Methods and systems for preparing mRNA

Abstract

Methods and systems for "one-pot" preparation of mRNA are provided, which do not include transfers, centrifugations, or purification between steps. High quality mRNA is produced in relatively short time, using any chemistry or capping reagent.

Description

[0001] Attorney Docket No. TERR-001 / 01WO; 40647 / 5 METHODS AND SYSTEMS FOR PREPARING MRNA RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63 / 690,671, U.S. Provisional Application No.63 / 690,683, and U.S. Provisional Application No.63 / 690,689, each of which was filed September 4, 2024 and is incorporated by reference herein in its entirety. BACKGROUND Messenger RNA (mRNA) is a single-stranded molecule of RNA made from a DNA template during the process of transcription. mRNA plays a critical role in gene expression, and is increasingly important for a number of biopharma applications. mRNA is used for research and development (R&D) work in many areas of bioscience. However, existing processes for producing mRNA are not efficient. Improved methods and systems for preparing mRNA are needed in the art. SUMMARY The present invention provides, in various embodiments, methods and systems for “one- pot” preparation of mRNA, with no transfers, centrifugations, or purification between steps. Features of embodiments of the present invention include, for example: one day turnaround from plasmid DNA; full automation for reproducible results; throughput of hundreds of mRNAs per week; support for any chemistry or capping reagent; high quality mRNA (e.g., integrity up to 99%, >99% capping, dsDNA below limit of detection, endotoxin <0.5EU / mL, minimal residual DNA / protein); and ability to scale to support pre-clinical batches (hundreds of mg). In some embodiments, the invention provides a one-pot method of preparing mRNA from circular plasmid DNA, comprising: linearizing the circular plasmid DNA into linear plasmid DNA; performing in vitro transcription (IVT) of the linear plasmid DNA into mRNA; digesting residual linear and circular plasmid DNA; quenching enzymatic activity; and Attorney Docket No. TERR-001 / 01WO; 40647 / 5 precipitating the mRNA, wherein the method is performed in a single pot with no transfers, centrifugations, or purification between steps. In some embodiments, the method is performed in 96-well plate format using automated liquid handlers. In some embodiments, the method further comprises adding about 0.2 µL to about 3 µL of water to each well of the 96-well plate at a frequency of about every one minute to about every 15 minutes. In some embodiments, adding comprises adding about 1 µL of water to each well of the 96-well plate about every 7 minutes. In some embodiments, the linearizing comprises incubating the circular plasmid DNA with a restriction enzyme for about 10 minutes to about 60 minutes at about 30°C to about 50°C. In some embodiments, the linearizing comprises incubating the circular plasmid DNA with a restriction enzyme for about 15 minutes at about 37°C. In some embodiments, the linearizing further comprises assessing linearization completeness by comparing migration using gel or capillary electrophoresis. In some embodiments, the performing the IVT comprises adding a solution comprising a plurality of nucleotide triphosphates (NTPs) to the result of the linearizing; and incubating the resulting mixture for about 30 minutes to about 90 minutes at about 25°C to about 50°C. In some embodiments, the performing the IVT comprises adding a solution comprising a plurality of nucleotide triphosphates (NTPs) to the result of the linearizing; and incubating the resulting mixture for about 60 minutes at about 37°C. In some embodiments, the solution includes at least one of a modified NTP, a capping reagent, an RNase inhibitor, and pyrophosphatase. In some embodiments, the digesting comprises adding DNase I to the result of the IVT; and incubating the resulting mixture for about 15 minutes to about 90 minutes at about 25°C to about 50°C. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the digesting comprises adding DNase I to the result of the IVT; and incubating the resulting mixture for about 60 minutes at about 37°C. In some embodiments, the digesting further comprises adding Proteinase K to the result of the IVT. In some embodiments, the quenching comprises adding EDTA to the result of the digesting. In some embodiments, the precipitating comprises adding LiCl to the result of the quenching. In some embodiments, the method further comprises purifying the mRNA, wherein the purifying comprises cooling the result of the precipitating; centrifuging the precipitated mRNA to form an RNA pellet; aspirating liquid off the RNA pellet; and washing the RNA pellet with ethanol. In some embodiments, the cooling is performed at about -90°C to about 10°C for about 60 minutes to overnight. In some embodiments, the cooling is performed at about -20°C for about one hour. In some embodiments, the centrifuging is performed at about 0°C to about 25°C for about 5 minutes to about 30 minutes. In some embodiments, the centrifuging is performed at about 4°C for about 10 minutes. In some embodiments, the centrifuging is performed at about 4000 RPM. In some embodiments, the washing is performed at least two times. In some embodiments, the purifying further comprises heating the washed RNA pellet at about 20°C to about 50°C for about 5 minutes to about 60 minutes. In some embodiments, the purifying further comprises heating the washed RNA pellet at about 37°C for about 30 minutes. In some embodiments, the purifying further comprises resuspending the RNA pellet in water or buffer. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the resuspending comprises mixing the RNA pellet with the water or buffer using pipetting action for about 15 minutes to about one hour at about 4°C to about 50°C. In some embodiments, the resuspending comprises mixing the RNA pellet with the water or buffer using pipetting action for about 15 minutes at about 37°C. In some embodiments, the mRNA produced by the method has a value for A260 / 280 greater than or equal to about 1.95. In some embodiments, the mRNA produced by the method has greater than or equal to about 90% integrity as measured by capillary gel electrophoresis. In some embodiments, the invention provides a method of preparing mRNA from circular plasmid DNA, comprising: conducting a plurality of reactions that result in product of mRNA from a circular plasmid DNA, wherein the method is performed in a single pot with no transfers, centrifugations, or purification between steps. In some embodiments, the invention provides a multi-enzymatic composition for preparing mRNA from circular plasmid DNA, comprising: circular plasmid DNA; a restriction enzyme; an RNA polymerase; a capping reagent; an RNase inhibitor; pyrophosphatase; and a DNA endonuclease, wherein the circular plasmid DNA, the restriction enzyme, the RNA polymerase, the capping reagent, the RNase inhibitor, the pyrophosphatase, and the DNA endonuclease are each added to a single pot with no transfers, centrifugations, or purification between additions. In some embodiments, the circular plasmid DNA is supercoiled. In some embodiments, the restriction enzyme linearizes the circular plasmid DNA into linear plasmid DNA. In some embodiments, the restriction enzyme is BbsI, BspQI, or XbaI. In some embodiments, the RNA polymerase performs in vitro transcription of the linear plasmid DNA into mRNA. In some embodiments, the RNA polymerase is T7, SP6, or T3 RNA polymerase. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the composition further comprises a plurality of nucleotide triphosphates (NTPs). In some embodiments, the plurality of NTPs includes at least one modified NTP. In some embodiments, the at least one modified NTP comprises N1-methyl-pseudo-UTP. In some embodiments, the capping reagent is a co-transcriptional capping reagent or a post-transcriptional capping reagent. In some embodiments, the capping reagent comprises a Cap 1 analog or a Cap 0 analog. In some embodiments, the capping reagent comprises a compound having the following structure: In some the following structure:

