Cell-free production of ribonucleic acid

Abstract

To provide a cell-free production method for biosynthesis of ribonucleic acid (RNA).SOLUTION: A cell-free production method comprises the steps of: (a) incubating a cell lysate mixture including, RNA, and at least one enzyme activity selected from the group consisting of enzyme activity which depolymerizes the RNA, heat resistant kinase activity, and heat resistant RNA polymerase activity, for producing a cell lysate mixture including a nucleoside-phosphoric acid; (b) heating the cell lysate mixture to a temperature at which endogenous nuclease and phosphatase are deactivated or partially deactivated, without completely deactivating the heat resistant kinase activity and heat resistant polymerase activity, for producing a cell lysate mixture; and (c) incubating the cell lysate mixture in presence of an energy supply source and a deoxyribonucleic acid (DNA) mold for coding RNA of interest, for producing a cell lysate mixture including the RNA of interest.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 319,220, filed April 6, 2016, and U.S. Provisional Application No. 62 / 452,550, filed January 31, 2017, each of which is incorporated by reference in its entirety. [Background technology]

[0002] Ribonucleic acid (RNA) is ubiquitous in life. RNA serves as an important messenger of information within cells, carrying instructions from DNA for the regulation and synthesis of proteins. RNA is of interest in biotechnology because synthetic regulation of mRNA levels within cells (either positively by the introduction of mRNA or negatively by the introduction of siRNA or dsRNA) has applications in areas such as crop protection, anticancer therapy, and vaccines. For example, RNA interference (RNAi) refers to a cellular mechanism that uses the gene's DNA sequence to "turn off" a gene - a process termed "silencing". In a wide variety of organisms, including animals, plants, and fungi, RNAi is induced by double-stranded RNA (dsRNA). Functional single-stranded (e.g., mRNA) and double-stranded RNA molecules have been produced in living cells and in vitro using purified recombinant enzymes and purified nucleotide triphosphates (see, e.g., European Patent No. 1631675 and US Patent Application Publication No. 2014 / 0271559A1, each of which is incorporated herein by reference). However, production of RNA on a scale that would allow widespread commercial application is currently cost-prohibitive. Summary of the Invention

[0003] Provided herein are methods, compositions, cells, constructs, and systems for the production (biosynthesis) of RNA. Generally, multimeric RNA from biomass material is enzymatically depolymerized into its component monomers, which are then phosphorylated (by a series of kinases) into their cognate triphosphorylated variants, which are then polymerized into multimeric RNA using the corresponding nucleic acid (e.g., DNA) template.

[0004] In some embodiments, the disclosed methods, compositions, cells, constructs, and systems are used for the production of RNA under cell-free conditions, for example, using at least one cell lysate, a combination of purified proteins, or a combination of cell lysate(s) and purified protein(s). In some embodiments, the disclosure is based on the conversion of RNA from biomass (e.g., endogenous RNA of cells) into desired synthetic RNA (e.g., synthetic single-stranded or double-stranded RNA) using cell lysate. First, RNA from biomass (e.g., endogenous RNA), such as messenger RNA (mRNA), transfer RNA (tRNA), and / or ribosomal RNA (rRNA) (e.g., present in cell lysate), is depolymerized by one or more nucleases into its monomeric form, 5'-nucleoside monophosphate (NMP) (Figure 1, reaction 1). Next, the nucleases, as well as native nucleases and phosphatases, are inactivated or partially inactivated (e.g., by heat inactivation), and the NMPs are phosphorylated to ribonucleotide triphosphates (NTPs) by a series of thermostable kinase activities (Figure 1, reaction 2). Finally, the NTPs are polymerized by an RNA polymerase (e.g., a thermostable RNA polymerase) using a nucleic acid (e.g., DNA) template to form the desired RNA (Figure 1, reaction 3). The desired synthetic RNA can optionally be purified from a cell lysate.

[0005] Therefore, some aspects of the present disclosure provide a cell-free method for producing (biosynthesizing) ribonucleic acid (RNA), the method comprising: (a) incubating at least one cell lysate mixture comprising (i) RNA and (ii) at least one enzymatic activity selected from the group consisting of an enzymatic activity that depolymerizes RNA, a thermostable kinase activity, and a thermostable RNA polymerase activity, under conditions that result in depolymerization of the RNA to produce a cell lysate mixture comprising nucleoside monophosphates; (b) incubating the cell lysate mixture produced in step (a) with endogenous nucleases without completely inactivating the thermostable kinase activity and the thermostable RNA polymerase activity. and (c) heating the cell lysate mixture produced in step (b) to a temperature (e.g., 50-80° C.) that inactivates or partially inactivates the nuclease and phosphatase to produce a cell lysate mixture containing heat-inactivated nuclease and phosphatase; and (c) incubating the cell lysate mixture produced in step (b) in the presence of an energy source (e.g., an ATP regenerating system) and a deoxyribonucleic acid (DNA) template (e.g., containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest) under conditions that result in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates to produce a cell lysate mixture containing an RNA of interest.

