 6.51, 6.52, 6.53, 6.6 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0142] For example, the restriction endonucleases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave one or more bonds of a polynucleotide substrate in a buffer having a redox potential of less than about -150 mV with an activity at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the restriction endonucleases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave one or more bonds of a polynucleotide substrate in a buffer having an oxidation-reduction potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV, less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV The voltage at about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, or less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, or less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV, less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV, less than about -600 mV, less than about 80 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV or less than about -600 mV, and the activity of the restriction endonuclease is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about .6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0143] In some embodiments, according to the present invention, reverse transcriptase can be prepared and used with the diselenide bond between two selenocysteine ​​residues. In some embodiments, reverse transcriptase can include one or more non-standard amino acids. In some embodiments, reverse transcriptase can include one or more selenocysteine ​​residues. In some embodiments, reverse transcriptase can include the diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, reverse transcriptase can include one or more diselenide bonds. In some embodiments, the enzyme activity that the reverse transcriptase comprising one or more non-standard amino acids has under harsh conditions (for example, in reducing buffer or lysis buffer) is higher than the corresponding reverse transcriptase without the non-standard amino acids under the same conditions.

[0144] For example, the reverse transcriptases provided herein that contain one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can synthesize cDNA from RNA with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5.6, 5.7, 5.8, 6.9, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0145] For example, the reverse transcriptases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can synthesize cDNA from RNA in a buffer with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, , 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0146] For example, the reverse transcriptase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can synthesize cDNA from RNA in a buffer comprising a detergent, a reducing agent, and / or a reductase (e.g., a reductase) with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.2, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.10, 4.20, 4.30, 4.40, 4.50, 4.60, 4.70, 4.80, 4.90, 5.10, 5.11, 5.12, 5.13, 5.14, 5.15, 5.16, 5.17, 5.18, 5.19, 6.11, 6.12, 6.13, 6.14, 6.15, 6.16, 6.17, 6.18, 6.19, 7.20, 7.21, 7.22, 7.23, 7.24, 7.25, 7.26, 7.27, 7.28, 7.29, 8. .1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0147] For example, the reverse transcriptase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can synthesize cDNA from RNA in a buffer having a redox potential of less than about -150 mV, and the activity is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5 4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the reverse transcriptase provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can synthesize cDNA from RNA in a buffer having an oxidation-reduction potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV 80 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV or less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV or less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV 80 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV or less than about -600 mV, and the activity of the reverse transcriptase is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.65, 2.70, 2.80 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about .7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0148] In some embodiments, according to the present invention, protease can be prepared and used with the diselenide bond between two selenocysteine ​​residues. In some embodiments, protease can comprise one or more non-standard amino acids. In some embodiments, protease can comprise one or more selenocysteine ​​residues. In some embodiments, protease can comprise the diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, protease can comprise one or more diselenide bonds. In some embodiments, the enzymatic activity that the protease comprising one or more non-standard amino acids has under harsh conditions (for example, in reducing buffer or lysis buffer) is higher than the corresponding protease without the non-standard amino acids under the same conditions.

[0149] For example, the proteases provided herein that comprise one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of a polypeptide substrate with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 50, 60, 70, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0150] For example, the proteases provided herein that contain one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of a polypeptide substrate in a buffer with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0151] For example, the proteases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of a polypeptide substrate in a buffer comprising a detergent, a reducing agent, and / or a reductase (e.g., a reductase) with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 4.5 ... , 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0152] For example, the proteases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of a polypeptide substrate in a buffer having a redox potential of less than about -150 mV with an activity at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.1, 8.2, 8.3, 8.4, 8.5 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the proteases provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can cleave bonds of a polypeptide substrate in a buffer having an oxidation-reduction potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV, less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV, less than about -600 mV, less than about mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV or less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV or less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV 0 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV or less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV or less than about -600 mV, and the activity of the protease is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.65, 2.70, 2.80, 2.90, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.70, 2.80, 2.90, 2.17, 2.18, 2.19, 2.20, 2.21, 2.22 7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0153] In some embodiments, according to the present invention, enzymes comprising one or more catalytic cysteine ​​residues (i.e., cysteine ​​that participates in the catalytic reaction, such as active site cysteine) can be prepared and used by replacing one or more of these catalytic cysteine ​​residues with one or more selenocysteine ​​residues. One or more selenocysteine ​​substitutions can increase or change the enzyme activity in the reaction environment.

[0154] In some embodiments, according to the present invention, carbon capture enzymes (e.g., carbon fixation enzymes) can be prepared and used with a diselenide bond between two selenocysteine ​​residues. In some embodiments, carbon capture enzymes (e.g., carbon fixation enzymes) can include one or more non-standard amino acids. In some embodiments, carbon capture enzymes (e.g., carbon fixation enzymes) can include one or more selenocysteine ​​residues. In some embodiments, carbon capture enzymes (e.g., carbon fixation enzymes) can include a diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, carbon capture enzymes (e.g., carbon fixation enzymes) comprising one or more non-standard amino acids have an enzyme activity under harsh conditions (e.g., in a reducing buffer or a lysis buffer) that is higher than the corresponding carbon capture enzymes (e.g., carbon fixation enzymes) without the non-standard amino acids under the same conditions. For example, in some embodiments, carbon capture enzymes (e.g., carbon fixation enzymes), such as dehydratase (e.g., β-carbonic anhydrase) can include one or more catalytic selenocysteine ​​substitutions.

[0155] For example, the carbon capture enzymes (e.g., carbon fixation enzymes) provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can capture or fix carbon with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4.6, 5.8, 6.9, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 8.11, 8.12, 8.13, 8.14, 8.15, 8.16, 8.17, 8.18, 8.19, 9.10, 9.11, 9.12, 9.13, 9.14, 9.15 , 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times. For example, an enzyme provided herein comprising one or more non-standard active site amino acids (e.g., one or more active site selenocysteine ​​residues), e.g., a carbon capture enzyme (e.g., a carbon fixation enzyme) can capture or fix carbon with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.10, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.11, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.12, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.13, 7. .5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0156] For example, the carbon capture enzymes (e.g., carbon fixation enzymes) provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can capture or fix carbon in a buffer or environment with an activity that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.1, 7.2, 7.3, 7.4, 7.5 .5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0157] For example, the carbon capture enzymes (e.g., carbon fixation enzymes) provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can capture or fix carbon in a buffer comprising a detergent, a reductant, and / or a reductase (e.g., a reductase) or in a reducing environment, and the activity is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.10, 2.20, 2.30, 2.40, 2.50, 2.60, 2.70, 2.80, 2.90, 3.90, 4.10, 4.11, 4.12, 4.13, 4.14, 4.15, 4.16, 4.17, 4.18, 4.19, 5.20, 5.21, 5.22, 5.23, 5.24, 5.25, 5.26, 5.27, 5.28, 5.29, 6.30, 6.31, 6.32, 6.33, 6.34, 6.35, 6.36, 6.37, 6.38, 6.39, 7.40, 7.41, 7.42, 7.43, 7.44, 7.45, 7.46, 7.47, 7.48, 7.49, 8.50, 8.51, 8.52, 8.53, 8.54, 8.55, 8.56, 8.57, 8.58, 8.59, 8.6 .7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0158] For example, the carbon capture enzyme (e.g., carbon fixation enzyme) provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can capture or fix carbon in a buffer or environment with a redox potential of less than about -150 mV, and the activity is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.10, 3.11, 3.12, 3.13, 3.14, 3.15, 3.16, 3.17, 3.18, 3.19, 4.5 ... 70, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.For example, the carbon capture enzymes (e.g., carbon fixation enzymes) provided herein comprising one or more non-standard amino acids (e.g., one or more selenocysteine ​​residues) can capture or fix carbon in a buffer or environment having a redox potential of less than about -160 mV, less than about -170 mV, less than about -180 mV, less than about -190 mV, less than about -200 mV, less than about -210 mV, less than about -220 mV, less than about -230 mV, less than about -240 mV, or less than about -250 mV, less than about -260 mV, less than about -270 mV, less than about -280 mV, less than about -290 mV, less than about -300 mV, less than about -310 mV, less than about -320 mV, less than about -330 mV, less than about -340 mV, less than about -350 mV, less than about -360 mV, less than about -370 mV, less than about -380 mV, less than about -390 mV, less than about -400 mV, less than about -410 mV, less than about -420 mV, less than about -430 mV, less than about -440 mV, less than about -450 mV, less than about -460 mV, less than about -470 mV, less than about -480 mV, less than about -490 mV, less than about -500 mV, less than about -510 mV, less than about -520 mV, less than about -530 mV, less than about -540 mV, less than about -550 mV, less than about -560 mV, less than about -570 mV, less than about -580 mV, less than about -590 mV, less than about - The present invention relates to a method for producing a high-performance liquid crystal display device having a high-performance liquid crystal display device and a low-performance liquid crystal display device having a high-performance liquid crystal display device and a low-performance liquid crystal display device having a low ... V, less than about -490mV, less than about -500mV, less than about -510mV, less than about -520mV, less than about -530mV, less than about -540mV or less than about -550mV, less than about -560mV, less than about -570mV, less than about -580mV, less than about -590mV or less than about -600mV, the activity of the carbon capture enzyme (e.g., carbon fixation enzyme) is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.1, 4.2, 4.3, 4.4, 4.5, 5.6, 5.7, 5.8, 5.9, 6.9, 7.10, 7.20, 7.30, 7.40, 7.50, 7.60, 7.70, 7.80, 7.90, 7. 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times.

[0159] In some aspects, a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof is provided herein, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids. In some embodiments, according to the present invention, the stabilized DNase I polypeptide can be prepared and used with a diselenide bond between two selenocysteine ​​residues. In some embodiments, the stabilized DNase I polypeptide can comprise one or more non-standard amino acids. In some embodiments, the stabilized DNase I polypeptide can comprise one or more selenocysteine ​​residues. In some embodiments, the stabilized DNase I polypeptide can comprise a diselenide bond between two selenocysteine ​​residues. The diselenide bond can be intramolecular or intermolecular. In some embodiments, the stabilized DNase I polypeptide can comprise one or more diselenide bonds. In some embodiments, the stabilized DNase I polypeptide can comprise one or more catalytic selenocysteine ​​substitutions.

[0160] In some aspects, provided herein is a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids, wherein the stabilized DNase I polypeptide has a higher endonuclease activity on a DNA substrate in an environment than a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids. For example, the endonuclease activity of the stabilized DNase I polypeptide on a DNA substrate in an environment can be higher than that of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids. I polypeptide, its functional fragment or its variant has an endonuclease activity against the DNA substrate that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9 .5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 or more times.

[0161] In some aspects, a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof is provided herein, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids, wherein the stabilized DNase I polypeptide is not destabilized in an environment that does not comprise the destabilization of the corresponding DNase I polypeptide, its functional fragment, or a variant thereof of the one or more non-standard amino acids. The destabilization can be achieved by contacting the corresponding DNase I polypeptide with one or more destabilizing agents. The destabilization can be achieved by placing the corresponding DNase I polypeptide in a destabilized environment. The environment that destabilizes the corresponding DNase I polypeptide is described in other parts of this article.

[0162] In some aspects, provided herein is a composition comprising a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized deoxyribonuclease I (DNase I) polypeptide comprises one or more non-standard amino acids, wherein the stabilized DNase I polypeptide has a melting temperature (Tm) that is lower than the Tm of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids. m At least 5°C higher.

[0163] In some embodiments, the composition may comprise a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized DNase I polypeptide comprises one or more non-standard amino acids, wherein the melting temperature (T m ) can be compared to the T of the corresponding DNase I polypeptide, its functional fragment or its variant that does not contain the one or more non-standard amino acids. m In some embodiments, the composition may comprise a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof, wherein the stabilized DNase I polypeptide comprises one or more non-standard amino acids, wherein the melting temperature (T) of the stabilized DNase I polypeptide is at least 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, or 36°C. m ) can be compared to the T of the corresponding DNase I polypeptide, its functional fragment or its variant that does not contain the one or more non-standard amino acids. mLess than 1°C high.

[0164] In some embodiments, at least one, two, three, four or more of the one or more non-standard amino acids are selenocysteine. In some embodiments, at least two of the one or more non-standard amino acids are directly connected by a bond.

[0165] In some embodiments, at least four of the one or more non-standard amino acids can be directly connected by a key, wherein the first pair of the at least four of the one or more non-standard amino acids can be directly connected by a key, and the second pair of the at least four of the one or more non-standard amino acids can be directly connected by a key. In some embodiments, the key is a diselenide bond. In some embodiments, the diselenide bond can be an intermolecular bond or an intramolecular bond.