[0002] Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the capping reagent comprises a Vaccinia capping enzyme. In some embodiments, the DNA endonuclease digests residual linear and circular plasmid DNA. In some embodiments, the DNA endonuclease comprises a salt-tolerant variant of DNaseI. In some embodiments, the DNA endonuclease comprises micrococcal nuclease or nuclease P1. In some embodiments, the composition further comprises a protease. In some embodiments, the protease is a serine protease. In some embodiments, the serine protease comprises Proteinase K. In some embodiments, the invention provides a buffer system for preparing mRNA from circular plasmid DNA, comprising: a first buffer formulation; a second buffer formulation; and a third buffer formulation, wherein the first buffer formulation, the second buffer formulation, and the third buffer formulation support linearization of circular plasmid DNA, in vitro transcription of linear plasmid DNA, and digestion of residual linear and circular plasmid DNA, respectively; and wherein the first buffer formulation, the second buffer formulation, and the third buffer formulation are prepared sequentially in a single pot. In some embodiments, the first buffer formulation comprises restriction enzyme reaction buffer components. In some embodiments, the first buffer formulation comprises potassium acetate, Tris- acetate, magnesium acetate, and bovine serum albumin (BSA). In some embodiments, the first buffer formulation comprises 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 µg / mL BSA and has a pH of about 7.9 at 25°C. In some embodiments, the second buffer formulation is prepared by adding an RNA polymerase reaction buffer solution to the first buffer formulation. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the RNA polymerase reaction buffer solution comprises Tris-HCl, MgCl2, DTT, and spermidine. In some embodiments, the RNA polymerase reaction buffer solution comprises 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine and has a pH of about 7.9 at 25°C. In some embodiments, the second buffer formulation comprises potassium acetate, Tris- acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, and spermidine. In some embodiments, the second buffer formulation comprises 20 mM potassium acetate, 8 mM Tris-acetate, 4 mM magnesium acetate, 40 µg / mL BSA, 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine and has a pH of about 7.9 at 25°C. In some embodiments, the third buffer formulation is prepared by adding a DNA endonuclease reaction buffer solution to the second buffer formulation. In some embodiments, the DNA endonuclease reaction buffer solution comprises Tris- HCl, MgCl2, and CaCl2. In some embodiments, the DNA endonuclease reaction buffer solution comprises 10 mM Tris-HCl, 2.5 mM MgCl2, and 0.5 mM CaCl2, and has a pH of about 7.6 at 25°C. In some embodiments, the third buffer formulation comprises potassium acetate, Tris- acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, spermidine, and CaCl2. In some embodiments, the third buffer formulation comprises 16.67 mM potassium acetate, 6.67 mM Tris-acetate, 3.33 mM magnesium acetate, 33.33 µg / mL BSA, 41.67 mM Tris- HCl, 7.08 mM MgCl2, 0.83 mM DTT, 1.67 mM spermidine, and 0.42 mM CaCl2. In some embodiments, the invention provides a buffer composition for performing in vitro transcription (IVT) of linear DNA into mRNA, wherein the buffer composition comprises restriction enzyme reaction buffer components and RNA polymerase reaction buffer components. In some embodiments, the buffer composition comprises potassium acetate, Tris-acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, and spermidine. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the buffer composition comprises 20 mM potassium acetate, 8 mM Tris-acetate, 4 mM magnesium acetate, 40 µg / mL BSA, 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine and has a pH of about 7.9 at 25°C. In some embodiments, the invention provides a buffer composition for digesting residual plasmid DNA following in vitro transcription, wherein the buffer composition comprises restriction enzyme reaction buffer components, RNA polymerase reaction buffer components, and DNA endonuclease reaction buffer components. In some embodiments, the buffer composition comprises potassium acetate, Tris-acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, spermidine, and CaCl2. In some embodiments, the buffer composition comprises 16.67 mM potassium acetate, 6.67 mM Tris-acetate, 3.33 mM magnesium acetate, 33.33 µg / mL BSA, 41.67 mM Tris-HCl, 7.08 mM MgCl2, 0.83 mM DTT, 1.67 mM spermidine, and 0.42 mM CaCl2. Additional features and advantages of embodiments of the present invention are described further below. This summary section is meant merely to illustrate certain features of embodiments of the invention, and is not meant to limit the scope of the invention in any way. The failure to discuss a specific feature or embodiment of the invention, or the inclusion of one or more features in this summary section, should not be construed to limit the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS The foregoing summary, as well as the following detailed description of certain embodiments of the invention, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the systems and methods of the present application, there are shown in the drawings preferred embodiments. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown. In the drawings: FIG.1 shows an example of a standard method of RNA production; Attorney Docket No. TERR-001 / 01WO; 40647 / 5 FIG.2 shows an example of an improved reaction design according to various embodiments of the present invention; FIG.3 shows a schematic of firefly luciferase (FLuc) mRNA produced according to embodiments of the present invention; FIG.4 shows fragment analyzer results for FLuc mRNA production according to embodiments of the present invention; FIG.5 shows an exemplary portion of computer script (left) and an example apparatus (right) for computer-implemented processes according to embodiments of the present invention; FIG.6 shows fragment analyzer results for automated FLuc mRNA production according to embodiments of the present invention; FIG.7 shows a schematic of Cas9 (CRISPR-associated protein 9) mRNA produced according to embodiments of the present invention; and FIG.8 shows fragment analyzer results for Cas9 mRNA production according to embodiments of the present invention. DETAILED DESCRIPTION Existing processes for producing mRNA have a number of drawbacks. As illustrated, for example, in FIG.1, standard RNA production involves multiple transfers and centrifugations. Significant attritions occur at each step. Standard RNA production also requires in-process controls (UV, etc.). It is a relatively long (two day) process, which is labor intensive and hard to automate. In contrast, embodiments of the present invention provide an improved reaction design, as illustrated, for example, in FIG.2. Methods and systems according to embodiments of the present invention provide