[0006] The cell lysate mixture may comprise a single cell lysate obtained from a cell that contains RNA and expresses at least one enzyme (including at least one fusion enzyme) that acts as a ribonuclease, acts as a kinase, and / or acts as an RNA polymerase. Alternatively, the cell lysate mixture may comprise at least two (e.g., at least three, four, five, or six) cell lysates, where at least one cell lysate is obtained from a cell that contains RNA and at least one cell lysate (e.g., at least two, three, four, or five) is obtained from a cell that expresses at least one enzyme that acts as a nuclease, acts as a kinase, and / or acts as an RNA polymerase.

[0007] An enzyme or fusion enzyme is considered to "act as a nuclease" if the enzyme in the fusion enzyme exerts nuclease activity (cleaves or depolymerizes nucleic acids; e.g., RNase R). An enzyme or fusion enzyme is considered to "act as a kinase" if the enzyme in the fusion enzyme exerts kinase activity (catalyzes the transfer of a phosphate group from one molecule to another; e.g., polyphosphate kinase). An enzyme or fusion enzyme is considered to "act as a polymerase" if the enzyme in the fusion enzyme exerts polymerase activity (assembles nucleotides to produce nucleic acids; e.g., RNA polymerase).

[0008] In some embodiments, the RNA in step (a) is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA).

[0009] In some embodiments, the cell lysate mixture comprises at least one ribonuclease, at least one thermostable kinase, and / or at least one RNA polymerase (e.g., thermostable RNA polymerase).The use of fusion enzymes is also encompassed by the present disclosure.For example, the cell lysate mixture can comprise a fusion of ribonuclease and kinase or a fusion of multiple kinases.Other fusion enzymes are encompassed by the present disclosure.

[0010] Other aspects of the present disclosure provide modified cells, cell lysates, and cell lysate mixtures comprising at least one nucleoside monophosphate kinase (e.g., a thermostable nucleoside monophosphate kinase), at least one nucleoside diphosphate kinase (e.g., a thermostable nucleoside diphosphate kinase), and at least one polyphosphate kinase (e.g., a thermostable polyphosphate kinase). In some embodiments, the cells may also comprise at least one ribonuclease and / or at least one RNA polymerase (e.g., a thermostable RNA polymerase).

[0011] In some embodiments, the method of producing (biosynthesizing) RNA comprises (a) the step of: (a) translating RNA (e.g., mRNA, tRNA, and / or rRNA), RNase R, a thermostable kinase (e.g., PfPyrH, TthAdk, TthCmk, PfGmk, AaNdk, TePpk, and / or PPK2 (see, e.g., Table 6), and a thermostable T7 (b) incubating the cell lysate produced in step (a) under conditions that result in depolymerization of RNA to 5'-NMP, thereby producing a cell lysate containing 5'-NMP; (c) heating the cell lysate produced in step (b) to 60-80°C to inactivate endogenous nucleases and phosphatases without completely inactivating the thermostable kinase and the thermostable RNA polymerase, thereby producing a cell lysate containing heat-inactivated nucleases and phosphatases; and (d) incubating the cell lysate produced in step (c) in the presence of an energy source (e.g., an ATP regenerating system including polyphosphate) and a modified nucleic acid (e.g., DNA) template (e.g., containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest) under conditions that result in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates to produce the RNA of interest.

[0012] In some embodiments, the RNA, RNase R, thermostable kinase, and thermostable T7 RNA polymerase are contained within a single line of cultured cells (e.g., engineered cells). In other embodiments, cultured cells (e.g., engineered cells) containing a subset of the above activities / components are lysed and the lysates are combined to generate a cell lysate mixture containing all of the enzymatic activities described in step (a) above. In some embodiments, the enzymatic activities are added to the lysate described in step (a) above in the form of purified enzymes. In some embodiments, the lysate and / or purified protein are combined before the heat inactivation step described in step (c) above. In other embodiments, the lysate and / or purified protein are combined after the heat inactivation step described in step (c) above.

[0013] The RNA of interest can be any form of RNA, including single-stranded RNA and double-stranded RNA.For example, the RNA of interest can be messenger RNA (mRNA), antisense RNA, microRNA, short interfering RNA (siRNA) or short hairpin RNA (shRNA).Other RNA interference (RNAi) molecules are included herein.

[0014] The details of certain aspects of the invention are set forth in the accompanying drawings and detailed description. Other features, objects, and advantages of the invention will be apparent from the description and claims. [Brief description of the drawings]

[0015] [Figure 1]Figure 1 shows a schematic diagram of the cell-free RNA production described herein. Cells containing RNA (e.g., endogenous RNA), nucleases, thermostable kinases, and / or thermostable RNA polymerases from biomass are lysed (or lysed in combination), and the resulting cell lysate(s) are incubated under conditions that result in depolymerization of RNA. The cell lysate is then heated to inactivate nucleases and any endogenous phosphatases (without inactivating thermostable kinases and thermostable RNA polymerases). The cell lysate is then incubated in the presence of a modified DNA template encoding an RNA of interest under conditions that result in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates, thereby producing the RNA of interest (e.g., ssRNA or dsRNA). Alternatively, individual purified pathway enzymes (e.g., RNA polymerases such as thermostable RNA polymerases) can be added to the cell lysate after the heat inactivation step. Therefore, in some cases, the modified cells used to produce the cell lysate do not express one or more of the enzymatic activities described above, such as a nuclease, a thermostable kinase, and / or a thermostable RNA polymerase.