[0166] In some embodiments, the half-life of the stabilized DNase I polypeptide, its functional fragment or its variant can be at least 1.1 times higher than the half-life of the corresponding DNase I polypeptide, its functional fragment or its variant that does not contain the one or more non-standard amino acids. In some embodiments, the half-life of the stabilized DNase I polypeptide, its functional fragment or its variant can be at least 1.1 times higher than the half-life of the corresponding DNase I polypeptide, its functional fragment or its variant that does not contain the one or more non-standard amino acids. 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 times or more. In some embodiments, the half-life of the stabilized DNase I polypeptide, its functional fragment or its variant can be less than 1.1 times higher than the half-life of the corresponding DNase I polypeptide, its functional fragment or its variant that does not comprise the one or more non-standard amino acids.

[0167] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof may have at least 70%, 75%, 80%, 85%, 90%, 95% sequence identity with SEQ ID NO: 1. In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof may have at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity with SEQ ID NO: 1. In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof may have less than 10% sequence identity with SEQ ID NO: 1.

[0168] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof may include a sequence having at least 70%, 75%, 80%, 85%, 90%, 95% sequence identity to at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 261 contiguous amino acids of SEQ ID NO: 1. In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof may include a sequence having at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 261 contiguous amino acids of SEQ ID NO: 1. In some embodiments, the stabilized DNase I polypeptide, its functional fragment or its variant may include a sequence having less than 10% sequence identity with at least 25, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 261 consecutive amino acids of SEQ ID NO:1.

[0169] In some embodiments, the DNase I comprises an amino acid sequence that is similar to ALKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGUESUGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADUSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNUAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT (SEQ ID97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 , 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 1 86, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of 9, 250, 260, or 261 consecutive amino acids wherein U is a non-standard amino acid, such as selenocysteine.

[0170] In some embodiments, the DNase I comprises an amino acid sequence that is similar to ALKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGUESUGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADUSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNUAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT (SEQ ID NO: 1) has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein U is a non-standard amino acid, such as selenocysteine.

[0171] In some embodiments, the DNase I comprises an amino acid sequence that is the same as RGTRLMGLLLALAGLLQLGLSLKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGCESCGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADCSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNCAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT (SEQ ID NO:2) has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, wherein U is a non-standard amino acid, such as selenocysteine.

[0172] In some embodiments, the DNase I comprises an amino acid sequence that is the same as RGTRLMGLLLALAGLLQLGLSLKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGCESCGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADCSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNCAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT (SEQ ID100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 1 96, 197, 198, 199, 200, 201, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity of 3, 274, 275, 276, 277, 278, 279, 280, or 281 consecutive amino acids wherein U is a non-standard amino acid, such as selenocysteine.

[0173] In some embodiments, the DNase I further comprises at least one affinity tag. In some embodiments, the affinity tag of DNase I is a C-terminal affinity tag. In some embodiments, the affinity tag of DNase I is an N-terminal affinity tag. In some embodiments, the first affinity tag of DNase I is an N-terminal affinity tag, and the second affinity tag of DNase I is a C-terminal affinity tag. In some embodiments, the first affinity tag of DNase I is a first N-terminal affinity tag, and the second affinity tag of DNase I is a second N-terminal affinity tag. In some embodiments, the first affinity tag of DNase I is a first C-terminal affinity tag and the second affinity tag of DNase I is a second C-terminal affinity tag.

[0174] For example, the DNase I may include a poly-histidine tag, a poly-histidine-glycine tag, a poly-arginine tag, a poly-aspartic acid tag, a poly-cysteine ​​tag, a poly-phenylalanine, a c-myc tag, a herpes simplex virus glycoprotein D (gD) tag, a FLAG tag, a KT3 epitope tag, a tubulin epitope tag, a T7 gene 10 protein peptide tag, a streptavidin tag, a streptavidin binding protein (SPB) tag, a Strep-tag, a Strep-tag II, an albumin binding protein (ABP) tag, an alkaline phosphatase (AP) tag, a bluetongue virus tag, or a 5-mercaptoethanol tag. tag, B-tag), calmodulin binding peptide (CBP) tag, chloramphenicol acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSV tag, ketosteroid isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3. TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tag, protein C tag, S1-tag, S-tag, biotin carboxyl carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, V5 tag, or a combination thereof.

[0175] In some embodiments, the DNase I further comprises at least two affinity tags. For example, the DNase I may comprise at least two affinity tags, and the affinity tags are selected from: poly-histidine tag, poly-histidine-glycine tag, poly-arginine tag, poly-aspartic acid tag, poly-cysteine ​​tag, poly-phenylalanine, c-myc tag, herpes simplex virus glycoprotein D (gD) tag, FLAG tag, KT3 epitope tag, tubulin epitope tag, T7 gene 10 protein peptide tag, streptavidin tag, streptavidin binding peptide (SPB) tag, Strep-tag, Strep-tag II, albumin-binding protein (ABP) tag, alkaline phosphatase (AP) tag, blue tongue virus tag (B-tag), calmodulin binding peptide (CBP) tag, chloramphenicol Acetyltransferase (CAT) tag, choline-binding domain (CBD) tag, chitin-binding domain (CBD) tag, cellulose-binding domain (CBP) tag, dihydrofolate reductase (DHFR) tag, galactose-binding protein (GBP) tag, maltose-binding protein (MBP), glutathione-S-transferase (GST), Glu-Glu (EE) tag, human influenza hemagglutinin (HA) tag, horseradish peroxidase (HRP) tag, NE-tag, HSV tag, sterol isomerase (KSI) tag, KT3 tag, LacZ tag, luciferase tag, NusA tag, PDZ domain tag, AviTag, calmodulin-tag, E-tag, S-tag, SBP-tag, Softag 1, Softag 3, TC tag, VSV-tag, Xpress tag, Isopeptag, SpyTag, SnoopTag, Profinity eXact tag, Protein C tag, S1-tag, S-tag, biotin carboxyl carrier protein (BCCP) tag, green fluorescent protein (GFP) tag, small ubiquitin-like modifier (SUMO) tag, tandem affinity purification (TAP) tag, HaloTag, Nus-tag, thioredoxin-tag, Fc-tag, CYD tag, HPC tag, TrpE tag, ubiquitin tag, VSV-G epitope tag, and V5 tag.

[0176] In some embodiments, the DNase I comprises an affinity tag, and the affinity tag is GST. In some embodiments, the DNase I comprises an affinity tag, and the affinity tag is a poly-histidine tag, such as a 6x-His tag. In some embodiments, the DNase I comprises an affinity tag, and the affinity tag is MBP. In some embodiments, the DNase I comprises an affinity tag, and the affinity tag is a strep-tag, such as two strep tags.

[0177] In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, the first affinity tag is GST, and the second affinity tag is a poly-histidine tag, such as a 6x-His tag. In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, the first affinity tag is GST, and the second affinity tag is a strep tag. In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, the first affinity tag is a strep tag, such as two strep tags, and the second affinity tag is a poly-histidine tag, such as a 6x-His tag. In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, the first affinity tag is MBP, and the second affinity tag is a poly-histidine tag, such as a 6x-His tag. In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, the first affinity tag is MBP, and the second affinity tag is a strep tag, such as two strep tags.

[0178] In some embodiments, the DNase I comprises a first affinity tag, a second affinity tag, and a third affinity tag, wherein the first affinity tag is GST, the second affinity tag is a poly-histidine tag, such as a 6x-His tag, and the third affinity tag is a strep tag, such as two strep tags. In some embodiments, the DNase I comprises a GST tag, a His tag, and two strep tags. In some embodiments, the DNase I comprises a first affinity tag, a second affinity tag, and a third affinity tag, wherein the first affinity tag is MBP, the second affinity tag is a poly-histidine tag, such as a 6x-His tag, and the third affinity tag is a strep tag, such as two strep tags. In some embodiments, the DNase I comprises an MBP tag, a His tag, and two strep tags.

[0179] In some embodiments, the DNase I comprises an amino acid sequence that is similar to MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDGGSGSAALKIAAFNIRT FGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGUESUGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAP SDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADUSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNUAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLT(SEQ %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0180] In some embodiments, the DNase I comprises an amino acid sequence that is consistent with ALKIAAFNIRTFGETKMSNATLASYIVRIVRRYDIVLIQEVRDSHLVAVGKLLDYLNQDDPNTYHYVVSEPLGRNSYKERYLFLFRPNKVSVLDTYQYDDGUESUGNDSFSREPAVVKFSSHSTKVKEFAIVALHSAPSDAVAEINSLYDVYLDVQQKWHLNDVMLMGDFNADUSYVTSSQWSSIRLRTSSTFQWLIPDSADTTATSTNUAYDRIVVAGSLLQSSVVPGSAAPFDFQAAYGLSNEMALAISDHYPVEVTLTGSHHHHHHGSGGGSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 1034) ID NO:4) having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0181] In some embodiments, the DNase I comprises an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (SEQ ID NO:5).

[0182] In some embodiments, the DNase I comprises an affinity tag, wherein the affinity tag comprises an amino acid sequence that is identical to MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSD (SEQ ID NO:6) has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0183] In some embodiments, the DNase I comprises an affinity tag comprising an amino acid sequence having at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to GSHHHHHHGSGGGSGGSAWSHPQFEKGGGSGGGSGGSAWSHPQFE K (SEQ ID NO:7).

[0184] In some embodiments, the DNase I comprises an affinity tag comprising the amino acid sequence HHHHHH (SEQ ID NO: 8). In some embodiments, the DNase I comprises an affinity tag comprising the amino acid sequence AWSHPQFEK (SEQ ID NO 9).

[0185] In some embodiments, the DNase I comprises an affinity tag, wherein the DNase and the affinity tag are separated by a joint. In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, wherein the DNase and the first affinity tag are separated by a joint, and the DNase and the second affinity tag are separated by a joint. In some embodiments, the DNase I comprises a first affinity tag and a second affinity tag, wherein the first affinity tag and the second affinity tag are separated by a joint. In some embodiments, the DNase I comprises a first affinity tag, a second affinity tag and a third affinity tag, wherein the first affinity tag, the second affinity tag and the third affinity tag are each separated by a joint. In some embodiments, the DNase I comprises a first affinity tag, a second affinity tag, a third affinity tag and a fourth affinity tag, wherein the first affinity tag, the second affinity tag, the third affinity tag and the fourth affinity tag are each separated by a joint. In some embodiments, the joint comprises an amino acid sequence of (GS)n, (GGS)n or (GGGS)n or a combination thereof, wherein n is an integer of 1-10. In some embodiments, the linker comprises the amino acid sequence of GSGGGSGGS (SEQ ID NO: 10). In some embodiments, the linker comprises the amino acid sequence of GGGSGGGSGGS (SEQ ID NO: 11). In some embodiments, the linker comprises the amino acid sequence of GS (SEQ ID NO: 12). In some embodiments, the linker comprises the amino acid sequence of GGSGSA (SEQ ID NO 13). In some embodiments, the linker comprises the amino acid sequence of GGSGSAA (SEQ ID NO 14).

[0186] In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1, position 105 of SEQ ID NO: 1, position 174 of SEQ ID NO: 1, or position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1, position 105 of SEQ ID NO: 1, position 174 of SEQ ID NO: 1, and position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1 and position 105 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1 and position 174 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1 and position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 105 of SEQ ID NO: 1 and position 174 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 105 of SEQ ID NO: 1 and position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 174 of SEQ ID NO: 1 and position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1, position 105 of SEQ ID NO: 1, and position 174 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1, position 105 of SEQ ID NO: 1, and position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 102 of SEQ ID NO: 1, position 105 of SEQ ID NO: 1, and position 210 of SEQ ID NO: 1. In some embodiments, the one or more non-standard amino acids may be located at position 105 of SEQ ID NO: 1, position 174 of SEQ ID NO: 1, and position 210 of SEQ ID NO: 1.

[0187] In some embodiments, the non-standard amino acid at position 102 may be directly linked by a bond to the non-standard amino acid at position 105. In some embodiments, the non-standard amino acid at position 174 may be directly linked by a bond to the non-standard amino acid at position 210. In some embodiments, the non-standard amino acid at position 102 may be directly linked by a bond to the non-standard amino acid at position 174. In some embodiments, the non-standard amino acid at position 102 may be directly linked by a bond to the non-standard amino acid at position 210. In some embodiments, the non-standard amino acid at position 105 may be directly linked by a bond to the non-standard amino acid at position 174. In some embodiments, the non-standard amino acid at position 105 may be directly linked by a bond to the non-standard amino acid at position 210.