preparation of mRNA from circular DNA plasmid in single pot, with no transfers, centrifugations, or purification between steps. As shown in FIG.2, in methods and systems according to embodiments of the present invention, restriction enzyme (RE) digestion (linearization) of circular double-stranded DNA (dsDNA), in vitro transcription (IVT) of linear Attorney Docket No. TERR-001 / 01WO; 40647 / 5 dsDNA, digestion of residual DNA (e.g., using DNase I), quenching of enzymatic activity (e.g., using EDTA), and precipitation of mRNA (e.g., using LiCl) are all performed in the same “pot.” No purification is performed between steps. No in-process controls are needed. The reaction design is automation friendly. Purification meets the needs for R&D and preclinical studies, and yields a quality that is identical / comparable to standard RNA production. Methods according to embodiments of the present invention require only about 2-3 hours total reaction time. Overnight precipitation is optional. Only water and ethanol (EtOH) are needed as solvents. Reagent costs are relatively low. Improved mRNA Protocol The reactions according to embodiments of the present invention are performed in stages, as described further below. In certain preferred embodiments, these reactions may be performed in 96-well plate format using automated liquid handlers (producing 96 different mRNAs, or up to about 50 mg of any combination of mRNAs desired). All stages except for centrifugation can be performed “on deck.” In some embodiments, assembly of gene inserts may also be performed on deck and transformations / preparations may also be added to the protocol. When using a 96-well liquid handler, the present inventors have recognized that the plate upon which the reactions are run is at elevated temperature (e.g., 37 °C) for an extended time (e.g., several hours), which results in significant undesired evaporation. It would be possible to seal then unseal the plate between additions, but that would require a hands-on intervention, which is undesirable. Instead, the solution provided by embodiments of the present invention is to periodically (e.g., about every 1-15 minutes, preferably about every 7-8 minutes) add a small amount (e.g., 0.2-3 µL) of water to each well of the reaction plate. For example, in some embodiments, 1µL of water is added to each well of the reaction plate about every 7 minutes. These additions have been found by the inventors to be sufficient to keep the volume constant during the duration of the mRNA production. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 The existing standard protocol separates each of the below-described stages into separate “pots” with purifications and in-process testing performed between stages 1, 2, and 3. The present inventors have unexpectedly found that both the separation and the in-process testing / purification between steps can be avoided, while maintaining high standards for the final product. As described further below, the concentrations of buffers in this improved protocol are different from the standard protocol. However, the concentration of each “stage appropriate” enzyme is identical to the standard protocol. For example, the concentration of polymerase in the IVT step is identical to the standard protocol. It should be noted that because no purification is performed in this improved protocol, the enzymes from each previous stage are still in solution and presumed to still be active. For example, the restriction enzyme from stage 1 may still be active throughout stages 2 and 3, etc. Stage 1: DNA Linearization This step affords the complete linearization of the circular plasmid DNA into linear DNA ready for stage 2. The linearization is driven by the enzymatic activity of an appropriate restriction enzyme in an appropriate buffer matrix. Standard methods typically involve purification of template DNA prior to IVT (e.g., using phenol-chloroform extraction). Notably, in embodiments of the present invention, no purification is required post linearization. If desired, linearization completeness can be assessed by comparing migration using gel or capillary electrophoresis. Linearization can be completed with any desired restriction enzyme (such as, but not limited to, BbsI, BspQI, and XbaI). Restriction enzymes suitable for use embodiments of the present invention specifically include, for example, those listed in Table 1 (which includes restriction enzymes sold by New England Biolabs (NEB) under the indicated trademarks). This step may be performed at a temperature of about 30°C to about 50°C, for a duration of about 10 minutes to about 60 minutes. For example, in some embodiments, linearization is performed at 37°C for about 15 minutes. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 Table 1 AatII BcoDI BsrFI-v2 EcoRV-HF® MseI Acc65I BfaI BsrGI-HF® Esp3I MslI AccI BfuAI BsrI FatI MspA1I AciI BglI BssHII FauI MspI AclI BglII BssSI-v2 Fnu4HI MspJI AcuI BlpI BstAPI FokI MwoI AfeI BmgBI BstBI FseI NaeI AflII BmrI BstEII-HF® FspI NarI AflIII BmtI-HF® BstNI HaeII Nb.BbvCI AgeI-HF® BpmI BstUI HaeIII Nb.BsmI AhdI Bpu10I BstXI HgaI Nb.BsrDI AleI-v2 BpuEI BstYI HhaI Nb.BssSI AluI BsaAI BstZ17I-HF® HincII Nb.BtsI AlwI BsaBI Bsu36I HindIII NciI AlwNI BsaHI BtgI HindIII-HF® NcoI ApaI BsaI-HF®v2 BtgZI HinfI NcoI-HF® ApaLI BsaJI BtsCI HinP1I NdeI ApeKI BsaWI BtsIMutI HpaI NgoMIV ApoI-HF® BsaXI BtsI-v2 HpaII NheI-HF® AscI BseRI Cac8I HphI NlaIII AseI BseYI ClaI Hpy166II NlaIV AsiSI BsgI AvaI BsiEI AvaII HF® AvrII HF® BaeGI DpnI HpyCH4III NsiI BaeI DpnII HpyCH4IV NsiI-HF® BamHI DraI HpyCH4V NspI BamHI-HF® BsmBI-v2 DraIII-HF® I-CeuI Nt.AlwI BanI BsmFI DrdI I-SceI Nt.BbvCI BanII BsmI EaeI KasI Nt.BsmAI BbsI BsoBI EagI-HF® KpnI-HF® Nt.BspQI BbsI-HF® Bsp1286I EarI MboI Nt.BstNBI BbvCI BspCNI EciI MboII Nt.CviPII BbvI BspDI Eco53kI MfeI-HF® WarmStart® BccI BspEI EcoNI MluCI Nt.BstNBI BceAI BspHI EcoO109I MluI-HF® PacI BcgI BspMI EcoP15I MlyI PaeR7I BciVI BspQI EcoRI MmeI PaqCI® BclI BsrBI EcoRI-HF® MnlI PciI BclI-HF BsrDI EcoRV MscI PflFI Attorney Docket No. TERR-001 / 01WO; 40647 / 5 PflMI PstI Sau96I SpeI-HF® TspMI PI-PspI PstI-HF® SbfI-HF® SphI TspRI PI-SceI PvuI-HF® ScaI-HF® SphI-HF® Tth111I PleI PvuII ScrFI SrfI XbaI PluTI PvuII-HF® SexAI SspI-HF® XcmI PmeI RsaI SfaNI StuI XhoI PmlI RsrII SfcI StyD4I XmaI PpuMI SacI-HF® SfiI StyI-HF® XmnI PshAI SacII SfoI SwaI ZraI PsiI-v2 SalI SgrAI TaqI-v2 PspGI SalI-HF® SmaI TfiI PspOMI SapI SmlI TseI PspXI Sau3AI SnaBI Tsp45I Stage 2: In Vitro Transcription (IVT) A new set of buffer reagents is added to the reaction mixture from stage 1 to achieve a new set of buffer concentrations (see Table 2 below). During the same addition, nucleotide triphosphates (NTPs), capping reagent, and polymerase (e.g., T7 RNA polymerase) are added to the matrix to begin the reaction. This step can utilize other polymerases including variants of T7 or any other polymerase compatible with IVT (such as SP6, T3, etc.). This step may be performed at a temperature of about 25°C to about 50°C, for a duration of about 30 minutes to about 90 minutes. For example, in some