[0016] [Diagram 2]Figure 2A shows a schematic diagram of a polyphosphate-dependent kinase pathway for energy generation. Figure 2B shows a schematic diagram of an additional exemplary energy conversion pathway for use in the methods and systems of the present disclosure. UMP kinase (e.g., from Pyrococcus furiosus) and polyphosphate kinase (e.g., from Thermosynechococcus elongatus, Caldilinea aerophila, Deinococcus geothermalis, Meiothermus ruber, Meiothermus silvanus, Deinococcus geothermalis, Anaerolinea thermophila, Chlorobaculum tepidum, Oceanithermus profundus, Roseiflexus castenholzii, Roseiflexus sp., or Truepera radiovctrix) can be used to convert UMP to UDP, and NDP kinase (e.g., encoded by Aquifex aeolicus ndk gene) and polyphosphate kinase can be used to convert UDP to UTP. CMP kinase (e.g., from Thermus thermophilus) and polyphosphate kinase can be used to convert CMP to CDP, and NDP kinase and polyphosphate kinase can be used to convert CDP to CTP. GMP kinase (e.g., from Thermotoga maritima) and polyphosphate kinase can be used to convert GMP to GDP, and NDP kinase and polyphosphate kinase can be used to convert GDP to GTP. AMP kinase (e.g., from Thermus thermophilus) and polyphosphate kinase can be used to convert AMP to ADP, and NDP kinase (e.g., encoded by the Aquifex aeolicus ndk gene) and polyphosphate kinase can be used to convert ADP to ATP. Alternatively, class III PPK2 enzymes (see, e.g., Table 6) can be used to convert AMP to ATP.

[0017] [Figure 3A-B] FIG. 3A shows a schematic diagram of an example of a DNA template used for the biosynthesis of double-stranded RNA. The DNA template encoded as part of a plasmid contains a single coding region and includes a promoter and one or more terminators operably linked to the coding region of interest. After transcription, the RNA folds into a hairpin structure by intramolecular nucleotide base pairing. The DNA template, either alone or encoded as part of a plasmid, contains two complementary domains (1 and 3) separated by domain 2. FIG. 3B shows a schematic diagram of another example of a DNA template used for the biosynthesis of double-stranded RNA. The DNA template contains a convergent promoter sequence on the complementary strand. The RNA sequences transcribed from each template strand anneal after transcription. [Figure 3C] Figure 3C shows a schematic diagram of another example of a DNA template used for the biogenesis of double-stranded RNA. The DNA template, encoded as part of a plasmid, contains a convergent promoter sequence operably linked to a coding region of interest on the complementary strand and one or more terminator sequences to prevent read-through transcription. [Figure 3D] Figure 3D shows a schematic diagram of another example of a DNA template used for the biogenesis of double-stranded RNA. The DNA template, encoded as part of a plasmid, contains independent cassettes, each of which includes a promoter operably linked to a coding region of interest and one or more terminators, driving the transcription of complementary sequences, which anneal after transcription. [Figure 3E] Figure 3E shows a schematic diagram of another example of a DNA template used for the biogenesis of double-stranded RNA: a DNA-dependent RNA polymerase is used to produce a ssRNA template, and an RNA-dependent RNA polymerase is used to produce double-stranded RNA.

[0018] [Figure 4]FIG. 4 shows a schematic diagram of another example of the cell-free RNA production method of the present disclosure. The process begins with a single fermentation vessel in which modified cells are produced, using standard fermentation techniques. The biomass produced by fermentation is optionally concentrated, for example, by microfiltration (MF), and then lysed by mechanical homogenization. The lysate is then pumped into a second fermentation vessel, where the expressed nuclease enzyme converts the RNA into its monomeric components. The entire reaction is heated to inactivate any endogenous phosphatase or nuclease (e.g., RNase) activity, as well as any other exogenous / introduced cellular (e.g., nuclease) activity that may be detrimental to RNA product stability and / or fidelity. After heat inactivation, polyphosphate is supplied to the reaction as a high-energy phosphate source for phosphorylation of NMPs to NTPs by a series of thermostable kinases, followed by polymerization into dsRNA. Downstream processing can be used to increase the purity to as much as 99% by weight (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%) dsRNA. For example, processing can be used to increase the purity to 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 70-80%, 70-90%, or 70-95%. An exemplary downstream process begins with the addition of a protein precipitant (e.g., ammonium acetate), followed by removal of proteins, lipids, and some DNA from the product stream by disk centrifugation (DSC) or tangential flow filtration (TFF). Ultrafiltration is then performed to remove salts and reduce volume. Addition of lithium chloride to the product stream results in precipitation of the dsRNA product, which is then separated from the bulk liquid using a disk centrifuge to obtain an 80% pure dsRNA product stream. Further chromatographic polishing yields a 99% pure product (Nilsen, TW. Cold Spring Harb Protoc. 2012 Dec 1;2012(12)).