[0188] In some embodiments, the bond can be a diselenide bond. In some embodiments, the diselenide bond can be in the position of a disulfide bond in a corresponding recombinant enzyme without the one or more non-standard amino acids.

[0189] In some embodiments, the Tm of the corresponding stabilized DNase I polypeptide, its functional fragment or variant thereof may be less than 37° C. In some embodiments, the Tm of the stabilized DNase I polypeptide, its functional fragment or variant thereof may be greater than 37° C., 40° C., 45° C., 50° C., 55° C., 60° C. or 65° C. In some embodiments, the Tm of the stabilized DNase I polypeptide, its functional fragment or variant thereof may be at least 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C. or greater.

[0190] In some embodiments, the Tm of the stabilized DNase I polypeptide, its functional fragment or variant thereof may be at least 10°C higher than the Tm of the corresponding recombinase, its functional fragment or variant thereof. In some embodiments, the Tm of the stabilized DNase I polypeptide, its functional fragment or variant thereof may be at least 15°C higher than the Tm of the corresponding recombinase, its functional fragment or variant thereof. In some embodiments, the Tm of the stabilized DNase I polypeptide, its functional fragment or variant thereof may be at least 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or more higher than the Tm of the corresponding recombinase, its functional fragment or variant thereof. In some embodiments, the Tm of the stabilized DNase I polypeptide, its functional fragment or variant thereof may be less than 10°C higher than the Tm of the corresponding recombinase, its functional fragment or variant thereof.

[0191] In some embodiments, the half-life of the stabilized DNase I polypeptide, its functional fragment or its variant in the environment can be longer than that of the corresponding DNase I polypeptide not comprising the one or more non-standard amino acids. 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000-fold or more greater half-life of the I polypeptide, its functional fragment or variant thereof in said environment.

[0192] In some embodiments, the half-life of the DNase I polypeptide, its functional fragment or its variant in the environment can be greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours or longer. In some embodiments, the half-life of the DNase I polypeptide, its functional fragment or its variant in the environment can be less than 1 hour.

[0193] In some embodiments, the half-life of the DNase I polypeptide, its functional fragment or its variant in the environment can be greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days or longer. In some embodiments, the half-life of the DNase I polypeptide, its functional fragment or its variant in the environment can be less than 1 day.

[0194] In some embodiments, the stabilized DNase I polypeptide can have an endonuclease activity on a DNA substrate in an environment that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof on the DNA substrate that does not comprise the one or more non-standard amino acids. In some embodiments, the stabilized DNase I polypeptide can have an endonuclease activity on a DNA substrate in an environment that is less than 1.1 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof on the DNA substrate that does not comprise the one or more non-standard amino acids.

[0195] In some embodiments, the stabilized DNase I polypeptide can have an endonuclease activity on a DNA substrate after being present in an environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids after being present in the environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes. In some embodiments, the stabilized DNase I polypeptide can have an endonuclease activity on a DNA substrate after being present in an environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes, which is less than 1.1 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids after being present in the environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 minutes.

[0196] In some embodiments, the stabilized DNase I polypeptide can have an endonuclease activity on a DNA substrate after being present in an environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids after being present in the environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours. In some embodiments, the stabilized DNase I polypeptide can have an endonuclease activity on a DNA substrate after being present in an environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours that is less than 1.1 times higher than the endonuclease activity of a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids after being present in the environment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18, or 24 hours.

[0197] In some embodiments, the DNA substrate may be genomic DNA. In some embodiments, the DNA substrate may be single-stranded DNA, double-stranded DNA, circular DNA or cell-free DNA.

[0198] In some embodiments, the environment may be an environment with a temperature of 4° C.-98° C. In some embodiments, the environment may be an environment with a temperature of 5° C.-97° C., 6° C.-96° C., 7° C.-95° C., 8° C.-94° C., 9° C.-93° C., 10° C.-92° C., 11° C.-91° C., 12° C.-90° C., 13° C.-89° C., 14° C.-88° C., 15° C.-85° C., 20° C.-75° C., 25° C.-70° C., 30° C.-65° C., 35° C.-60° C., 40° C.-55° C., or 45° C.-50° C. In some embodiments, the environment can be an environment with a temperature of at least 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C or more. In some embodiments, the environment can be an environment with a temperature of less than 4°C.

[0199] In some embodiments, the environment can be an environment containing a lysis buffer. In some embodiments, the lysis buffer can be an NP-40 lysis buffer, a RIPA (radioimmunoprecipitation assay) lysis buffer, an SDS (sodium dodecyl sulfate) lysis buffer or an ACK (ammonium-chloride-potassium) lysis buffer.

[0200] In some embodiments, the environment can be an environment containing a detergent with a concentration of 0.01% to 20%. In some embodiments, the environment can be an environment containing a detergent with a concentration of 0.01% to 20%, 0.05% to 19.5%, 0.1% to 19%, 0.2% to 18.5%, 0.3% to 18%, 0.4% to 17.5%, 0.5% to 17%, 0.6% to 16.5%, 0.7% to 16%, 0.8% to 15%, 0.8% to 14%, 0.9% to 13% or 1% to 12%. In some embodiments, the environment can be an environment containing a detergent with a concentration less than 0.01%. In some embodiments, the environment can be an environment containing a detergent with a concentration greater than 20%.

[0201] In some embodiments, the detergent can be a nonionic detergent. The nonionic detergent can include Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside and octyl thioglucosidoside. In some embodiments, the detergent can be an ionic detergent. The ionic detergent can include sodium dodecyl sulfate (SDS). In some embodiments, the detergent can be a cationic detergent. The cationic detergent can be ethyl trimethyl ammonium bromide. In some embodiments, the detergent can be a zwitterionic detergent. The zwitterionic detergent can be CHAPS (3-[(3-cholamidopropyl) dimethylammonium]-1-propane sulfonate).

[0202] In some embodiments, the environment may include divalent cations at a concentration of 0.01mM to 100mM. In some embodiments, the environment may include divalent cations at a concentration of 0.01mM to 100mM, 0.05mM to 95mM, 0.1mM to 90mM, 0.5mM to 85mM, 1mM to 80mM, 5mM to 75mM, 10mM to 70mM, 15mM to 65mM, 20mM to 60mM, 25mM to 55mM, 30mM to 50mM or 35mM to 45mM. In some embodiments, the environment may include divalent cations at a concentration of less than 0.01mM. In some embodiments, the environment may include divalent cations at a concentration greater than 100mM. In some embodiments, the divalent cations are selected from the group consisting of Mg, 2+ , Mn 2+ , Ca 2+ 、Co 2+ and Zn 2+ group.

[0203] In some embodiments, the environment may include a reducing agent having a concentration of 0.01 mM to 100 mM. The reducing agent may be glutathione, albumin or thioredoxin. In some embodiments, the environment may include a reducing agent having a concentration of 0.01 mM to 100 mM, 0.05 mM to 95 mM, 0.1 mM to 90 mM, 0.5 mM to 85 mM, 1 mM to 80 mM, 5 mM to 75 mM, 10 mM to 70 mM, 15 mM to 65 mM, 20 mM to 60 mM, 25 mM to 55 mM, 30 mM to 50 mM or 35 mM to 45 mM. In some embodiments, the environment may include a reducing agent having a concentration less than 0.01 mM. In some embodiments, the environment may include a reducing agent having a concentration greater than 100 mM.

[0204] In some embodiments, the environment may have a pH of 5-9. In some embodiments, the environment may have a pH of 6-8. In some embodiments, the environment may have a pH of 7-8. In some embodiments, the environment may have a pH of less than 5. In some embodiments, the environment may have a pH greater than 9.

[0205] In some embodiments, the environment can have a salt concentration of 10mM to 1M. In some embodiments, the environment can have a salt concentration of 15mM to 950mM, 20mM to 900mM, 30mM to 850mM, 40mM to 800mM, 50mM to 750mM, 60mM to 700mM, 70mM to 650mM, 80mM to 600mM, 90mM to 550mM, 100mM to 500mM, 150mM to 450mM or 200mM to 400mM. In some embodiments, the environment can have a salt concentration less than 10mM. In some embodiments, the environment can have a salt concentration greater than 1M.

[0206] In some embodiments, the environment can be in a droplet. In some embodiments, the environment can be the blood circulation system. In some embodiments, the environment can be any environment in which the stabilised DNase I polypeptide, its functional fragment or its variant can have enzymatic activity.

[0207] In some embodiments, the environment can have a reduction potential that is less than -150 mV, -160 mV, -170 mV, -180 mV, -190 mV, -200 mV, -210 mV, -220 mV, -230 mV, -240 mV, or -250 mV, -260 mV, -270 mV, -280 mV, -290 mV, -300 mV, -310 mV, -320 mV, -330 mV, -340 mV, or -350 mV. In some embodiments, the environment may have a reduction potential greater than -150 mV.

[0208] In some embodiments, the stabilization DNase I polypeptide, its functional fragment or its variant can be a recombinant. In some embodiments, the recombinant can utilize recombinant DNA technology to produce, for example, by the stabilization DNase I polypeptide, its functional fragment or its variant expressed by phage or yeast expression system. In other embodiments, the recombinant can produce by the DNA molecule of the stabilization DNase I polypeptide, its functional fragment or its variant of the synthetic coding.

[0209] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or variant thereof can be bovine DNase I. The stabilized DNase I polypeptide, its functional fragment or variant thereof can be any other type of DNases I, including but not limited to Escherichia coli DNase I, Microcella alkaliphila DNase I, Lactobacillus algidus DNase I, Vibrio cholerae DNase I, Bifidobacterium longum DNase I, Homo sapiens DNase I and Raoultella ornithinolytica DNase I.

[0210] In some embodiments, the composition may include a polynucleotide encoding a composition disclosed herein. In some embodiments, the polynucleotide may be a vector. The vector may be a fragment of a nucleic acid molecule. The vector may be taken from a virus, a plasmid, or a cell of a higher organism. The vector may be stably maintained in an organism. The vector may be inserted together with an exogenous nucleic acid fragment for cloning purposes. The vector may include features that allow the nucleic acid fragment to be easily inserted into or removed from the vector. The vector may be a genetically engineered plasmid.

[0211] In some embodiments, a bond directly linking two of the one or more non-standard amino acids of the stabilized DNase I polypeptide may not be broken in an environment, while a bond directly linking two of the one or more standard amino acids of the corresponding DNase I polypeptide may be broken in the same environment.

[0212] In some embodiments, the method of preparing the compositions disclosed herein may include expressing the amino acid sequence of the stabilized DNase I polypeptide. In some embodiments, the expression may include expression in a cell or in vitro.

[0213] In some embodiments, the cell may be a bacterial cell. In some embodiments, the cell may be a cell whose genome is recoded. In some embodiments, the cell may not be a bacterial cell. The cell may be obtained or separated from a subject. The cell may be obtained or separated from a tissue. The subject may be an animal, such as a human, mouse, rat, pig, dog, rabbit, sheep, horse, chicken or other animal. The cell may be a neuron. The cell may be one of the cells in the blood-brain barrier system. The cell may be a cell line, such as a neuronal cell line. The cell may be a primary cell, such as a cell obtained from the brain of a subject. The cell may be a cell population separated from a subject, such as a tissue biopsy, a cytology sample, a blood sample, a fine needle aspirate (FNA) sample or a combination thereof. The cells can be obtained from body fluids, such as urine, milk, sweat, lymph, blood, sputum, amniotic fluid, aqueous humor, vitreous humor, bile, cerebrospinal fluid, chyle, chyme, exudate, endolymph, perilymph, gastric acid, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum, serous fluid, smegma, sputum, tears, vomitus or other body fluids. The cells may include cancer cells, non-cancerous cells, tumor cells, non-tumor cells, healthy cells or any combination thereof.

[0214] In some embodiments, the cell can include a reassigned codon recognized by a stabilized non-standard amino acid tRNA comprising an anti-codon corresponding to the reassigned codon.

[0215] In some embodiments, the amino acid sequence of the DNase I polypeptide of the stabilization can be encoded by a polynucleotide sequence comprising at least one codon for a natural amino acid, which codon can be replaced by the codon of the reallocation. In some embodiments, the amino acid sequence of the DNase I polypeptide of the stabilization can be encoded by a polynucleotide sequence comprising at least one, two or three codons for a natural amino acid, which codon can be replaced by the codon of the reallocation.

[0216] In some embodiments, the stabilized non-standard amino acid tRNA can be a selenocysteine ​​tRNA.