embodiments, IVT proceeds at 37°C for about 60 minutes. Stage 3: Residual DNA digestion A new set of buffer reagents is added to the reaction mixture from stage 1 to achieve a new set of buffer concentrations (see Table 2 below). Next, DNase I enzyme is added to the mixture and the reaction proceeds to ensure the complete digestion of plasmid DNA into smaller fragments or monomers. This step prevents the IVT reaction from continuing. In certain preferred embodiments, the DNase I is a salt-tolerant variant of DNase I. In other embodiments, other known DNA digesting enzymes may be used, such as, but not limited to, Micrococcal Attorney Docket No. TERR-001 / 01WO; 40647 / 5 Nuclease and Nuclease P1. This step may be performed at a temperature of about 25°C to about 50°C, for a duration of about 15 minutes to about 90 minutes. For example, in some embodiments, the residual DNA digestion reaction proceeds at 37°C for about 60 minutes. In some embodiments, a protease may be optionally added at the end of the reaction to digest all other proteins in solution. In some embodiments, the protease is a serine protease, such as, but not limited to, Proteinase K. It works to reduce the overall residual protein concentration, and may be used, for example, at a concentration of about 2.5 µM, which does not change the buffer system (which is diluted only about 0.5% by the addition of the enzyme). Table 2 shows stage specific buffer compositions for stages 1, 2, and 3 according to certain exemplary embodiments of the present invention (e.g., as detailed in Table 4). The stage specific buffer compositions may vary in different embodiments (e.g., according to specific enzymes used at each stage), as described further below. However, it should be noted that in each embodiment, the buffer compositions, like the enzyme compositions, are additive – i.e., the buffer from stage 1 is still present throughout stages 2 and 3, etc. For example, the stage 2 buffer includes both the stage 1 buffer (restriction enzyme buffer) and the IVT buffer. Similarly, the stage 3 buffer includes the buffer from stages 1 and 2, as well as the DNase reaction buffer. Table 2 Stage Component Concentration Unit pH .9 .9 .9 Attorney Docket No. TERR-001 / 01WO; 40647 / 5 Magnesium Acetate 3.33 mM BSA 33.33 ug / mL Step 4: Quenching and precipitation The reaction is quenched by the addition of EDTA, which sequesters available magnesium, preventing further enzymatic activity. Next, LiCl is added to the mixture to selectively precipitate RNA such that it “crashes out” of solution. The other reaction components (including proteins, salts, small molecules, and DNA) are expected to remain in solution. Step 5: Purification and resuspension The reaction mixture is cooled and then centrifuged for purification. In various embodiments, the mixture is cooled to a temperature of about 10°C to about -90°C for at least about 60 minutes (e.g., from about 60 minutes to overnight), and then spun at a temperature of about 0°C to about 25°C for at least about 5 minutes (e.g., about 5 minutes to about 30 minutes). For example, in some embodiments, the reaction mixture is cooled to -20°C for 1 hour before being spun at about 4000 RPM (or whatever maximum can be achieved) for 10 min at 4°C. The plate is then returned to the deck and the reaction mixture is aspirated off of the RNA pellet. Ethanol is then gently added on top of the pellet and aspirated off again. This is repeated a second time and then the plate is heated to remove excess ethanol. In various embodiments, heating to remove ethanol is performed at a temperature of about 20°C to about 50°C for at least about 5 minutes (e.g., about 5 minutes to about 60 minutes). For example, in some embodiments the plate is heated at 37°C for 30 minutes to remove excess ethanol. At this point, the RNA can be left dry or it can be resuspended in water or buffer. If resuspended, water or buffer is added to achieve a desired concentration and then the pellet is mixed using pipetting action at a Attorney Docket No. TERR-001 / 01WO; 40647 / 5 temperature of about 4°C to about 50°C for at least about 15 minutes (e.g., about 15 minutes to 1 hour). For example, in some embodiments, the pellet is mixed using pipetting action for 15 minutes at 37°C. Exemplary Materials Table 3 shows a list of materials suitable for use in various embodiments of the present invention. The list in Table 3 is one example; suitable materials may vary in different embodiments, as described further below. Table 3 Stock Component Concentration Unit Vendor Catalog # 1 As used herein, “MolBio Water” refers to a DNase / RNase-Free Distilled Water sold by Thermo Fisher Scientific and others under the trademark UltraPure™ from Invitrogen for use in molecular biology applications. It is 0.1-μm membrane-filtered and tested for DNase and RNase Attorney Docket No. TERR-001 / 01WO; 40647 / 5 activity. In other embodiments, other types of nuclease-free water (e.g., available commercially or produced in-house) can be used. As used herein, “CutSmart buffer” refers to a 10X buffer solution sold by New England Biolabs under the trademark rCutSmart™ (1X Buffer Components: 50 mM Potassium Acetate; 20 mM Tris-acetate; 10 mM Magnesium Acetate; 100 µg / ml Recombinant Albumin; pH 7.9 at 25°C). In other embodiments, other types of 10X buffer solutions may be used for linearization, for example depending on the specific restriction enzyme used (which may vary as described above). Other types of buffer solutions suitable for linearization include, but are not limited to, other 10X buffers sold by New England Biolabs under the trademark NEBuffer™, such as NEBuffer™ r1.1 (1X Buffer Components: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 100 μg / ml Recombinant Albumin, pH 7.0 at 25°C), NEBuffer™ r2.1 (1X Buffer Components: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml Recombinant Albumin, pH 7.9 at 25°C), and NEBuffer™ r3.1 (1X Buffer Components: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg / ml Recombinant Albumin, pH 7.9at 25°C). In some embodiments, the 10X IVT buffer, NTPs (ATP, CTP, GTP), and Polymerase Mix are from the T7 High Yield RNA Synthesis Kit sold by New England Biolabs under the trademark HiScribe®, where the 10X IVT buffer is T7 Reaction Buffer (1X Buffer Components: 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, 2 mM spermidine, pH 7.9 at 25°C). In other embodiments, other RNA polymerases (SP6, T3, etc.) and / or IVT buffers may be used. In some embodiments, the IVT reaction mix includes at least one modified NTP. In certain embodiments, the at least one modified NTP comprises N1-methyl-pseudo-UTP (N1- N1mePsU). In other embodiments, other modified NTPs may be used, such as, but not limited to, 5-Methylcytidine Triphosphate (5-methyl-CTP), Pseudouridine Triphosphate (Ψ-TP), and 2- Thiouridine Triphosphate (s2UTP). Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the IVT reaction mix includes a capping reagent, which may be a co-transcriptional or a post-transcriptional capping reagent. In some embodiments, the capping reagent comprises a trinucleotide cap analog. In some embodiments, the capping reagent is a co- transcriptional capping reagent from TriLink sold under the trademark CleanCap®, such as CleanCap® Reagent AG, which comprises a compound having the following structure: or CleanCap® structure: CleanCap® AG are of 5’ capped mRNA resulting in a Cap 1 structure. In various embodiments, other Cap 1 or Cap 0 capping agents may be used, such as, but not limited to, an Anti-Reverse Cap Analog (ARCA) such as 3'-O-Me- 7mG(5')ppp(5')G, a Cap 0 / m7G Cap Analog such as m7G(5')ppp(5')G, and a Vaccinia capping system including a methyl-transferase (e.g., a three-component enzyme that includes RNA triphosphatase, guanylyltransferase, and (guanine-N7)-methyltransferase activities). Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the IVT reaction mix includes an RNase inhibitor. Non-limiting examples of RNase inhibitors that may be used in the present invention include murine RNase inhibitor, human placenta RNase inhibitor, porcine RNase inhibitor, etc. In some embodiments, the inorganic phosphatase is isolated from a recombinant source (e.g., an E. coli strain that carries the Ribonuclease Inhibitor gene from mouse). An RNase inhibitor is optionally included in the reaction mixture during in vitro transcription at a concentration of around 1unit / µL. In some embodiments, the IVT reaction mix includes pyrophosphatase (inorganic). Pyrophosphatase catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form two orthophosphates, and can enhance RNA synthesis by degrading the PPi generated during the IVT reaction. In some embodiments, the inorganic phosphatase is isolated from a recombinant source (e.g., E. coli). The pyrphosphatase enzyme is included during IVT at a concentration around 0.01 to 0.1 units / µL. Non-limiting examples of DNase enzymes that may be used in the present invention include DNase I-XT (NEB M0570), Micrococcal Nuclease (NEB M0247), Nuclease P1 (NEB M0660). In some embodiments, the DNase I may be a salt-tolerant variant of DNase I, such as DNase I-XT from New England Biolabs. In some embodiments, the 10X DNase reaction buffer used may be a buffer compatible with standard DNase I (1X Buffer Contents: 10 mM Tris-HCl, 2.5 mM MgCl2, 0.5 mM CaCl2, pH 7.6 at 25°C), which can minimize total salt in the stage 3 buffer. In other embodiments, the 10X DNase reaction buffer used may be a buffer compatible with another DNase enzyme, such as Micrococcal Nuclease (1X Buffer Contents: 50 mM Tris- HCl, 5 mM CaCl2, pH 7.9 @ 25°C). In some embodiments, Proteinase K is optionally added at the end of the DNase I reaction to digest all other proteins in solution. Proteinase K is a subtilisin-related serine protease that hydrolyzes a variety of peptide bonds. In other embodiments, other proteases may be used to clean up the enzymatic reactions described herein. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 In some embodiments, the EDTA is a molecular biology grade 0.5 M solution of EDTA, pH 8.0, RNase-free. In other embodiments, EDTA solutions of different concentrations and / or pH may be used. In some embodiments, the LiCl is a DNase-free, RNase-free LiCl precipitation solution having a stock concentration of 7.5 M. In other embodiments, LiCl solutions of different concentrations may be used. Exemplary Protocol Table 4 lists details of an exemplary protocol for preparing mRNA according to methods and systems of the present invention. Values are given for four reactions (two plasmids each in duplicate); however, these may be scaled for any desired number of reactions (e.g., for 96-well plates as described above, with multiple constructs). Additionally, components, concentrations, times, and temperatures may be varied in different embodiments as detailed above. Referring to Table 4, Plasmid ID, Undigested pDNA (plasmid DNA) Concentration, and Undigested pDNA Mass are values entered by the user. “MM” refers to a Master Mix prepared as indicated and added to each reaction at the specified volume. Non-MM components include supercoiled pDNA (stage 1) and reaction mix from stage 1 (stage 2). Table 4 Plasmid Details A1 B1 C1 C2 Final Final Final Final Linearization Recipe Stock Conc. Conc. Conc. Conc.Conc. MolBio Water n / a n / a n / a n / a n / a CutSmart Buffer (10x) 10 1 1 1 1 BspQI (10U / uL) 10 1 1 1 1 Attorney Docket No. TERR-001 / 01WO; 40647 / 5 IVT Details Template concentration Expected Yield (mg) 0.125 0.125 0.125 0.125 Final Final Final Final IVT Recipe Stock Conc. Conc. Conc. Conc.Conc.10 b ff 10 1 1 1 1 CTP (mM) 100 8 8 8 8 GTP (mM) 100 8 8 8 8 N1mePsU (mM) 100 8 8 8 8 Cleancap AG (mM) 100 6 6 6 6 Polymerase Mix (X) 10 1 1 1 1 Linear Plasmid (ug / ul) 935 0.04 0.04 0.04 0.04 Volume Volume Volume Volume Stage 1: DNA Linearization Component (uL) (uL) (uL)(uL)Ste 1: Add Master Mix (MM) oume MM Component(uL)Recipe MolBio Water 7 7 7 7 35.2 CutSmart Buffer 1 1 1 1 8.8 BspQI 1 1 1 1 8.8 Supercoiled pDNA 1 1 1 1 MM to add to Step 2: Incubate at 37C for 15 minutes Total Volume Note: There is no purification / testing before IVTNote: 1uL water added every 7.5min to slow evaporation on deckStage 2: In Vitro Volume Volume Volume Volume Transcription (IVT) Component (uL) (uL) (uL)(uL) Volume MM Component(uL)Recipe MolBio Water 0.5 0.5 0.5 0.5 2.2 10x IVT buffer 2.5 2.5 2.5 2.5 11 ATP 2.0 2.0 2.0 2.0 8.8 CTP 2.0 2.0 2.0 2.0 8.8 GTP 2.0 2.0 2.0 2.0 8.8 N1mePsU 2.0 2.0 2.0 2.0 8.8 Cleancap AG 1.5 1.5 1.5 1.5 6.6 Attorney Docket No. TERR-001 / 01WO; 40647 / 5 IVT Enzyme Mix 2.5 2.5 2.5 2.5 11 Reaction mix from stage 1 10.0 10.0 10.0 10.0 MM to add to plasmid 15.0 15.0 Step 2: Incubate for 60 minutes at 37C Total Volume 25.0 25.0 Note: 1uL water added every 7.5minStage 3: Residual DNA Volume Volume Volume Volume digestion Component (uL) (uL) (uL)(uL) MM 7.5min Component(uL)Recipe DNase buffer 10x 2.5 2.5 2.5 2.5 11.00 DNase 2.5 2.5 2.5 2.5 11.00 Total Volume 30.0 30.0 30.0 30.0 Step 2: Incubate for 60 min at 37CStep 4: Quenching and Volume Volume Volume Volume precipitation Component (uL) (uL) (uL)(uL) Component(uL)EDTA (500mM) 4.5 4.5 4.5 4.5 Total Volume 34.5 34.5 34.5 34.5 Step 2: Add LiClVolume Component(uL)LiCl (7.5M) 34.5 34.5 34.5 34.5 Total Volume 69.0 69.0 69.0 69.0 Step 2: Cool to -20C for one hourStep 5: Purification and Volume Volume Volume Volume resuspension Component (uL) (uL) (uL)(uL) Step 2: Ethanol wash 1 Ethanol 69.0 Step 3: Ethanol wash 2 Ethanol 69.0 Step 4 (optional): Rehydrate pellet in water or buffer Attorney Docket No. TERR-001 / 01WO; 40647 / 5 Product Analysis In various embodiments, one or more of the following may be determined for the final product (purified, resuspended mRNA): final concentration (ng / µL mRNA); final A260 / A280 (unitless); expected final concentration (ng / µL mRNA); final yield (mg mRNA); expected productivity (mg mRNA / mL IVT); actual productivity (mg mRNA / mL IVT); %expected productivity (expected productivity / actual productivity); full length product (%FLP); IFN-β in A459 (pg / mL IFN- β). In some embodiments, the expected final concentration is 2000 ng / µL mRNA, and the expected