[0019] [Figure 5A]Figures 5A-5B show a comparison of ribonuclease activity upon digestion of purified E. coli RNA (Figure 5A). Release of acid-soluble nucleotides (mononucleotides and short oligonucleotides) upon nuclease treatment was most rapid with Benzonase, RNase A, RNase R, and Nuclease P1. [Figure 5B] LC-MS analysis of the reaction products demonstrated the release of NMPs from RNA by RNase R and nuclease P1 treatment.

[0020] [Figure 6] Figure 6 is a graph showing the depolymerization of lysate RNA with exogenous RNase R, and the products were analyzed by UPLC to specifically identify 5'-NMPs. In the absence of RNase R, the lysate exerted endogenous RNase activity, which resulted in a slow accumulation of 5'-NMPs (dark grey solid line). Addition of exogenous RNase R resulted in rapid 5'-NMP release (dark grey dotted line) without affecting the rate of 2' or 3'NMP accumulation (light grey line). Hence, overexpression of RNase R accelerates the rate of conversion of multimeric RNA to 5'-NMPs and reduces the deleterious effects of phosphatase / nuclease activity present in the extract. The experiment was performed at a final concentration of 50% lysate.

[0021] [Figure 7A-B]Figures 7A-7C show the results of RNase R overexpression in 1 L bioreactor cultures grown in batch phase. (Figure 7A) SDS-PAGE analysis of protein expression in the two cultures. The empty vector culture contained an empty protein expression vector (pETDuet-1). The RNase R culture contained E. coli rnr cloned into pETDuet-1. The sample from the induced culture (+) showed strong expression of RNase R (MW 92.9 kDa with a C-terminal hexahistidine tag) indicated by the right arrow. (Figure 7B) Growth kinetics (including growth before and after induction) of the empty vector (dark grey) and RNase R expressing strains (light grey) demonstrated that RNase R overexpression was not detrimental to cell growth. The dotted lines represent exponential curve fits. [Figure 7C] Overexpressed RNase R was active in lysates of batch-grown biomass and liberated acid-soluble nucleotides. In the empty vector strain (dark grey solid line), adding exogenous RNase R increased the rate of nucleotide release (dark grey dotted line). In contrast, the strain expressing RNase R showed rapid nucleotide release upon lysis (light grey solid line). Adding exogenous RNase R did not increase the rate of nucleotide release or the final nucleotide yield (light grey dotted line). Experiments were performed at a final concentration of 50% lysate.

[0022] [Figure 8] Figure 8 is a graph depicting the effect of chelating Mg2+ on depolymerization rates in high density lysates. Lysates prepared from biomass containing empty vector (dark grey) were insensitive to EDTA. Lysates with overexpressed RNase R (light grey) showed rapid RNA depolymerization upon Mg2+ removal, with 8 mM EDTA providing the greatest depolymerization rate. Experiments were performed at a final concentration of 90% lysate.

[0023] [Figure 9A-B]Figures 9A-9D show graphs demonstrating the stability of exogenous isotope-labeled "heavy" NMPs (hNMPs) in lysates: (Figure 9A) hAMP was relatively stable in lysates, with 90% remaining after 1 hour incubation at 37°C. (Figure 9B) hCMP was degraded in lysates, with 70% remaining after approximately 30 minutes. Addition of 10 mM sodium orthovanadate (dotted line), an inhibitor of several phosphatases and kinases, significantly improved stability. [Figure 9C-D] (Figure 9C) hUMP was degraded in the lysate with 70% remaining after approximately 20 min. Sodium phosphate (150 mM) (dotted line) and sodium orthovanadate (dotted line) significantly improved stability. (Figure 9D) hGMP was degraded in the lysate with 70% remaining after approximately 10 min. Sodium orthovanadate significantly improved stability with 70% hGMP remaining after 30 min.

[0024] [Figure 10] Figure 10 is a graph demonstrating the effect of heat inactivation on the stability of exogenous NTPs in lysates. After preincubation of lysates at 70°C, the temperature was lowered to 37°C and an equimolar mixture of NTPs (ATP, CTP, UTP, and GTP) was added. Preincubation times are listed in the legend to the right. Control lysates (not subjected to heat inactivation) rapidly consumed NTPs (T=0 min). Increasing preincubation times stabilized NTPs, and 15 min at 70°C eliminated NTPase activity (T=15 min).

[0025] [Figure 11]11A-11B are graphs demonstrating the effect of heat inactivation on the stability of NMPs and dsRNA in lysates. (FIG. 11A) Heat inactivation stabilized NMPs in lysates. Lysates were treated with exogenous RNase R (lysate+RNase R) at 37° C. (t=0 min-5 min) to release NMPs and then heat inactivated at 70° C. (t=5 min to 25 min). The temperature was then lowered to 37° C. and the reactions were incubated for an additional 60 min. NMPs were generally stable in lysates after heat inactivation. (FIG. 11B) Heat inactivation stabilized the reactants and products of the transcription reaction in lysates. Lysates were preincubated at the indicated temperatures for 15 min, then the temperature was lowered to 37° C. and the transcription reactions were added. Heat inactivation at 70°C and 80°C, but not 60°C, stabilized the substrate and product sufficiently to produce detectable transcripts similar to the positive control (no lysate).