[0217] In some embodiments, the method may include culturing the cell under conditions where the amino acid sequence of the stabilized DNase I polypeptide can be expressed. In some embodiments, the reassigned codon may be UAG, UAA, UGA, or a combination thereof.

[0218] In some aspects, a method is provided herein, the method comprising contacting a DNA substrate that may be in a buffer, in a reaction environment, or on a solid surface with a stabilized deoxyribonuclease I (DNase I) polypeptide, a functional fragment thereof, or a variant thereof comprising one or more non-standard amino acids; wherein the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof can catalyze the cleavage or fragmentation of the DNA substrate at a higher rate than a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids. In some embodiments, the stabilized DNase I polypeptide, a functional fragment thereof, or a variant thereof can catalyze the cleavage or fragmentation of the DNA substrate at a higher rate than a corresponding DNase I polypeptide, a functional fragment thereof, or a variant thereof that does not comprise the one or more non-standard amino acids. 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 times or more of the I polypeptide, its functional fragment or variant thereof.

[0219] In some embodiments, the stabilized DNase I polypeptide, its functional fragment or its variant can be a stabilized DNase I polypeptide, its functional fragment or variant disclosed in other parts of this paper. In some embodiments, the DNA substrate can be genomic DNA. In some embodiments, the DNA substrate is from a single cell. The single cell (or cell) is described in other parts of this paper. In some embodiments, the method can include forming a plurality of containers, each container including a single cell in a plurality of cells; the stabilized DNase I polypeptide, its functional fragment or its variant; and a lysis buffer. In some embodiments, the method can further include cracking the single cell, thereby releasing the DNA substrate from the single cell.

[0220] In some embodiments, the method may further include barcoding the DNA substrate or its fragment. The barcode on the DNA substrate may be a natural or synthetic nucleic acid sequence contained in a polynucleotide to clearly identify the polynucleotide and other sequences contained in the polynucleotide with the barcode sequence. The barcode may uniquely identify a subject, a sample (e.g., a cell-free sample), a nucleic acid sequence (e.g., a sequence of bases with one or more epigenetic modifications), or any combination thereof. The barcode may be associated with a DNA substrate or a complementary strand. The DNA substrate may include a single barcode. The DNA substrate may include one or more barcodes, such as a first barcode and a second barcode. In some cases, the first barcode may be different from the second barcode. In some cases, each barcode in a plurality of barcodes may be a unique barcode. In some cases, the barcode may include a sample identification barcode. For example, the first barcode may include a unique barcode, and the second barcode may include a sample identification barcode.

[0221] In some embodiments, the method may further include amplifying the DNA substrate or a fragment thereof. The amplification may include polymerase chain reaction (PCR) amplification, loop-mediated isothermal amplification, nucleic acid sequence-based amplification, strand displacement amplification, multiple displacement amplification, rolling circle amplification, ligase chain reaction, helicase-dependent amplification, branching amplification, clonal amplification, or any combination thereof. In some cases, the amplification may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or more amplification cycles. In some embodiments, the amplification may include clonal amplification. In some cases, a separate DNA substrate or fragment may be amplified in situ on a support. In some cases, the amplification generates no more than about 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 15 or 10 20 Amplicon.

[0222] In some embodiments, the method further includes sequencing the DNA substrate or its fragment. The sequencing may include: no bisulfite sequencing, bisulfite sequencing, TET-assisted bisulfite (TAB) sequencing, ACE-sequencing, high-throughput sequencing, Maxam-Gilbert sequencing, large-scale parallel signature sequencing, Polony sequencing, 454 pyrophosphate sequencing, Sanger sequencing, Illumina sequencing, SOLiD sequencing, Ion Torrent semiconductor sequencing, DNA nanoball sequencing, Heliscope single molecule sequencing, single molecule real-time (SMRT) sequencing, nanopore DNA sequencing, shotgun sequencing, RNA sequencing, Enigma sequencing or any combination thereof. In some embodiments, the sequencing includes whole genome sequencing. In some embodiments, the sequencing includes high-throughput sequencing, large-scale parallel sequencing, Sanger sequencing or next generation sequencing.

[0223] In some embodiments, the multiple containers include solid supports. In some embodiments, the DNA substrate is not attached to the solid support in the container. In some embodiments, the DNA substrate can be attached to the solid support in the container. The support can be any solid or semi-solid product, on which reagents such as nucleic acids can be fixed. Nucleic acids can be immobilized on solid supports by any method, including but not limited to physical adsorption, formed by ionic bonds or covalent bonds, or a combination thereof. Solid supports can include polymers, glass or metal materials. Examples of solid supports include films, planar surfaces, microtiter plates, beads, filters, test strips, slides, coverslips and test tubes, meaning any solid phase material on which oligomers are synthesized, attached, connected or otherwise fixed. The support can be composed of organic polymers, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy (polyethyleneoxy) and polyacrylamide, as well as copolymers and grafts thereof. The support can also be inorganic, such as glass, silica, controlled-pore-glass (CPG) or reversed-phase silica. The configuration of the support can be in the form of a bead, sphere, microparticle, particle, gel or surface. The surface can be planar, substantially planar or non-planar. The support can be porous or non-porous and can have swelling or non-swelling properties. The support can be shaped to include one or more wells, depressions, or other vessels, containers, features or locations. Multiple supports can be configured into arrays at different locations.

[0224] In some embodiments, buffer, reaction environment or solid surface can include primers to the sequence specificity of the DNA substrate or its fragment. The primer can be a nucleic acid with a known or unknown sequence. The primer can be a single strand. In some cases, the primer can include a barcode (e.g., a unique identifier sequence). The primer can be an amplification primer that hybridizes with an adapter and extends in an amplification reaction using a target nucleic acid as a template. The primer can be a sequencing primer that hybridizes with an adapter and extends in a sequencing reaction using a target nucleic acid as a template.

[0225] In some embodiments, the plurality of cells may include at least 2, 3, 4, 5, 5.5, 6, 6.5 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 2 , 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000 , 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x108 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , 9x10 10 , 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , 9x10 11 , 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 or 9x10 12 cells.

[0226] In some embodiments, the multiple cells can be from one or more biological samples. The biological sample can be from a subject, such as a biopsy, a cytological specimen, a blood sample, a fine needle aspiration (FNA) sample or any combination thereof. The biological sample can be obtained from a body fluid, such as urine, milk, sweat, lymph, blood, sputum, amniotic fluid, aqueous humor, vitreous humor, bile, cerebrospinal fluid, chyle, chyme, exudate, endolymph, perilymph, gastric acid, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, rheumatism, saliva, sebum, serous fluid, smegma, sputum, tears, vomitus or other body fluids.

[0227] In some embodiments, the one or more biological samples include at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more samples. In some embodiments, the one or more biological samples may include samples from different subjects. In some embodiments, the one or more biological samples may include samples from the same subject.

[0228] In some embodiments, the one or more biological samples are from a subject suffering from a disease. The disease may include cancer, a neurological disorder, or an autoimmune disease. In some embodiments, the disease may include a neurological disorder. In some cases, neurological disorders can include: acquired epileptic aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, corpus callosum dysgenesis, agnosia, Aicardi syndrome, Alexander disease, Alpers disease, alternating paralysis, Alzheimer's disease, amyotrophic lateral sclerosis (see motor neuron disease), anencephaly, Angelman syndrome, angiomatosis, hypoxia, aphasia, apraxia, intracranial arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger syndrome, ataxia telangiectasia, attention deficit hyperactivity disorder, autism, auditory processing disorder, autonomic dysfunction, back pain, Deng's disease, Behcet's disease, Bell's facial palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial pressure heightened, bilateral frontoparietal polygyria, Binswanger disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus injury, brain abscess, brain injury, brain trauma, brain tumor, Brown-Sequard syndrome, Canavan disease, carpal tunnel syndrome (CTS), causalgia, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, head disease, brain aneurysm, cerebral arteriosclerosis, brain atrophy, cerebral gigantism, cerebral palsy, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic pain, chronic regional pain syndrome, Coffin-Lawley syndrome, Lowry syndrome, coma including persistent vegetative state, congenital diplegia, cortical-basal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative injury, Cushing syndrome, cytomegalovirus infection, Dandy-Walker syndrome, Dawson disease, Demosin Morsier syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, neurological movement disorders, diabetic neuropathy, diffuse sclerosis, autonomic dysfunction, dyscalculia, dysgraphia, dyslexia, dystonia, infantile epileptic encephalopathy, empty sella syndrome, encephalitis, encephalocele, cerebral trigeminal angiomatosis, fecal incontinence, epilepsy, Erb's palsy, erythromelalgia, essential tremor, Fabry disease, Fabry syndrome, syncope, familial spastic paralysis, febrile epilepsy, Fisher syndrome,Friedreich's ataxia, FART syndrome, Gaucher disease, Gerstmann syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, gray matter heterotopia, Guillain-Barré syndrome, HTLV-1-associated myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary ataxia polyneuritis, herpes zoster oticus, herpes zoster, Hirayama syndrome Syndrome, holoprosencephaly, Huntington disease, hydrocephalus, hydrocephalus, hypercortisolism, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinence pigmenti, phytanic acid storage disease of infancy, Refsum disease of infancy, infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbourne syndrome, Klippel syndrome Feil syndrome, Krabbe disease, Kugelberg-Weiland disease, Kuru, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, Wallenberg syndrome, learning deficits, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, dementia with Lewy bodies, lissencephaly, locked-in syndrome, Lou Gehrig's disease, Gehrig's disease, lumbar disc disease, Lyme disease-neurological sequelae, Mathew-Johnson disease (spinocerebellar ataxia type 3), megalencephaly, maple syrup urine disease, megalencephaly, Melkerson-Rosenthal syndrome, Meniere's disease, meningitis, Menkes disease, metachromatic leukodystrophy, microcephaly, migraine, Miller-Ferguson syndrome, mild stroke, mitochondrial myopathy, Moebius syndrome, monomelic muscular atrophy, motor neuron disease, motor skill impairment, moyamoya disease, mucopolysaccharidosis, multi-infarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, diffuse demyelinating sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy disease, myotonia congenita, narcolepsy, neurofibromatosis, neuroleptic malignant syndrome, neurologic manifestations of AIDS, neurologic sequelae of lupus, neuromyotonia, neuronal ceroid lipofuscinosis, neuronal migration disorder, Niemann-Pick disease, non-24-hour sleep-wake syndrome, nonverbal learning disorder, O'Sullivan-McLeod syndrome, occipital neuralgia, occult spinal canal dysraphism sequence, Ohtawara syndrome, olivopontocerebellar atrophy, strabismus-opsoclonus-myoclonus syndrome, optic neuritis, orthostatic hypotension, overuse syndrome, visual retention, paresthesia, Parkinson's disease, paramyotonia congenita, paraneoplastic disease, paroxysmal seizures,Parry-Romberg syndrome, Romberg syndrome, Pelizeu-Merzbach disease, periodic paralysis, peripheral neuropathy, persistent vegetative state, diffuse neurologic disorder, light-induced sneeze reflex, phytanic acid storage disease, Pick's disease, pinched nerve, pituitary tumor, PMG, poliomyelitis, polymicrogyria, polymyositis, cerebral punctate malformation, postpoliomyelitis syndrome, postherpetic neuralgia (PHN), postinfectious encephalomyelitis, postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive hemifacial atrophy (also called Romberg syndrome), progressive multifocal leukoencephalopathy, progressive sclerosis gray matter dystrophy, progressive supranuclear palsy, pseudotumor cerebri, Ramsay-Hunt syndrome (types I and II), Rasmussen encephalitis, reflex sympathetic dystrophy syndrome, Refsum disease, repetitive movement disorder, repetitive stress injury, restless legs syndrome, retrovirus-associated myelopathy, Rett syndrome, Reye's syndrome, Romberg syndrome, rabies, Saint Vitus chorea, Sandhoff disease, schizophrenia, Schilder disease, schizophrenia, sensory integration disorders, septo-optic dysplasia, shaken baby syndrome, herpes zoster, Shy-Drager syndrome, Sjogren syndrome, sleep apnea, sleep sickness, Snatiation, Sotos syndrome, spasticity, spina bifida, spinal cord injury, spinal cord tumor, spinal muscular atrophy, spinal stenosis, Steele-Richardson-Olszewski syndrome, see progressive supranuclear palsy, spinocerebellar ataxia, Stiff-person syndrome, stroke, Sturge-Weber syndrome, rge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial siderosis, Sydenham chorea, syncope, synesthesia, syringomyelia, tardive dyskinesia, Tartu-Sachs disease, temporal arteritis, tethered cord syndrome, Thomsen disease, thoracic outlet syndrome, trigeminal neuralgia, Todd's palsy, Tourette syndrome, transient ischemic attack, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremor, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, vasculitis including temporal arteritis, Von Hippel-Lindau disease (VHL), Viliuisk encephalomyelitis (VE), Wallenberg syndrome, Hoffmann disease,West syndrome, whiplash, Williams syndrome, Wilson disease, X-linked spinal bulbar muscular atrophy, and Zellweger syndrome.