productivity is 5 mg mRNA / mL IVT. Interferon Beta (IFN- β) in A459 cells may be measured by an enzyme-linked immunosorbent assay (ELISA) procedure or a similar immunoassay technique. The following is an exemplary description of the steps involved. Cell Culture: Grow A459 cells (a human lung carcinoma cell line) in appropriate culture medium until they reach the desired confluence. Sample Preparation: If measuring IFN-β secretion, collect the culture supernatant from the A459 cells after the treatment period (e.g., after exposure to a stimulus that induces IFN-β production). For intracellular IFN-β measurement, lyse the cells using a suitable lysis buffer to release intracellular proteins. ELISA Procedure: Coat a microplate (typically a 96-well plate) with an anti-IFN-β capture antibody specific for IFN-β. Incubate overnight at 4°C or at room temperature. Block non-specific binding sites with a blocking buffer (e.g., BSA or milk) to prevent background signal. Add the collected cell supernatant or lysate to the wells. Include standards of known IFN-β concentrations to create a calibration curve. Add a detection antibody specific to IFN-β, which is usually conjugated to an enzyme (e.g., horseradish peroxidase). This antibody binds to the IFN-β that was captured by the first antibody. Add a substrate that reacts with the enzyme to produce a colorimetric signal. The intensity of the color is proportional to the amount of IFN-β in the sample. Measure the absorbance of each well using a plate reader. Compare the absorbance values of the samples to the standard curve to determine the concentration of IFN-β. Analyze the data using appropriate software to quantify the IFN-β levels in the samples based on the standard curve. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 Table 5 shows an example of an analysis panel that may be performed to verify quality. In other embodiments, additional attributes (e.g., residual protein, dsRNA, endotoxin) or fewer attributes (e.g., just A260 / A280 and integrity via FA) may be assayed. Table 5 The values listed in Table 5 are targets and are normally exceeded using both the standard and this improved protocol. In practice, dsRNA content is below the limit of detection. The standard protocol will normally afford mRNA with 90% integrity as measured by capillary gel electrophoresis (Fragment Analyzer; FA). The improved methods described herein have achieved integrity >99% for the same mRNA. DNA FA Protocol: In certain preferred embodiments, FA for DNA is performed at 1ng / µL. Linearization reactions are at 100ng / µL. FA is run using 24µL at 1ng / µL in 1X TE buffer (1X Buffer Contents: 10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). This solution may be prepared, for example, by diluting 1µL of the linearization reaction mixture in 9µL buffer, and adding 2.4µL of that diluted mixture to 21.6µL buffer. RNA FA Protocol: In certain preferred embodiments, samples comprise 100ng of mRNA in 25µL marker diluent. This solution may be prepared, for example, by diluting 2µL of the mRNA in 38µL water, and adding 1µL of the 1:20 dilution solution to 24µL water. Attorney Docket No. TERR-001 / 01WO; 40647 / 5 EXAMPLES Example 1: FLuc In this example, the target mRNA encodes firefly luciferase (FLuc), illustrated schematically in FIG.3. When expressed in cells, FLuc catalyzes a two-step chemical reaction comprising: (1) luciferin + ATP → luciferyl adenylate + PPi; and (2) luciferyl adenylate + O2 → oxyluciferin + AMP + light. FLuc mRNA was produced using methods and systems of the present invention as described above. The mRNA is about 2000 nucleotides in length and incorporates a Cap 1 structure, N1-Methylpseudouridine (N1MepU), and 100A tail. Fragment analysis results for the FLuc example are shown in FIG.4. Complete digestion of 15µg pDNA was achieved in 15 minutes.2.02mg final yield of capped and tailed mRNA was achieved in 3 hours. A260 / 280 was 1.97. Purity was about 90-95% FLP. Example 2: Automation Unlike standard RNA production methods, the improved reaction design described herein is amenable to automation. Various embodiments of the present invention provide systems configured for applications in 96-well plate format using automated liquid handlers. The systems also preferably include one or more processors configured, in accordance with instructions stored on a non-transitory computer readable medium, to perform steps of the improved mRNA protocol described above. FIG.5 shows an example of computer-implemented instructions (left) and an example apparatus (right) for automation according to embodiments of the present invention. These automated embodiments feature minimal touch points. In some embodiments, the operator prepares master mixes and centrifuges the plate, nothing else. In other embodiments, those steps may also be automated. In certain exemplary embodiments, run time for a process automated as described above is about 3 hours (plus 30 minutes precipitation at -20°C). Purification (aspiration, EtOH washes, resuspension) is performed on deck. Testing was performed on 8 samples, with 5 deck slots open for expansion. FIG.6 shows fragment analyzer Attorney Docket No. TERR-001 / 01WO; 40647 / 5 results for automated FLuc mRNA production according to embodiments of the present invention. Automation was successful, evidenced by about 99% full-length product. Example 3: Cas9 In this example, the target mRNA encodes Cas9 (CRISPR-associated protein 9), illustrated schematically in FIG.7. Cas9 is an RNA-guided DNA endonuclease that can unwind and cleave dsDNA at sites complementary to its 20-nucleotide guide RNA (gRNA) sequence, approximately three bases from the PAM (protospacer adjacent motif) sequence. Cas9 mRNA was produced using methods and systems of the present invention as described above. The mRNA is about 5000 nucleotides in length and incorporates a Cap 1 structure, N1- Methylpseudouridine (N1MepU), and 100A tail. Fragment analysis results for the Cas9 example are shown in FIG.8. Purity was >95% FLP as determined by Fragment Analyzer. Actual productivity (mg mRNA / mL IVT) was about 7-8 mg / mL. In this example, 1µg of circular plasmid DNA yielded 180µg of mRNA. While there have been shown and described fundamental novel features of the invention as applied to the preferred and illustrative embodiments thereof, it will be understood that omissions and substitutions and changes in the form and details of the disclosed invention may be made by those skilled in the art without departing from the spirit of the invention. Moreover, as is readily apparent, numerous modifications and changes may readily occur to those skilled in the art. For example, various features and structures of the different embodiments discussed herein may be combined and interchanged. Hence, it is not desired to limit the invention to the exact construction and operation shown and described and, accordingly, all suitable modification equivalents may be resorted to falling within the scope of the invention as claimed. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Claims