[0026] [Figure 12] Figure 12 is a graph demonstrating the temperature-dependent activity of UMP kinase from P. furiosus (PfPyrH), quantified by luciferase assay for ATP consumption. The specific activity of purified PfPyrH was largely insensitive to incubation temperature.

[0027] [Figure 13] Figure 13 is a graph demonstrating the temperature-dependent activity of AMP kinase from T. thermophilus (TthAdk) compared to Adk from E. coli (EcAdk), as measured by luciferase. Purified EcAdk was active at temperatures below 60°C. TthAdk had a higher specific activity, with a maximum at 70°C.

[0028] [Figure 14]Figure 14 is a graph demonstrating the temperature-dependent activity of CMP kinase from T. thermophilus (TthCmk), as measured by luciferase. Purified TthCmk was relatively insensitive to temperature, with high activity between 37 and 80°C.

[0029] [Figure 15] Figure 15 is a graph demonstrating the temperature-dependent activity of GMP kinases from E. coli (EcGmk), T. thermophilus (TthGmk), and T. maritima (TmGmk), as measured by luciferase. Purified EcGmk (dark grey) was more active at lower temperatures, while TthGmk (light grey) and TmGmk (medium grey) were most active at 70°C.

[0030] [Figure 16] Figure 16 is a graph of data demonstrating the activity of purified NDP kinase (AaNdk) from A. aeolicus, as measured by luciferase. Purified AaNdk was highly active between 37 and 80°C using ATP and GDP as substrates, with optimal activity at 50°C.

[0031] [Figure 17] Figure 17 is a graph demonstrating the activity of purified polyphosphate kinase 1 (PPK1) enzymes from E. coli (EcPpk), Thermosynechococcus elongatus (TePpk), and Thermus thermophilus (TthPpk), as measured by luciferase. EcPpk was most active at temperatures ≦60° C., while TePpk was optimally active at 70° C. TthPpk showed relatively low activity.

[0032] [Figure 18]Figure 18 is a graph demonstrating the activity of commercially available T7 RNA polymerases in buffer using conditions recommended by their respective manufacturers at 37° C. ThermoT7 and MegaScript polymerases showed higher specific activity than NEB polymerase under the conditions tested with a double-stranded DNA template (e.g., in FIG. 3B).

[0033] [Figure 19] Figure 19 is a graph comparing T7 RNA polymerase activity in diluted lysates at 37°C and 50°C under standardized reaction conditions with double-stranded DNA template. At 37°C, Thermo T7 showed the highest specific activity. At 50°C, only Thermo T7 had detectable activity, producing >10g / L / hr of dsRNA.

[0034] [Figure 20] Figure 20 is a graph demonstrating ThermoT7 activity in buffer and high density heat inactivated lysates. ThermoT7 activity was highest in lysates clarified by centrifugation after heat inactivation. Polymerase activity in unclarified matrix exceeded that of buffer alone, but omitting the clarification step resulted in a 60% decrease in activity.

[0035] [Figure 21] Figure 21 is a graph demonstrating the tolerance of ThermoT7 to elevated temperatures. Preincubating ThermoT7 at 50°C had no effect on subsequent polymerase activity assayed at 37°C. Preincubation at 60°C and 70°C resulted in rapid, irreversible inhibition of enzyme function.

[0036] [Figure 22]Figure 22A is an image of an SDS-PAGE gel showing expression and solubility data for A. thermophila PPK2 in E. coli strain GL16-170. MW: unstained protein standard broad range (New England Biolabs Cat#P7704). -: pre-induced cultures. +: induced cultures at harvest. L: soluble protein in clarified lysate. A. thermophila PPK2: 33 kDa. Figure 22B is a graph showing ATP production of A. thermophila PPK2 in heat inactivated lysate. Closed circles represent ATP production from ADP. Open circles represent ATP production from AMP. For both substrates, A. thermophila PPK2 produces ATP at a rate in excess of 400 mM / hr.

[0037] [Diagram 23] FIG. 23 is an image of an agarose gel demonstrating the application of thermostable class III PPK2 for energy generation in cell-free dsRNA production. The left lane contains a positive control, demonstrating dsRNA synthesis from NTPs. The middle lane contains a positive control, demonstrating dsRNA synthesis from NMPs in a nucleotide kinase-expressing lysate using exogenous ATP as the energy source. The right lane contains reactions demonstrating dsRNA synthesis from NMPs and HMPs using nucleotide kinase and C. aerophila Ppk-expressing lysate. In each case, the cell-free RNA synthesis reactions are Mn2+ independent. Reactions without polymerase are included as negative controls to illustrate the background nucleic acid content of each lysate-containing reaction.