[0229] In some cases, the disease may include an autoimmune disease. In some cases, the autoimmune disease may include: acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome (APS), autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune Epidemiological thyroid disease, axonal & neuronal neuropathy, Balo disease, Behcet's disease, bullous pemphigoid, cardiomyopathy, Castlemen's disease, lipid diarrhea (non-tropical), Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan syndrome, cold agglutinin disease, congenital heart block, coxsackie myocarditis, Klay's disease, essential mixed cryoglobulinemia, apoptosis Myelinating neuropathy, dermatomyositis, Devic disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evan syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, Graves disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henock-Schoniein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, immune regulation Lipoprotein, inclusion body myositis, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus erythematosus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic), neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism,PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococci), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, parsplantis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndromes type I, II & III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, postpericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Rett syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Slogren syndrome, sperm and testicular autoimmunity, stiff-man syndrome, subacute bacterial endocarditis (SBE), sympathetic ophthalmia, Takayasu arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TPP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCT), D), uveitis, vasculitis, bullous dermatosis, vitiligo or Wegener's granulomatosis, chronic active hepatitis, primary biliary cirrhosis, cadilated cardiomyopathy, myocarditis, autoimmune polyglandular syndrome type I (APS-I), cystic fibrosis vasculitis, acquired hypoparathyroidism, coronary artery disease, pemphigus foliaceus, pemphigus vulgaris, Rasmussen's encephalitis, autoimmune gastritis, insulin-induced hypoglycemia syndrome (Hirata disease), type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE), pernicious anemia, treatment-resistant Lyme arthritis, polyneuropathy, demyelinating disease, atopic dermatitis, autoimmune hypothyroidism, Vitiligo, thyroid-related eye disease, autoimmune diarrhea, ACTH deficiency, dermatomyositis, Sjogren's syndrome, systemic sclerosis, progressive systemic sclerosis, morphea, primary antiphospholipid antibody syndrome, chronic idiopathic urticaria, connective tissue syndrome, necrotizing crescentic glomerulonephritis (NCGN), systemic vasculitis, Raynaud's syndrome, chronic liver disease, visceral leishmaniasis, autoimmune C1 deficiency, membranoproliferative glomerulonephritis (MPGN), prolonged clotting time, immunodeficiency, atherosclerosis, neuron disease, paraneoplastic pemphigus, paraneoplastic stiff-man syndrome, paraneoplastic encephalomyelitis, subacute autonomic neuropathy, cancer-related retinopathy, paraneoplastic opsoclonus, myoclonus, ataxia,Lower motor neurone syndrome and Lambert-Eaton myasthenic syndrome.

[0230] In some cases, the disease may include: AIDS, anthrax, botulism, brucellosis, chancroid, chlamydia, cholera, coccidioidomycosis, cryptosporidiosis, cyclosporiasis, diphtheria, ehrlichiosis, arboviral encephalitis, enterohemorrhagic Escherichia coli, giardiasis, gonorrhea, dengue fever, Haemophilus influenzae, Hansen's disease (leprosy), Hantavirus pulmonary syndrome, hemolytic uremic syndrome, hepatitis A, hepatitis B, hepatitis C, human immunodeficiency virus, Legionnaires' disease, listeriosis, Lyme disease, malaria, measles, meningococcal disease, mumps, pertussis (whooping cough), plague, paralytic poliomyelitis , psittacosis, Q fever, rabies, Rocky Mountain spotted fever, rubella, congenital rubella syndrome (SARS), shigellosis, smallpox, streptococcal disease (invasive group A), streptococcal toxic shock syndrome, streptococcal pneumonia, syphilis, tetanus, toxic shock syndrome, trichinosis, tuberculosis, tularemia, typhoid fever, vancomycin intermediate-resistant Staphylococcus aureus, varicella, yellow fever, variant Creutzfeldt-Jakob disease (vCJD), Ebola hemorrhagic fever, echinococcosis, Hendra virus infection, human monkeypox, influenza A, H5N1, Lassa fever, Marburg hemorrhagic fever, Nipah virus, Oneonian fever, Rift Valley fever, Venezuelan equine encephalitis, and West Nile virus.

[0231] In some cases, the disease can include cancer. In some cases, the cancer can include thyroid cancer, adrenocortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, central nervous system (CNS) cancer, peripheral nervous system (PNS) cancer, breast cancer, Castleman's disease, cervical cancer, childhood non-Hodgkin's lymphoma, lymphoma, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's family of tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, childhood leukemia, chronic lymphocytic leukemia, Baxter's leukemia, chronic myeloid leukemia, liver cancer, lung carcinoid tumor, non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disorders, nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer, non-melanoma skin cancer, stomach cancer, testicular cancer, thymic cancer, uterine cancer (e.g., uterine sarcoma), vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia.

[0232] In some cases, the disease may include a hyperproliferative disorder. Malignant hyperproliferative disorders may be divided into risk groups, such as a low risk group and a medium to high risk group. Hyperproliferative diseases may include, but are not limited to, cancer, hyperplasia, or neoplasia.In some cases, the hyperproliferative cancer can be breast cancer, such as ductal carcinoma of the ductal tissue in the breast, medullary carcinoma, colloid carcinoma, ductal carcinoma, and inflammatory breast cancer; ovarian cancer, including ovarian epithelial tumors, such as adenocarcinoma in the ovary and adenocarcinoma that has metastasized from the ovary to the abdominal cavity; uterine cancer; cervical cancer, such as adenocarcinoma in the cervical epithelium (including squamous cell carcinoma and adenocarcinoma); prostate cancer, such as a prostate cancer selected from the group consisting of adenocarcinoma, or adenocarcinoma that has metastasized to the bone; pancreatic cancer, such as epithelioid carcinoma in the pancreatic ductal tissue and adenocarcinoma in the pancreatic duct; bladder cancer, such as transitional cell carcinoma in the bladder, urothelial carcinoma (transitional cell carcinoma), urothelial cell tumors in the bladder, squamous cell carcinoma, adenocarcinoma, and small cell carcinoma; leukemia, such as acute myeloid leukemia (AML); myeloid leukemia (AML), chronic myeloid leukemia (CML), mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and myelodysplastic syndrome (MDS); bone cancer; lung cancer such as non-small cell lung cancer (NSCLC), which can be divided into squamous cell carcinoma, adenocarcinoma, and large cell undifferentiated carcinoma), and small cell lung cancer; skin cancer such as basal cell carcinoma, melanoma, squamous cell carcinoma, and actinic keratosis (which can be a skin condition that sometimes develops into squamous cell carcinoma); ocular retinoblastoma; skin or intraocular (eye) cancer. melanoma; primary hepatocellular carcinoma (benign cancer in the liver); renal cancer; autoimmune deficiency syndrome (AIDS)-related lymphomas such as diffuse large B-cell lymphoma, B-cell immunoblastic lymphoma, and small non-lytic cell lymphoma; Kaposi's sarcoma; virus-induced cancers including hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatocellular carcinoma; human lymphotropic virus type 1 (HTLV-1) and adult T-cell leukemia / lymphoma; and human papillomavirus (HPV) and cervical cancer; central nervous system (CNS) cancers such as primary brain tumors, including gliomas (astrocytomas, anaplastic astrocytomas, or glioblastoma multiforme), oligodendrogliomas, ependymomas, brain peripheral nervous system (PNS) cancers such as acoustic neuroma and malignant peripheral nerve sheath tumors (MPNST), including neurofibromas and schwannomas, malignant fibroblastomas, malignant fibrous histiocytomas, malignant meningiomas, malignant mesotheliomas, and malignant mixed Müllerian tumors; oral and oropharyngeal cancers such as hypopharyngeal cancer, laryngeal cancer, nasopharyngeal cancer, and oropharyngeal cancer; gastric cancers such as lymphomas, gastric stromal tumors, and carcinoid tumors; testicular cancers such as germ cell tumors (GCTs), which include seminoma and non-seminoma, and gonadal stromal cell tumors, which include Leydig cell tumors and Sertoli cell tumors; thymic cancers such as thymoma, thymic carcinoma, Hodgkin's disease, non-Hodgkin lymphoma carcinoids, or carcinoid tumors; rectal cancer; and colon cancer.In some cases, the diseases graded, classified, characterized or diagnosed according to the methods of the present disclosure include, but are not limited to, thyroid diseases, such as benign thyroid diseases, including, but not limited to, follicular adenoma, Hurthle cell adenoma, lymphocytic thyroiditis and thyroid hyperplasia. In some cases, the diseases graded, classified, characterized or diagnosed according to the methods of the present disclosure include, but are not limited to, malignant thyroid diseases, such as follicular carcinoma, follicular variants of papillary thyroid carcinoma, medullary carcinoma and papillary carcinoma.

[0233] The disease may include a hereditary disease. A hereditary disease may be a condition caused by an abnormality in a gene or chromosome. Hereditary diseases may be divided into two categories: single gene diseases, and multifactorial diseases and multigenic (complex) diseases. Single gene diseases may be caused by a single mutated gene. Hereditary single gene diseases may include, but are not limited to, autosomal dominant inheritance, autosomal recessive inheritance, X-linked dominant inheritance, X-linked recessive inheritance, and Y-linked mitochondrial inheritance. For a person suffering from an autosomal dominant disease, only one mutated copy of the gene is required. Examples of autosomal dominant types of conditions may include, but are not limited to, Huntington's disease, neurofibromatosis 1, Marfan syndrome, hereditary non-polyposis colorectal cancer, or hereditary multiple exostoses. In autosomal recessive diseases, two copies of the gene must be mutated for a subject suffering from an autosomal recessive disease. Examples of this type of disease may include, but are not limited to, cystic fibrosis, sickle cell disease (partial sickle cell disease), familial Tay-Sachs disease, Niemann-Pick disease, or spinal muscular atrophy. X-linked dominant diseases are caused by gene mutations on the X chromosome, such as X-linked hypophosphatemic rickets. Some X-linked dominant diseases such as Rett syndrome, pigment disorder type 2 and Aicardi syndrome may be fatal. X-linked recessive diseases are also caused by gene mutations on the X chromosome. Examples of this type of disease may include, but are not limited to, hemophilia A, Duchenne muscular dystrophy, red-green color blindness, muscular dystrophy and androgenic alopecia. Y-linked diseases are caused by mutations on the Y chromosome. Examples may include, but are not limited to, male infertility and hirsutism. Mitochondrial inherited genetic diseases, also known as maternal inheritance, may be applicable to genes in mitochondrial DNA, such as in Leber's hereditary optic neuropathy.

[0234] In some embodiments, the plurality of cells may include a plurality of bacterial cells or fungal cells. In some embodiments, the plurality of bacterial cells or fungal cells may include at least 2, 3, 4, 5, 5.5 6, 6.5 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 2 , 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000 , 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9 , 5x10 9 , 6x10 9 , 7x10 9 , 8x109 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , 9x10 10 , 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , 9x10 11 , 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 or 9x10 12 cells.

[0235] In some embodiments, the plurality of cells comprises a plurality of immune cells. In some embodiments, the plurality of immune cells may comprise at least 2, 3, 4, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 2 , 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000 , 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7 、3x10 7 、4x10 7 、5x10 7 、6x10 7 、7x10 7 、8x10 7 、9x10 7 、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、8x10 11 、9x10 11 、1x10 12 、2x10 12 、3x10 12 、4x10 12 、5x10 12 、6x10 12 、7x1012 , 8x10 12 or 9x10 12 cells.