Attorney Docket No. TERR-001 / 01WO; 40647 / 5 CLAIMS 1. A one-pot method of preparing mRNA from circular plasmid DNA, comprising: linearizing the circular plasmid DNA into linear plasmid DNA; performing in vitro transcription (IVT) of the linear plasmid DNA into mRNA; digesting residual linear and circular plasmid DNA; quenching enzymatic activity; and precipitating the mRNA, wherein the method is performed in a single pot with no transfers, centrifugations, or purification between steps.

2. The method of claim 1, wherein the method is performed in 96-well plate format using automated liquid handlers.

3. The method of claim 2, further comprising adding about 0.2 µL to about 3 µL of water to each well of the 96-well plate at a frequency of about every one minute to about every 15 minutes.

4. The method of claim 3, wherein the adding comprises adding about 1 µL of water to each well of the 96-well plate about every 7 minutes.

5. The method of claim 1, wherein the linearizing comprises incubating the circular plasmid DNA with a restriction enzyme for about 10 minutes to about 60 minutes at about 30°C to about 50°C.

6. The method of claim 1, wherein the linearizing comprises incubating the circular plasmid DNA with a restriction enzyme for about 15 minutes at about 37°C.Attorney Docket No. TERR-001 / 01WO; 40647 / 5 7. The method of claim 1, wherein the linearizing further comprises assessing linearization completeness by comparing migration using gel or capillary electrophoresis.

8. The method of claim 1, wherein the performing the IVT comprises adding a solution comprising a plurality of nucleotide triphosphates (NTPs) to the result of the linearizing; and incubating the resulting mixture for about 30 minutes to about 90 minutes at about 25°C to about 50°C.

9. The method of claim 1, wherein the performing the IVT comprises adding a solution comprising a plurality of nucleotide triphosphates (NTPs) to the result of the linearizing; and incubating the resulting mixture for about 60 minutes at about 37°C.

10. The method of claim 8, wherein the solution includes at least one of a modified NTP, a capping reagent, an RNase inhibitor, and pyrophosphatase.

11. The method of claim 1, wherein the digesting comprises adding DNase I to the result of the IVT; and incubating the resulting mixture for about 15 minutes to about 90 minutes at about 25°C to about 50°C.

12. The method of claim 1, wherein the digesting comprises adding DNase I to the result of the IVT; and incubating the resulting mixture for about 60 minutes at about 37°C.

13. The method of claim 11, wherein the digesting further comprises adding Proteinase K to the result of the IVT.

14. The method of claim 1, wherein the quenching comprises adding EDTA to the result of the digesting.Attorney Docket No. TERR-001 / 01WO; 40647 / 5 15. The method of claim 1, wherein the precipitating comprises adding LiCl to the result of the quenching.

16. The method of claim 1, further comprising purifying the mRNA, wherein the purifying comprises cooling the result of the precipitating; centrifuging the precipitated mRNA to form an RNA pellet; aspirating liquid off the RNA pellet; and washing the RNA pellet with ethanol.

17. The method of claim 16, wherein the cooling is performed at about -90°C to about 10°C for about 60 minutes to overnight.

18. The method of claim 16, wherein the cooling is performed at about -20°C for about one hour.

19. The method of claim 16, wherein the centrifuging is performed at about 0°C to about 25°C for about 5 minutes to about 30 minutes.

20. The method of claim 16, wherein the centrifuging is performed at about 4°C for about 10 minutes.

21. The method of claim 16, wherein the centrifuging is performed at about 4000 RPM.

22. The method of claim 16, wherein the washing is performed at least two times.

23. The method of claim 16, wherein the purifying further comprises heating the washed RNA pellet at about 20°C to about 50°C for about 5 minutes to about 60 minutes.Attorney Docket No. TERR-001 / 01WO; 40647 / 5 24. The method of claim 16, wherein the purifying further comprises heating the washed RNA pellet at about 37°C for about 30 minutes.

25. The method of claim 16, wherein the purifying further comprises resuspending the RNA pellet in water or buffer.

26. The method of claim 25, wherein the resuspending comprises mixing the RNA pellet with the water or buffer using pipetting action for about 15 minutes to about one hour at about 4°C to about 50°C.

27. The method of claim 25, wherein the resuspending comprises mixing the RNA pellet with the water or buffer using pipetting action for about 15 minutes at about 37°C.

28. The method of claim 1, wherein the mRNA produced by the method has a value for A260 / 280 greater than or equal to about 1.

95.

29. The method of claim 1, wherein the mRNA produced by the method has greater than or equal to about 90% integrity as measured by capillary gel electrophoresis.

30. A method of preparing mRNA from circular plasmid DNA, comprising: conducting a plurality of reactions that result in product of mRNA from a circular plasmid DNA, wherein the method is performed in a single pot with no transfers, centrifugations, or purification between steps.

31. A multi-enzymatic composition for preparing mRNA from circular plasmid DNA, comprising: circular plasmid DNA;Attorney Docket No. TERR-001 / 01WO; 40647 / 5 a restriction enzyme; an RNA polymerase; a capping reagent; an RNase inhibitor; pyrophosphatase; and a DNA endonuclease, wherein the circular plasmid DNA, the restriction enzyme, the RNA polymerase, the capping reagent, the RNase inhibitor, the pyrophosphatase, and the DNA endonuclease are each added to a single pot with no transfers, centrifugations, or purification between additions.