[0038] [Figure 24]FIG. 24 is an image of an agarose gel demonstrating the application of thermostable class III PPK2 for energy generation in cell-free dsRNA production. The left lane contains a positive control, demonstrating dsRNA synthesis from NTPs. The middle lane contains a positive control, demonstrating dsRNA synthesis from NMPs in nucleotide kinase-expressing lysate using exogenous ATP as the energy source. The right lane contains reactions demonstrating dsRNA synthesis from NMPs and HMPs using nucleotide kinase and C. aerophila Ppk-expressing lysate. In C. aerophila PPK2, dsRNA synthesis proceeds in the absence of AMP kinase and exogenous ADP. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] In some aspects, the present specification provides methods, compositions, cells, constructs, and systems for cell-free production (biosynthesis) of nucleic acids (e.g., RNA or DNA). In some embodiments, a single type of organism (e.g., a population of bacterial cells) can be modified to express at least one nuclease, at least one thermostable kinase, and at least one thermostable polymerase (e.g., RNA or DNA polymerase). The modified cells are grown (cultured) under conditions that result in enzyme expression. In some embodiments, the modified cells can be grown to a desired cell density, and then the expression of certain enzymes can be induced (activated). Thus, the transcription of certain enzymes can be under the control of an inducible promoter. The cells (e.g., modified and / or unmodified cells) are then lysed (e.g., mechanically, chemically, or enzymatically disrupted) to produce a cell lysate that contains the enzyme activity required for cell-free production of RNA (e.g., ssRNA or dsRNA). In some embodiments, cells containing multimeric RNA (e.g., mRNA, tRNA, and / or rRNA) are mixed with modified cells containing pathway enzymes prior to a cell lysis step. In other embodiments, cell lysate(s) obtained from cells containing multimeric RNA are combined (mixed) with cell lysate(s) obtained from modified cells containing pathway enzymes. In yet other embodiments, one or more purified pathway enzymes are combined (mixed) with cell lysate(s) obtained from modified cells. A "pathway enzyme" is an enzyme required to biosynthesize an RNA of interest (e.g., starting from a multimeric RNA).

[0040] To synthesize RNA, the cell lysate (or cell lysate mixture) is incubated under conditions that result in nuclease-mediated (e.g., RNase-mediated) depolymerization of the host-derived (endogenous) RNA into a desired yield of 5'-nucleoside monophosphates (NMPs or nucleoside monophosphates). In some embodiments, the cell lysate (or cell lysate mixture) is then heated to inactivate most of the host-derived enzymes, including phosphatases and nucleases (e.g., RNases), and any exogenous nuclease(s) that were previously added to the cell lysate to promote depolymerization of the host-derived RNA. After the heat inactivation step, the cell lysate is incubated under conditions that result in phosphorylation of NMPs to NTPs (nucleoside triphosphates) by a thermostable kinase (e.g., a thermostable nucleoside monophosphate kinase and a nucleoside diphosphate kinase), for example, using a thermostable polyphosphate kinase and the addition of polyphosphate as an energy source. The resulting NTPs are then polymerized into RNA by an RNA polymerase (e.g., a thermostable RNA polymerase) using a modified template (e.g., a DNA template) present in the lysate (e.g., either expressed by the modified cells and included as an intracellular component of the cell lysate or later added to the cell lysate). Cell-free production

[0041] "Acellular production" is the use of biological processes for the synthesis of biomolecules or chemicals without the use of living cells. Cells are lysed and crude or partially purified parts, both of which contain enzymes, are used for the production of the desired product. In some embodiments, purified enzymes can be added to the cell lysate. As an example, cells are cultured, harvested, and lysed by high pressure homogenization or other cell lysis methods (e.g., chemical cell lysis). Acellular reactions can be performed in batch or fed-batch mode. In some cases, the enzyme pathway fills the available volume of the reactor and may be more diluted than the intracellular environment. Nevertheless, substantially all of the intracellular catalysts, including those that are membrane-bound, are provided. The inner membrane is fragmented during cell lysis, and the membrane fragments can form membrane vesicles. See, for example, Swartz, AIChE Journal, 2012, 58(1), 5-13, incorporated herein by reference.

[0042] The cell-free methods, compositions, and systems of the present disclosure utilize cell lysates (e.g., crude or partially purified cell lysates), which are discussed in more detail herein. For example, cell lysates prepared by mechanical means (e.g., shearing or grinding) are distinct from chemically permeabilized cells. As discussed above, in some embodiments, during cell lysis (e.g., mechanical cell lysis), intracellular membranes are fragmented, resulting in the formation of inverted membrane vesicles in the cell lysate. Such inverted membrane vesicles are not produced by chemical cell permeabilization methods. Lysed cells (e.g., at least 75%, 80%, 85%, 90%, or 95%) are no longer intact. Therefore, permeabilized cells, which are intact cells containing perforations (small holes), are not considered to be lysed cells.

[0043] Although the methods provided herein are generally acellular and use cell lysates, in some embodiments, it may be advantageous to use permeabilized cells for at least some steps of the methods. Thus, the present disclosure does not exclude the use of permeabilized cells for at least one step of the present RNA production methods.