[0236] In some embodiments, the plurality of cells comprises a plurality of diseased cells. In some embodiments, the plurality of diseased cells may comprise at least 2, 3, 4, 5, 5.5, 6, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 2 , 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000 , 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x10 6 , 3x10 6 , 4x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 , 1x10 7 , 2x10 7 , 3x10 7 , 4x10 7 , 5x10 7 , 6x10 7 , 7x10 7 , 8x10 7 , 9x10 7 , 1x10 8 , 2x10 8 , 3x10 8 , 4x10 8 , 5x10 8 , 6x10 8 , 7x10 8 , 8x10 8 , 9x10 8 , 1x10 9 , 2x10 9 , 3x10 9 , 4x10 9, 5x10 9 , 6x10 9 , 7x10 9 , 8x10 9 , 9x10 9 , 1x10 10 , 2x10 10 , 3x10 10 , 4x10 10 , 5x10 10 , 6x10 10 , 7x10 10 , 8x10 10 , 9x10 10 , 1x10 11 , 2x10 11 , 3x10 11 , 4x10 11 , 5x10 11 , 6x10 11 , 7x10 11 , 8x10 11 , 9x10 11 , 1x10 12 , 2x10 12 , 3x10 12 , 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 or 9x10 12 cells.

[0237] In some embodiments, the plurality of cells comprises a plurality of cancer cells. In some embodiments, the plurality of cancer cells may comprise at least 2, 3, 4, 5, 5.5 6, 6.5 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 15,000, 2 , 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000, 60,000, 70,000, 80,000, 90,000 , 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1x10 6 , 2x106 、3x10 6 、4x10 6 、5x10 6 、6x10 6 、7x10 6 、8x10 6 、9x10 6 、1x10 7 、2x10 7 、3x10 7 、4x10 7 、5x10 7 、6x10 7 、7x10 7 、8x10 7 、9x10 7 、1x10 8 、2x10 8 、3x10 8 、4x10 8 、5x10 8 、6x10 8 、7x10 8 、8x10 8 、9x10 8 、1x10 9 、2x10 9 、3x10 9 、4x10 9 、5x10 9 、6x10 9 、7x10 9 、8x10 9 、9x10 9 、1x10 10 、2x10 10 、3x10 10 、4x10 10 、5x10 10 、6x10 10 、7x10 10 、8x10 10 、9x10 10 、1x10 11 、2x10 11 、3x10 11 、4x10 11 、5x10 11 、6x10 11 、7x10 11 、8x10 11 、9x10 11 、1x10 12 、2x10 12 、3x10 12, 4x10 12 , 5x10 12 , 6x10 12 , 7x10 12 , 8x10 12 or 9x10 12 cells.

[0238] The enzymes described herein can be prepared in a host cell, or in vitro, in a cell-free synthesis system. The host cell can be any host cell that can be robustly recoded. These host cells can be bacterial cells with a well-developed genetic system, of which Escherichia coli is an example. Other bacterial species can also be used. The cell-free system for producing proteins can be a coupled transcription / translation system or a translation-only system. A significant aspect of the method of the present invention is the use of biosynthesis rather than chemical synthesis methods.

[0239] The recoded cells with the constructed nucleic acid sequence can be cultured by any method known in the art. The culture can be carried out in batches or continuously in a shaker flask or in a fermenter or fixed on a solid surface (e.g., small particles contained in a larger container). Typically, the culture medium is supplemented with a selenium source, such as Na2SeO3. As known in the art, the production of the desired protein variant can be carried out under the control of an inducer or a repressor. For construction and protein production, any such system known in the art can be selected.

[0240] The above disclosure generally describes the present invention. All documents disclosed herein are expressly incorporated herein by reference. A more comprehensive understanding can be achieved by reference to the following specific examples, which are provided herein only for illustrative purposes and are not intended to limit the scope of the present invention.

[0241] Example

[0242] Example 1 - Expression and purification

[0243] RTΔA 2X310K cells transformed with plasmid pUC-TetO-GST-DNase_Sec-HisAltStrep carrying the gene encoding DNase I (UAG codon directs selenocysteine ​​incorporation replacing all cysteine ​​codons) were diluted 1 / 250 in 1 L terrific broth supplemented with 1000 μg / mL carbenicillin, 12.5 μg / mL tetracycline and 25 μM Na2SeO3 and induced with (1 mM) 3,4-dihydroxybenzoic acid during mid-log phase and subsequently grown O / N at 37°C. Cells were collected and resuspended in 25 mL washing buffer (50 mM K HPO , 300 mM NaCl, 20 mM imidazole and 10% glycerol, pH 8.0) with 8000 x g centrifugation for 10 min, containing a protease inhibitor cocktail (cOmplete, mini EDTA-free, Roche) and lysozyme (0.5 mg / mL). After incubation for 20 min at 4 ° C with gentle agitation, cells were dissolved by ultrasonic treatment (500 type, Fisher Scientific), and clarified three times by centrifugation for 30 min at 35000 x g. Lysate was filtered through a 0.2 μm membrane and protein was recovered by the IMAC using Ni-NTA resin and gravity flow column. Eluate was concentrated to 3 mL and dialyzed with TBS of pH 7.5, then purified to significantly uniform by size exclusion FPLC. During dialysis, a significant proportion of the sample may precipitate, while another proportion of the sample may be present in the less stable / soluble dimer form.The final yield of soluble enzyme was determined.

[0244] The RTΔA 2X310K-T7 cells transformed by plasmid pUC-TetO-GST-DNase_Sec-HisAltStrep carrying the gene encoding DNase I (UAG codon guides selenocysteine ​​to be incorporated to replace all cysteine ​​codons) were diluted 1 / 250 in 1L high concentration broth, which was supplemented with 1000 μg / mL carbenicillin, 33 μg / mL chloramphenicol and 25 μM Na2SeO3, and induced with 200 ng / mL anhydrotetracycline during the middle logarithmic phase, then grown O / N at 30 ℃. Cells were collected and purified by IMAC as previously described. In order to reduce precipitation, the sample was dialyzed with a larger volume (>6 mL). Protein samples were purified to obvious homogeneity by size exclusion FPLC or anion exchange chromatography (HiTrap Q HP column). The final yield of soluble enzymes was determined.

[0245] Example 2 - Mass Spectrometry

[0246] Complete protein samples are analyzed by utilizing the aforementioned method. Utilizing a cutoff filter with a molecular weight of 10kDa, selenoprotein samples are buffer exchanged into LC-MS grade water. Once buffer exchange is complete, samples will be diluted to 20 μM in methanol / water / formic acid (50 / 49 / 1) solution. After dilution, the protein solution is injected into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Instruments, Bremen, Germany) at a rate of 3 μL / min via electrospray ionization. In order to confirm the incorporation of selenocysteine, complete mass analysis is performed at 240k resolution and an average of 20 scans. The characterization of protein sequences is performed by ultraviolet light dissociation (UVPD) using a 193nm excimer laser (Coherent, Inc.), which is connected to the aforementioned Orbitrap mass spectrometer. For each UVPD spectrum, two 2.5mJ laser pulses are used, and 250 scans are averaged. MS1 spectra were deconvoluted using the Xtract deconvolution algorithm (Thermo Fisher Scientific). UVPD mass spectra were deconvoluted using Xtract and then analyzed using ProsightPC 3.0. Proteins containing selenocysteine ​​were searched by adding a modification of 61.9146 Da to the serine residues at the incorporation site (including the subtraction of one hydrogen atom due to the formation of a diselenide bond). The incorporation efficiency was calculated by dividing the area of ​​the modified protein peak by the total area of ​​the unmodified protein peak and the modified protein peak. The peak area for each protein is the sum of the integrated areas of the five most abundant peaks from each isotope cluster.

[0247] Example 3 - UAG genome recoded organisms suitable for expressing polypeptides with non-standard amino acids

[0248] The genome recoded organism (GRO) in which the UAG codon translation function is completely eliminated will be used to clearly incorporate non-standard amino acids (NSAA) at UAG. The genome recoded organism can include one or more triplet codons of redistribution to promote the incorporation of non-standard amino acids (NSAA) such as selenocysteine. By utilizing methods known to those skilled in the art, triplet codons can be redistributed to incorporate non-standard amino acids such as selenocysteine. Alternatively, by utilizing methods known to those skilled in the art, quadruple codons can be used to incorporate non-standard amino acids. Orthogonal aminoacyl-tRNA synthetase (aaRS) / tRNA pairs have been developed, which are based on non-functional UAG triplet, specifically and effectively decode quadruple UAGA codons, so that non-standard amino acids, such as selenocysteine, are clearly incorporated into UAGA codons, and high protein yields are obtained. This quadruple codon can be used in the present method.

[0249] Example 4 - Selection of tRNA

[0250] A gene selection method was developed that was able to distinguish between different levels of selenocysteine ​​incorporation. In order to make the reporter protein specifically 'dependent' on selenocysteine ​​rather than serine, the NMC-Af3-lactamase from Enterobacter cloacae was used. This enzyme has a high sequence similarity to the SME-1f3-lactamase of Serratia marcescens, which has previously been shown to require a disulfide bond adjacent to the active site serine residue for activity, but this imposes a significant fitness cost on E. coli. First, a C69S mutant was constructed from NMC-A that was unable to confer resistance to ampicillin (MIC<50μg / mL), indicating that the disulfide bond is essential for activity. Then, cysteine ​​69 was replaced with an amber stop codon (X69) for library selection, assuming that the incorporation of selenocysteine ​​and the formation of a selenyl-thiol bond might restore activity.

[0251] In order to eliminate any interference between the tRNASec library and the endogenous selenocysteine ​​incorporation mechanism, selA, selB and selC genes (encoding SelA, SelB and tRNASec, respectively) were deleted from Escherichia coli DH10B (formulated as DHΔabc). Cells containing reporter plasmid pNMC-A C69X and auxiliary plasmid pRSF-eSelA (expressing SelA) were transformed with plasmid pMB1-ZU containing the tRNASec antideterminant library. The transformant was inoculated (plate) on a culture medium containing an ampicillin concentration gradient so that mutants capable of selenocysteine-specific inhibition were selected. The single colonies produced covered a range of ampicillin concentrations. About 12 colonies from each plate were sequenced, and three different tRNASec mutants were found: G7-C66:U49-G65:C50-U64 (GGAAGAT G 7 GTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGGGGCCGCC

[0252] AGCGGTCCCGG T 49 C 50 AGGTTCGACTCCT T 64 G 65 C 66 ATCTTCCGCCA(SEQ ID NO:15)), C7-G66:U49-G65:C50-U64(GGAAGATC 7 GTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGGGGCCGCCAGCGGTCCCGG T 49 C 50 AGGTTCGACTCCTT 64 G 65 G 66 ATCTTCCGCCA (SEQ ID NO:16)) and C7-U66:U49-A65:A50-Δ64 (GGAAGAT C 7GTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGGGGCCGCC AGCGGTCCCGG T 49 A 50 AGGTTCGACTCCT A 65 T 66 ATCTTCCGCCA (SEQ ID NO: 17)) (where underlined bases indicate changes from the parental antideterminant sequence). Among these tRNASec variants, only G7-C66:U49-G65:C50-U64 was detected at the two highest ampicillin concentrations (200 and 250 μg / mL).

[0253] The tRNASec variant containing the G7-C66:U49-G65:C50-U64 anti-determinant sequence is denoted as tRNASecUx and is compared with a previously designed chimera (tRNAUTu) and a tRNASec derivative designed to have an anti-determinant region that tightly binds to EF-Tu (tRNAUG). The parent tRNASec containing the CUA anticodon and tRNAUG cannot produce active β-lactamase. The hybrid tRNAUTu incorporates selenocysteine ​​and can grow at 75 μg / mL. In contrast, the expression of tRNASecUx resulted in significantly higher β-lactamase activity (up to 400 μg / mL), but only when co-expressed with SelA, it was confirmed that the activity was selenocysteine-dependent. In order to further confirm the reaction of tRNASecUx incorporating selenocysteine ​​to the amber stop codon, a standard colorimetric assay was used based on the activity of the endogenous Escherichia coli selenoprotein formate dehydrogenase H (FdhH). FdhH is expressed under anaerobic conditions and catalyzes the oxidation of formate to produce CO2, accompanied by the reduction of the electron acceptor benzyl viologen, resulting in the formation of a dark purple color. Formate oxidation by FdhH is strictly dependent on the selenocysteine ​​residue at position 140; the mutant FdhH U140S is completely inactive. Only tRNASecUx and tRNAUTu, when co-expressed with SelA, produce active FdhH.

[0254] The selected tRNA contains non-standard sequences at the junction that usually interacts with EF-Tu. In view of the fact that neither the base of the receiving stem nor the adjacent T-arm base pair is considered to play a role in the interaction between tRNASec and SelA, the results show that the selected U:C makes a stronger combination with EF-Tu compared with the wild-type tRNASec sequence. Based on the model developed for the standard (canonical) tRNA, it is not predicted that the unusual (unusual) C50-U64 base pair is strongly bound to EF-Tu, and the expression of the hybrid tRNAUG containing the strong EF-Tu binding region from the main E. coli tRNAGly does not lead to the production of active beta-lactamase, indicating that the non-standard sequence is functionally important. Therefore, it is possible that the combination of the part of the engineered tRNASec with EF-Tu may be different from that of the standard tRNA, which is not necessarily surprising in view of the fact that tRNASec usually interacts with SelB.