32. The composition of claim 31, wherein the circular plasmid DNA is supercoiled.

33. The composition of claim 31, wherein the restriction enzyme linearizes the circular plasmid DNA into linear plasmid DNA.

34. The composition of claim 33, wherein the restriction enzyme is BbsI, BspQI, or XbaI.

35. The composition of claim 33, wherein the RNA polymerase performs in vitro transcription of the linear plasmid DNA into mRNA.

36. The composition of claim 35, wherein the RNA polymerase is T7, SP6, or T3 RNA polymerase.

37. The composition of claim 31, further comprising a plurality of nucleotide triphosphates (NTPs).Attorney Docket No. TERR-001 / 01WO; 40647 / 5 38. The composition of claim 37, wherein the plurality of NTPs includes at least one modified NTP.

39. The composition of claim 38, wherein the at least one modified NTP comprises N1-methyl- pseudo-UTP.

40. The composition of claim 31, wherein the capping reagent is a co-transcriptional capping reagent or a post-transcriptional capping reagent.

41. The composition of claim 40, wherein the capping reagent comprises a Cap 1 analog or a Cap 0 analog.

42. The composition of claim 40, wherein the capping reagent comprises a compound having the following structure:Attorney Docket No. TERR-001 / 01WO; 40647 / 5 43. The composition of claim 40, wherein the capping reagent comprises a compound having the following structure:

44. The composition of claim 40, wherein the capping reagent comprises a Vaccinia capping enzyme.

45. The composition of claim 35, wherein the DNA endonuclease digests residual linear and circular plasmid DNA.

46. The composition of claim 45, wherein the DNA endonuclease comprises a salt-tolerant variant of DNase I.

47. The composition of claim 45, wherein the DNA endonuclease comprises micrococcal nuclease or nuclease P1.

48. The composition of claim 31, further comprising a protease.

49. The composition of claim 48, wherein the protease is a serine protease.Attorney Docket No. TERR-001 / 01WO; 40647 / 5 50. The composition of claim 49, wherein the serine protease comprises Proteinase K.

51. A buffer system for preparing mRNA from circular plasmid DNA, comprising: a first buffer formulation; a second buffer formulation; and a third buffer formulation, wherein the first buffer formulation, the second buffer formulation, and the third buffer formulation support linearization of circular plasmid DNA, in vitro transcription of linear plasmid DNA, and digestion of residual linear and circular plasmid DNA, respectively; and wherein the first buffer formulation, the second buffer formulation, and the third buffer formulation are prepared sequentially a single pot.

52. The buffer system of claim 51, wherein the first buffer formulation comprises restriction enzyme reaction buffer components.

53. The buffer system of claim 51, wherein the first buffer formulation comprises potassium acetate, Tris-acetate, magnesium acetate, and bovine serum albumin (BSA).

54. The buffer system of claim 53, wherein the first buffer formulation comprises 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, and 100 µg / mL BSA and has a pH of about 7.9 at 25°C.

55. The buffer system of claim 52, wherein the second buffer formulation is prepared by adding an RNA polymerase reaction buffer solution to the first buffer formulation.Attorney Docket No. TERR-001 / 01WO; 40647 / 5 56. The buffer system of claim 55, wherein the RNA polymerase reaction buffer solution comprises Tris-HCl, MgCl2, DTT, and spermidine.

57. The buffer system of claim 56, wherein the RNA polymerase reaction buffer solution comprises 40 mM Tris-HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine and has a pH of about 7.9 at 25°C.

58. The buffer system of claim 51, wherein the second buffer formulation comprises potassium acetate, Tris-acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, and spermidine.

59. The buffer system of claim 58, wherein the second buffer formulation comprises 20 mM potassium acetate, 8 mM Tris-acetate, 4 mM magnesium acetate, 40 µg / mL BSA, 40 mM Tris- HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine and has a pH of about 7.9 at 25°C.

60. The buffer system of claim 55, wherein the third buffer formulation is prepared by adding a DNA endonuclease reaction buffer solution to the second buffer formulation.

61. The buffer system of claim 60, wherein the DNA endonuclease reaction buffer solution comprises Tris-HCl, MgCl2, and CaCl2.

62. The buffer system of claim 61, wherein the DNA endonuclease reaction buffer solution comprises 10 mM Tris-HCl, 2.5 mM MgCl2, and 0.5 mM CaCl2, and has a pH of about 7.6 at 25°C.

63. The buffer system of claim 51, wherein the third buffer formulation comprises potassium acetate, Tris-acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, spermidine, and CaCl2.Attorney Docket No. TERR-001 / 01WO; 40647 / 5 64. The buffer system of claim 63, wherein the third buffer formulation comprises 16.67 mM potassium acetate, 6.67 mM Tris-acetate, 3.33 mM magnesium acetate, 33.33 µg / mL BSA, 41.67 mM Tris-HCl, 7.08 mM MgCl2, 0.83 mM DTT, 1.67 mM spermidine, and 0.42 mM CaCl2.

65. A buffer composition for performing in vitro transcription (IVT) of linear DNA into mRNA, wherein the buffer composition comprises restriction enzyme reaction buffer components and RNA polymerase reaction buffer components.

66. The buffer composition of claim 65, wherein the buffer composition comprises potassium acetate, Tris-acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, and spermidine.

67. The buffer composition of claim 66, wherein the buffer composition comprises 20 mM potassium acetate, 8 mM Tris-acetate, 4 mM magnesium acetate, 40 µg / mL BSA, 40 mM Tris- HCl, 6 mM MgCl2, 1 mM DTT, and 2 mM spermidine and has a pH of about 7.9 at 25°C.

68. A buffer composition for digesting residual plasmid DNA following in vitro transcription, wherein the buffer composition comprises restriction enzyme reaction buffer components, RNA polymerase reaction buffer components, and DNA endonuclease reaction buffer components.

69. The buffer composition of claim 68, wherein the buffer composition comprises potassium acetate, Tris-acetate, magnesium acetate, BSA, Tris-HCl, MgCl2, DTT, spermidine, and CaCl2.

70. The buffer composition of claim 69, wherein the buffer composition comprises 16.67 mM potassium acetate, 6.67 mM Tris-acetate, 3.33 mM magnesium acetate, 33.33 µg / mL BSA, 41.67 mM Tris-HCl, 7.08 mM MgCl2, 0.83 mM DTT, 1.67 mM spermidine, and 0.42 mM CaCl2.

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