[0044] Although many of the embodiments described herein refer to "lysing cultured cells" containing a particular enzyme, it should be understood that this phrase is intended to encompass lysing a clonal population of cells obtained from a single culture (e.g., containing all the enzymes required to synthesize RNA), as well as lysing more than one clonal population of cells obtained from different cell cultures (e.g., each containing one or more enzymes required to synthesize RNA and / or multimeric RNA substrates). For example, in some embodiments, a population of cells (e.g., modified cells) expressing one thermostable kinase can be cultured together and used to produce one cell lysate, and another population of cells (e.g., modified cells) expressing a different thermostable kinase can be cultured together and used to produce another cell lysate. These two cell lysates, each containing a different thermostable kinase, can then be combined for use in the RNA biogenesis method of the present disclosure. Depolymerization of ribonucleic acid to nucleoside monophosphates

[0045] The present disclosure is based on the conversion of RNA from biomass (e.g., endogenous RNA of cells) into desired synthetic RNA using cell lysate by a cell-free process involving a series of enzymatic reactions. First, RNA (e.g., endogenous RNA) present in cell lysate derived from host cells is converted into its component monomers by nucleases. RNA (e.g., endogenous RNA) from biomass typically includes ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), other RNA, or combinations thereof. Depolymerization or degradation of RNA results in a pool of 5'-nucleoside monophosphates (5'-NMPs), also simply referred to as "monomers". These monomers are used as starting materials for downstream polymerization / synthesis of the RNA of interest, which are converted into nucleoside diphosphates, which are converted into nucleoside triphosphates. In some embodiments, the RNA of interest is ssRNA (e.g., mRNA). In some embodiments, the RNA of interest is dsRNA.

[0046] The amount of RNA (e.g., endogenous RNA) required to synthesize an RNA of interest can vary and depends, for example, on the desired length and yield of the RNA of interest and the nucleotide composition of the RNA relative to the nucleotide composition of the RNA (e.g., endogenous RNA) of the cell (e.g., E. coli cell). Typically, in bacterial cells, for example, the RNA (e.g., endogenous RNA) content ranges from 5-50% of the total cell mass. The mass of starting material can be calculated, for example, using the following equation: (kilograms (kg) of RNA / kilograms of dry cell weight) x 100%.

[0047] Endogenous RNA can be depolymerized or degraded into its component monomers by chemical or enzymatic means. However, chemical hydrolysis of RNA typically produces 2'- and 3'-NMPs, which cannot be polymerized into RNA. Therefore, the methods, compositions, and systems provided herein primarily use enzymes for depolymerization of endogenous RNA. "RNA-depolymerizing enzymes" catalyze the hydrolysis of the phosphodiester bond between two nucleotides in RNA. Therefore, "RNA-depolymerizing enzymes" convert RNA (multimeric RNA) into its monomeric form, nucleoside monophosphates (NMPs). Depending on the enzyme, enzymatic depolymerization of RNA can produce 3'-NMPs, 5'-NMPs, or a combination of 3'-NMPs and 5'-NMPs. Since it is not possible to polymerize 3'-NTPs (converted from 3'-NDPs, which are converted from 3'-NMPs), an enzyme (e.g., RNase R) that produces 5'-NMPs (which are then converted to 5'-NDPs and then to 5'-NTPs) is preferred. In some embodiments, the enzyme that produces 3'-NMPs is removed from the genomic DNA of the modified cells to increase the efficiency of RNA production. In some embodiments, the enzyme used for RNA depolymerization is RNase R. In some embodiments, the concentration of RNase R used is 0.1-1.0 mg / mL (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL). In some embodiments, the concentration of RNase R used is 0.4-0.6 mg / mL. In some embodiments, the concentration of RNase R used is 0.5 mg / mL. In some embodiments, the concentration of RNase R used is greater than 1.0 mg / mL.

[0048] Examples of enzymes that depolymerize RNA include, without limitation, nucleases, including ribonucleases (RNases, e.g., RNase R), and phosphodiesterases. Nucleases catalyze the breakdown of nucleic acids into smaller components (e.g., monomers, also referred to as nucleoside monophosphates, or oligonucleotides). Phosphodiesterases catalyze the breakdown of phosphodiester bonds. These enzymes that depolymerize RNA can be encoded by full-length genes or gene fusions (e.g., DNA that includes at least two different genes (or fragments of genes) that encode two different enzymatic activities).

[0049] RNases function to control RNA maturation and turnover in cells. Each RNase has a specific substrate preference - dsRNA or ssRNA. Thus, in some embodiments, a combination of different RNases or a combination of different nucleases can be used to depolymerize multimeric RNA (e.g., endogenous RNA) from biomass in general. For example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, or 1-10 different nucleases can be used in combination to depolymerize RNA. In some embodiments, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different nucleases can be used in combination to depolymerize RNA. Non-limiting examples of nucleases for use provided herein are included in Table 1. In some embodiments, the nuclease used is RNase R. [Table 1]

[0050] Enzymes that depolymerize RNA (e.g., RNases) can be endogenous to the host cell (derived from the host) or they can be encoded by modified nucleic acids that have been exogenously introduced into the host cell (e.g., on an episomal vector or integrated into the genome of the host cell).

[0051] In some embodiments, the modified nucleic acid encoding the enzyme that depolymerizes RNA is operably linked to an inducible promoter. Thus, in some embodiments, the expression of the modified nucleic acid encoding the enzyme that depolymerizes RNA is controlled temporally or spatially. For example, the nucleic acid can be modified to encode an enzyme (e.g., RNase) that is translocated or sequestered in the periplasm of the host cell, so that the activity of the enzyme does not interfere with cell growth or other metabolic processes. Upon cell lysis, the translocated enzyme is released from the periplasm, contacts endogenous RNA, and depolymerizes RNA into monomeric form. See, for example, International Publication No. WO2011 / 140516, published November 10, 2011, which is incorporated herein by reference.