[0255] The development of engineered E. coli strains lacking the prfA gene encoding release factor 1 (RF1) has allowed for the efficient incorporation of a range of unnatural amino acids, and the development of the genome-engineered amber-free E. coli C321.ΔA provides an opportunity to determine whether proteins that efficiently incorporate selenocysteine ​​can be expressed. The selA, selB, and selC genes were deleted in C321.ΔA (denoted as strain RTΔA), and the cells were transformed with amber-containing NMC-A reporter plasmids and helper plasmids. Compared to prfA+DHΔabc cells that still contain RF1, β-lactamase activity was significantly increased in RF1-deficient cells. In addition, in the context of RF1-deficiency, tRNASecUx can support the formation of functional diselenide bonds (via amber-mediated incorporation of two selenocysteine ​​residues U69 and U238).

[0256] In order to further improve the incorporation efficiency of selenocysteine, a number of measures have been taken to improve the level of Sec-tRNASec relative to Ser-tRNASec, including: increasing the level of SelA, reducing the gene dosage of tRNASecUx, and co-expressing phosphoseryl-tRNASec kinase. In order to monitor the efficiency of selenocysteine ​​incorporation and prove the feasibility of protein engineering, Escherichia coli dihydrofolate reductase (DHFR) containing an engineered non-essential seleno-sulfhydryl bond was prepared. The top-down mass spectrometry showed nearly 100% selenocysteine ​​incorporation without detectable background corresponding to DHFR containing serine. It was also observed that the rationally designed tRNAUTu chimera incorporated selenocysteine ​​into DHFR containing P39X substitution, but resulted in much lower levels of selenocysteine ​​incorporation (38%) and significant serine incorporation (62%). No mass corresponding to the incorporation of other standard amino acids was observed in the mass spectrum. To further validate selenocysteine ​​incorporation, the Pseudomonas aeruginosa metalloprotein azurin, whose essential cysteine ​​(C112) was replaced by selenocysteine ​​and the human selenoprotein cellular glutathione peroxidase (GPx-1) were also expressed. For azurin, this chemical change had previously been shown to be possible only by expressed protein ligation, and the essential cysteine ​​could be biologically replaced with selenocysteine ​​with good efficiency, as determined by mass spectrometry of the intact protein.

[0257] Example 5 - Methods

[0258] Strain construction

[0259] The selAB and selC genes were deleted from E. coli DH10B using the λ-Red system adapted from Datsenko and Wanner (2000). The antibiotic resistance cassette was excised using FLP recombinase, generating strain DHAabc. Deletion of the entire fdhF open reading frame generated strain DHAabcF.

[0260] E. coli C321.ΔA was obtained from Addgene. A 12 kb genomic region containing λ-phage genes and the TEM-1 β-lactamase inserted during strain development was removed to promote stable growth at 37°C and restore sensitivity to β-lactam antibiotics. Subsequent deletion of the selAB and selC genes and excision of the antibiotic resistance cassette produced strain RTΔA. To increase recombinant protein production, the lon gene encoding the Lon protease was deleted, and the rne gene was truncated to remove 477 amino acids from the C-terminus of RNase E, resulting in RTΔA.2.

[0261] Reporter plasmid

[0262] All reporter plasmids were derived from pcat-pheS. A 3281 bp fragment of pcat-pheS containing the 15A replication origin and the tetA gene conferring tetracycline resistance was ligated to a 1158 bp synthetic DNA fragment containing the blaSME-1 gene from Serratia marcescens encoding the SME-1 3-lactamase flanked by endogenous promoter and terminator sequences. This plasmid (pSME-1) was observed to be highly toxic to E. coli host cells and cultured poorly. The blaSME-1 open reading frame was replaced with blaNMC-A from Enterobacter cloacae encoding the NMC-A β-lactamase, which has nearly 70% sequence identity with the SME-1 generated plasmid pNMC-A, which did not show any toxicity. pNMC-A variants with serine or amber codons at residues 69 and 238 were generated by QuikChange site-directed mutagenesis.

[0263] p15A-fdhF was constructed by ligating the fragment obtained from pcat-pheS with a 2886 bp fragment amplified from E. coli DH10B genomic DNA containing the fdhF gene, endogenous promoter and terminator sequences, and an upstream formate response element. U140S and U140TAG variants were generated by QuikChange site-directed mutagenesis.

[0264] Helper plasmid

[0265] The RSF1030 origin of replication and the kan cassette were amplified by PCR as a 1563 bp fragment from pRSFDuet-1 (Novagen). A 1562 bp fragment containing the E. coli selA gene and the 5′ region covering the endogenous promoter was amplified from E. coli DH10B genomic DNA. The two fragments were assembled to produce the plasmid pRSF-SelA. The plasmid pRSF-eSelA was obtained by replacing the endogenous weakly active promoter with a strong constitutively active EM7 promoter and a standard Shine-Dalgarno sequence. The SelA expression plasmid was verified by complementing the E. coli DH10B (DHΔa) lacking selA by benzyl viologen assay. Compared to pRSF-SelA, pRSF-eSelA induced a strong color change, and this variant was used for all further experiments.

[0266] pRSF-U-eSelA was constructed by adding NotI and NcoI restriction sites between the RSF1030 origin and the selA promoter and subcloning the NotI / NcoI fragment containing the selC gene from pMB1-ZU. The pRSF-U-eSelA variant containing the mutant tRNASec gene was constructed by enzymatic inverse PCR. The plasmid pRSF-U-ΔSelA containing the truncated selA gene was generated by QuikChange site-directed mutagenesis, introducing TGA and TAA stop codons at positions 167 and 168, respectively.

[0267] Variant tRNA sequences

[0268] tRNASecCUA-GGAAGATCGTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGG

[0269] GGCCGCCAGCGGTCCCGGGCAGGTTCGACTCCTGTGATTCTTCCGCCA (SEQ ID NO: 17).

[0270] tRNASecUx-GGAAGATGGTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGGGGCCGCCAGCGGTCCCGGTCAGGTTCGACTCCTTGCATCTTCCGCCA (SEQ ID NO: 18).

[0271] tRNASecUG-GGAAGATGGTCGTCTCCGGTGAGGCGGCTGGACTCTAAATCCAGTTGGGGCCGCCAGCGGTCCCGGCGAGGTTCGACTCCTCGTATCTTCCGCCA (SEQ ID NO: 19).

[0272] tRNAUTu–GGAAGATGTGGCCGAGCGGTTGAAGGCACCGGTCTCTAAAACCGGCGACCCGAAAGGGTTCCAGAGTTCGAATTCCTGCATCTTCCGCCA (SEQ ID NO: 20).

[0273] The plasmid pRSF-eSelAK for constitutive expression of SelA and PSTK was constructed by inserting a synthetic DNA fragment between the selA gene and the kan cassette, adding the luxI terminator 3′ of selA and the Methanococcus jannaschii pstK gene, which encodes O-phosphoseryl-tRNASec kinase (PSTK) codon optimized for expression in Escherichia coli and is flanked by the EM7 promoter and luxI terminator.

[0274] Expression plasmid

[0275] Some expression plasmids are from pRST.11. For pDHFR-P39X-AU, the wrs1 gene is replaced by an operon controlled by a constitutive EM7 promoter, which contains the E. coli folA gene (amplified from DH10B genomic DNA) encoding dihydrofolate reductase, wherein the C-terminal Strep II tag is connected by a serine / alanine linker, and the selA gene is separated by the sequence TAGGAGGCAGATC (SEQ ID NO:21), thereby providing a standard Shine-Dalgarno sequence. Sc-tRNATrpAmb is replaced by tRNASecUx and tRNAUTu to express tRNASec variants from a strong leuP promoter. TAG and AGC codons are introduced at position 39 by QuikChange site-directed mutagenesis. pAz-C112X-AU was constructed similarly by replacing the folA gene with a synthetic DNA fragment containing the azu gene from Pseudomonas aeruginosa encoding azurin codons, optimized for expression in E. coli, with a C-terminal His6-tag. TAG and AGC codons were introduced at position 112 by QuikChange site-directed mutagenesis. pGPx-U49-AU was constructed by replacing the folA gene with a synthetic DNA fragment containing the human gpx1 gene encoding cellular glutathione peroxidase (GPx-1) codons, optimized for expression in E. coli, with an N-terminal His6-tag.

[0276] Library construction and selection

[0277] The 1518bp fragment comprising the MB1 origin of replication and the rop gene was amplified from pETDuet-1 (Novagen). The synthetic DNA fragment comprising the ble gene of codon optimization and the MCS assembly comprising NotI and NcoI sites were assembled to produce plasmid pMB1-Z, which ble gene from Streptoalloteichus hindustan imparts Zeocin resistance, and the side edge has an EM7 promoter and an endogenous terminator sequence. The 410bp fragment comprising the selC gene and its promoter was amplified from E. coli DH10B genomic DNA, which has NotI and NcoI sites, and is connected to pMB1-Z to build pMB1-ZU. The functionality of the selC gene was verified by the E. coli DH10B (DHAc) supplementary deletion selC.

[0278] The tRNASec anti-determinant library is produced by the reverse PCR of enzyme using oligonucleotide primers, so that the six positions of the main anti-determinant combined with EF-Tu are randomized. After self-connection for 16 hours, DNA is ethanol precipitated with GlycoBlue (Ambion), and transformed into the Escherichia coli DHAabc comprising plasmid pNMC-A C69X and plasmid pRSF-eSelA by electroporation. Transformant is diluted in 200ml LB substratum, and incubated overnight, the substratum comprises 12.5 μ g / mL bleomycin, 6.25 μ g / mL tetracycline and 25 μ g / mL kanamycin. After overnight growth, cell is diluted 1 / 50 in LB substratum, and incubated for 1 hour, the substratum comprises 6.25 μ g / mL bleomycin, 3.75 μ g / mL tetracycline, 12.5 μ g / mL kanamycin, 1 μM Na2SeO3 and 20 μ g / mL L-cysteine. A series of 250 μ l aliquots of cells were inoculated on LB agar, which contained 6.25 μ g / mL bleomycin, 3.75 μ g / mL tetracycline, 12.5 μ g / mL kanamycin, 1 μ M Na SeO and 20 μ g / mL L-cysteine ​​and 50-300 μ g / mL ampicillin (50 μ g / mL increment). After 20 hours at 37°C, each bacterium colony was observed on a flat board containing 50-200 μ g / mL ampicillin. Plasmid DNA was separated from the colony selections of all plates, and tRNASec mutations were determined by Sanger sequencing.

[0279] Primer sequences - Oligonucleotide primers used for library construction.

[0280] selClibfwd–TGGACTGGTCTCCCAGTTGGGGCCGCCAGCGGTCCCGGNNAGGTTCGACTCCTNNNATCTTCCGCCAAAATGC (SEQ ID NO: 22).

[0281] selClibrev-GCTGGCGGTCTCaACTGGATTTAGAGTCCAGCCGCCTCACCGGAGACGACNATCTTCCGCGCCTCG (SEQ ID NO: 23).

[0282] Re-phenotype

[0283] The NotI / NcoI fragment containing tRNASecUx is subcloned into pRSF-eSelA to produce pRSF-UX-eSelA. The pRSF-U-eSelA variant is transformed into the E. coli DHAabc containing the reporter plasmid pNMC-A C69TAG. The DHAabc cells containing pNMC-A and pRSFDuet-1 are used as positive controls. The DHAabc cells containing pNMC-A C69S and pRSF-UX-eSelA and pNMC-A C69TAG and pRSF-UX-ASelA are used as controls for selenocysteine-dependent fl-lactamase activity. The transformant is cultured overnight in LB medium, which contains 6.25pg / mL tetracycline, 25pg / mL kanamycin, 1pM Na2SeO3 and 20pg / mL L-cysteine. After overnight culture, cell is diluted 1 / 10 in the LB substratum comprising antibiotic, selenite and L-cysteine, and hatched 3 hours.Culture is normalized to OD600=0.1, and 5pl aliquots are inoculated in triplicate on LB agar, and this agar comprises 3.75pg / mL tetracycline, 12.5pg / mL kanamycin, 1pM Na2SeO3, 20pg / mL L-cysteine ​​and ampicillin cross the gradient of 0,10,25,50,75,100,150,200,250,300,350,400,450 and 500pg / mL.Plate is incubated overnight at 37 ℃.Use identical assay conditions to repeat this experiment with Escherichia coli RTΔA.

[0284] Benzyl Viologen Analysis

[0285] The E. coli DH10B cells containing pRSFDuet-1 and pcat-pheS were used as positive controls. The DHAabcF cells containing p15A-fdhF U140S and pRSF-UX-eSelA and p15A-fdhF U140X and pRSF-UX-ASelA were used as controls for selenocysteine-dependent formate dehydrogenase activity. The transformants were cultured overnight at 37°C in LB medium supplemented with 12.5pg / mL tetracycline and 50pg / mL kanamycin. Overnight cultures were diluted 1 / 20 in a final volume of 2ml and incubated for 3 hours. Culture is normalized to OD600=0.5, and 5pl aliquots are dotted on LB agar plates, which contain 3.75pg / mL tetracycline, 12.5pg / mL kanamycin, 5mM sodium formate, 10pMNa2MoO4, 1pM Na2SeO3 and 20pg / mL L-cysteine. Flat plates are incubated for 3h under aerobic conditions at 37°C, then transferred to anaerobic conditions and continued for 60h at 37°C. Once removed from the anaerobic chamber, agar is immediately covered on the plate, which contains 1mg / mL benzyl viologen, 250mM sodium formate and 25mM KH2PO4 pH 7.0. Plates are photographed within 1 hour of covering.

[0286] Modification and protein purification

[0287] Initial attempts to produce selenoproteins in E. coli strain RTΔA.2 used a combination of a helper plasmid from pRSF-UX-eSelA and an expression plasmid in which the endogenous selC promoter was replaced with the highly active E. coli leuP promoter, containing the azu gene downstream of the strong tacI promoter. Mass spectrometry analysis of early selenoprotein samples revealed that serine was incorporated almost exclusively at amber codons, and a number of optimizations were made to increase the ratio of Sec-tRNASec to Ser-tRNASec, which is thought to be the main driver of incorporation efficiency. To increase the ratio of SelA to tRNASec, expression of tRNASec variants was reduced by moving the leuP cassette to a lower copy expression plasmid containing the MB1 replication origin and adding a second selA gene downstream of the target selenoprotein. In addition, to prevent rapid depletion of the Sec-tRNASec pool after induction, the tacI promoter driving selenoprotein expression was replaced with a constitutive EM7 promoter. These changes generated the expression plasmids pDHFR-P39X-AU and pAz-C112X-AU.

[0288] To further reduce the pool of Ser-tRNASec available for participation in standard translation, the pstK gene encoding O-phosphoseryl-tRNASec kinase was added to the helper plasmid pRSF-eSelA to generate pRSF-eSelAK. It has been previously reported that PSTK increases the incorporation of selenocysteine ​​into tRNAUTu by producing Sep-tRNASec, which is an efficient substrate for SelA but poorly recognized by E. coli EF-Tu. At the same time, the selenium concentration in the culture medium was increased and L-cysteine ​​was omitted for selenoprotein preparation.

[0289] RTΔA.2 transformants containing pDHFR-P39X-AU and pRSF-eSelAK were cultured O / N in LB medium containing 100 μg / mL ampicillin, 50 μg / mL kanamycin and 1 μM Na2SeO3. Overnight cultures were diluted 1 / 500 in a final volume of 2 L of LB medium and incubated with stirring at 37°C for 24 hours. The medium contained 50 μg / mL ampicillin, 25 μg / mL kanamycin and 5 μM Na2SeO3. Cells were collected by centrifugation at 8000 x g for 10 min and resuspended in 20 mL of wash buffer (100 mM Tris, 150 mM NaCl, 1 mM EDTA pH 8.0) containing a protease inhibitor cocktail (cOmplete, Mini EDTA-free, Roche solution) and lysozyme 1 mg / mL. After incubation at 4 ℃ for 20min, cells were dissolved by ultrasonic treatment (500 type, Fisher Scientific), and clarified three times by centrifugation for 30min at 35000x g. Lysate was passed through a 0.2 μm filter, and selenium-DHFR was recovered according to the manufacturer's instructions (GE Healthcare) using Strep-Tactin affinity chromatography. Eluate was concentrated to 3mL and dialyzed with 50mMNH4Ac pH 6.5, followed by size exclusion FPLC ( Selenium-DHFR was isolated by using tRNASecUx with a yield of 68 μg / L and 100% incorporation efficiency. Selenium-DHFR was produced by using tRNAUTu with a yield of 131 μg / L and 38.1% incorporation efficiency. DHFR containing serine at position 39 was produced at a yield of 225 μg / L.

[0290] As previously described, RTΔA.2 transformants containing pAz-C112X-AU and pRSF-eSelAK were cultured, except that 20 μM Na2SeO3 was added for 24 hours of incubation. Cells were collected by centrifugation and the periplasmic components were separated. Briefly, the cell pellets were resuspended in 50 mL of 100 mM Tris and 0.75 M sucrose at pH 7.5. After lysozyme was added to 1 mg / mL protease inhibitor cocktail, the cells were gently shaken at 4°C for 20 min. 50 mL of 1 mM EDTA was added, and the samples were incubated for another 20 minutes. During a further incubation of 20 min, EDTA was neutralized by adding 3.5 mL 0.5 M MgCl2. By centrifugation for 30min under 35000x g and spheroblast is removed, periplasmic component is passed through 0.2 μm filter, and mixed with imidazole stock solution and obtain the ultimate concentration of 20mM.Selenoazurin is reclaimed by IMAC, Ni-NTA resin and gravity flow column.The eluate is concentrated and dialyzed with 50mM NH4Ac pH 6.5, and then separated by size exclusion FPLC.Selenoazurin is produced by utilizing tRNASecUx with the productive rate of 50 μ g / L and the incorporation efficiency greater than 76%.This value may be lower than the actual level of selenocysteine ​​incorporation, because during purification and afterwards, selenoazurin is observed to form the complex of higher molecular weight, resulting in the loss in size exclusion chromatography process.For the azurin sample comprising only serine, no aggregation is observed.

[0291] RTΔA.2 transformants containing pGPx-U49-AU and pRSF-eSelAK were cultured as described above for azurin. Cells were collected by centrifugation and resuspended in 50 mL of buffer (50 mM potassium phosphate, 150 mM NaCl, 10% glycerol, 1 mM DTT, pH 8.0) and 1 mg / mL lysozyme. Cells were solubilized and clarified by ultrasonic treatment, and GPx-1 was recovered by IMAC. The eluate was concentrated and dialyzed with 100 mM phosphate buffer pH 8.0, 0.1% Tween 20 and 1 mM DTT, and GPx-1 was separated by anion exchange chromatography (Q HP column). GPx-1 was produced at a yield of 500 μg / L and close to 100% selenocysteine ​​incorporation efficiency.

[0292] Mass spectrometry

[0293] By utilizing the method described above, intact protein samples are analyzed. By utilizing a 10kDa molecular weight cut-off filter, azurin, DHFR and GPx-1 sample buffers are exchanged into LC-MS grade water. Once buffer exchange is complete, the sample is diluted to 20 μM in methanol / water / formic acid (50 / 49 / 1) solution. After dilution, the protein solution is injected into an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via electrospray ionization at a rate of 3 μL / min. In order to confirm the incorporation of selenocysteine, complete mass analysis is carried out at 240k resolution and an average of 20 scans. The characterization of the protein sequence is carried out by using the ultraviolet light dissociation (UVPD) of a 193nm excimer laser (Coherent, Inc.), which is connected to the aforementioned Orbitrap mass spectrometer. For each UVPD spectrum, two 2.5mJ laser pulses are used, and 250 scans are averaged. MS1 spectrum is deconvoluted by utilizing Xtract deconvolution algorithm (Thermo Fisher).UVPD mass spectrum is deconvoluted by utilizing Xtract, and then ProsightPC3.0 is utilized to analyze.For azurin, by adding the modification of 62.9216Da to the serine at position 112, or for DHFR, adding the modification of 61.9146Da to the serine at position 39 (because a selenium-sulfhydryl bond is formed when selenocysteine ​​exists, so a hydrogen atom is subtracted from the DHFR modification), the protein comprising selenocysteine ​​is searched.Incorporation efficiency is calculated by the area of ​​the protein peak modified, divided by the total area of ​​the protein peak of unmodified protein mass peak and modification.The peak area of ​​every kind of protein is the sum of the integral areas of the five peaks with the maximum abundance from each isotope cluster.

[0294] Example 6 - DNase I-Activity

[0295] Fluorescently labeled probes as part of the DNase I activity assay were prepared by amplification. Amplification was performed in a reaction mixture containing plasmid DNA, DNA polymerase, amplification buffer, each type of deoxynucleoside triphosphate (dNTP) and primers. The amplification was performed under the following conditions: initial denaturation at 94°C for 5 minutes, 35 cycles of 1 minute at 94°C, 1 minute at 55°C, and 1 minute at 72°C, with a final extension step of 10 minutes at 72°C. The resulting PCR product was purified by utilizing GeneJET PCR purification kit (Thermo Fisher Scientific, Waltham, MA).

[0296] DNase I activity was determined by incubating the sample with fluorescently labeled PCR fragments and then detecting the fluorescently labeled products by capillary electrophoresis on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems Corporation, Carlsbad, CA). The assay was performed in a reaction mixture containing the PCR product and the sample diluted 10× in saline. The negative control contained only the labeled fragments without a source of DNase I activity. The reaction mixture was incubated at room temperature for 2 minutes, after which the reaction was terminated by incubation at 75°C for 10 minutes. The reactants were purified using the GeneJET PCR purification kit (Thermo Fisher). Fragment analysis was performed on each sample. Capillary electrophoresis was performed using the default genotyping module of the G5 dye set using POP-7 polymer (Applied Biosystems). The results were analyzed using GeneMapper software version 4.0 (Applied Biosystems). The DNase activity of the sample was expressed as the percentage difference in signal intensity compared to a control material containing no DNase, which was assigned a value of 1 (100%).

[0297] Example 7 - DNase I-Activity

[0298] 30 ng of seleno-DNase (GST–seleno-DNase I fusion) and an equal mole of disulfide DNase (wild-type DNase I purified from bovine pancreas, Sigma DN25) were incubated at 37°C for 2 hours in reaction buffer (NEB B0303S) supplemented with 0 mM DTT (-DTT) or 50 mM DTT (+DTT). 50 ng of double-stranded DNA was added to the mixture and digested at 37°C for 30 min, followed by heat inactivation at 75°C for 10 min. Both enzymes digested the DNA in 0 mM DTT, but only seleno-DNase digested the DNA in 50 mM DTT ( Figure 1 ).

[0299] Although preferred embodiments of the present invention have been shown and described herein, it is apparent to those skilled in the art that such embodiments are provided only as examples. Without departing from the present invention, those skilled in the art will appreciate that many variations, changes and alternatives exist. It should be understood that various alternatives to the embodiments of the present invention described herein may be used to implement the present invention. The following claims are intended to define the scope of the present invention and thus cover methods and structures within the scope of these claims and equivalents.

Claims

1. A composition comprising a deoxyribonuclease I (DNase I) polypeptide having 100% sequence identity with the polypeptide of SEQ ID NO: 1, wherein the DNase I polypeptide comprises a selenocysteine ​​at the following positions: (a) corresponds to position 102 of SEQ ID NO: 1; (b) a position corresponding to position 105 of SEQ ID NO: 1; (c) a position corresponding to position 174 of SEQ ID NO: 1; and (d) The position corresponding to position 210 of SEQ ID NO:

1.

2. The composition according to claim 1, wherein at least one pair of selenocysteine ​​is linked by a diselenide bond.

3. The composition of claim 2, wherein the selenocysteine ​​pair is located at positions 102 and 105 of the polypeptide corresponding to SEQ ID NO:

1.

4. The composition of claim 2, wherein the selenocysteine ​​pair is located at positions 174 and 210 of the polypeptide corresponding to SEQ ID NO:

1.

5. compositions according to claim 4, wherein said DNaseI polypeptide comprises two pairs of selenocysteine ​​linked by diselenide bond.

6. compositions according to claim 5, wherein said DNaseI polypeptide comprises a first pair of selenocysteine ​​positioned at positions 102 and 105 of the polypeptide corresponding to SEQ ID NO:1 and a second pair of selenocysteine ​​positioned at positions 174 and 210 of the polypeptide corresponding to SEQ ID NO:

1.

7. A bacterial cell comprising the composition of any one of claims 1-6.

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