[0052] "Conditions that cause RNA depolymerization" are known in the art. Or can be determined by those skilled in the art, taking into account, for example, the optimum conditions of nuclease (e.g., RNase) activity, including pH, temperature, time length, and salt concentration of cell lysate, and any exogenous cofactors. Examples include those described previously (see, for example, Wong, CH et al. J. Am. Chem. Soc., 105: 115-117, 1983; EP1587947B1; Cheng ZF, Deutscher MP. J Biol Chem. 277:21624-21629, 2002).

[0053] In some embodiments, metal ions (e.g., Mg 2+ ) is depleted from the depolymerization reaction. In some embodiments, metal ions (e.g., Mg 2+) is 8 mM or less (e.g., less than 8 mM, less than 7 mM, less than 6 mM, less than 5 mM, less than 4 mM, less than 3 mM, less than 2 mM, less than 1 mM, less than 0.5 mM). 2+ ) is at a concentration of 0.1 mM-8 mM, 0.1 mM-7 mM, or 0.1 mM-5 mM.

[0054] The pH of the cell lysate during the RNA depolymerization reaction may have a value of 3.0 to 8.0. In some embodiments, the pH value of the cell lysate is 3.0 to 8.0, 4.0 to 8.0, 5.0 to 8.0, 6.0 to 8.0, 7.0 to 8.0, 3.0 to 7.0, 4.0 to 7.0, 5.0 to 7.0, 6.0 to 7.0, 3.0 to 6.0, 4.0 to 6.0, 5.0 to 6.0, 3.0 to 5.0, 3.0 to 4.0, or 4.0 to 5.0. In some embodiments, the pH value of the cell lysate is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, the pH value of the cell lysate is 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. The pH value of the cell lysate may be adjusted as required.

[0055] The temperature of the cell lysate during the RNA depolymerization reaction can be 15°C to 70°C. In some embodiments, the temperature of the cell lysate during the RNA depolymerization reaction is 15-60°C, 15-50°C, 15-40°C, 15-30°C, 25-70°C, 25-60°C, 25-50°C, 25-40°C, 30-70°C, 30-60°C, or 30-50°C. In some embodiments, the temperature of the cell lysate during the RNA depolymerization reaction is 37°C. In some embodiments, the temperature of the cell lysate during the RNA depolymerization reaction is 15°C, 25°C, 32°C, 37°C, 40°C, 42°C, 45°C, 50°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, or 70°C.

[0056] The cell lysate during the RNA depolymerization reaction may be incubated for 5 minutes (min) to 72 hours (hr). In some embodiments, the cell lysate during the RNA depolymerization reaction is incubated for 5-10 min, 5-15 min, 5-20 min, 5-30 min, or 5 min-48 hr. For example, the cell lysate during the RNA depolymerization reaction may be incubated for 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 11 hr, 12 hr, 18 hr, 24 hr, 30 hr, 36 hr, 42 hr, or 48 hr. In some embodiments, the cell lysate during the RNA depolymerization reaction is incubated at a temperature of 37° C. for 24 hours. In some embodiments, the cell lysate during the RNA depolymerization reaction is incubated at a temperature of 37° C. for 5-10 min. In some embodiments, the cell lysate during the RNA depolymerization reaction has a pH of 7.0 and is incubated at a temperature of 37° C. for 15 minutes. In some embodiments, the cell lysate during the RNA depolymerization reaction can be incubated under conditions that result in greater than 65% conversion of RNA to 5′-NMP. In some embodiments, RNA is converted to 5′-NMP at (or at least) a rate of 50 mM / hr, 100 mM / hr, or 200 mM / hr.

[0057] In some embodiments, salt is added to the cell lysate, for example, to prevent enzyme aggregation. For example, sodium chloride, potassium chloride, sodium acetate, potassium acetate, or a combination thereof can be added to the cell lysate. The concentration of salt in the cell lysate during the RNA depolymerization reaction can be 5 mM to 1 M. In some embodiments, the concentration of salt in the cell lysate during the RNA depolymerization reaction is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M. In some embodiments, the cell lysate comprises a mixture including 40-60 mM potassium phosphate, 1-5 mM MnCl2, and / or 10-50 mM MgCl2 (e.g., 20 mM MgCl2).

[0058] In some embodiments, a buffer is added to the cell lysate, for example to achieve a particular pH value and / or salt concentration. Examples of buffers include, without limitation, phosphate buffer, Tris buffer, MOPS buffer, HEPES buffer, citrate buffer, acetate buffer, malate buffer, MES buffer, histidine buffer, PIPES buffer, bis-tris buffer, and ethanolamine buffer.

[0059] Depolymerization of RNA results in the production of 5'-NMPs, including 5'-AMP, 5'-UMP, 5'-CMP, and 5'-GMP. While NMPs may be present in relatively equimolar amounts in cell lysates, depolymerization of RNA does not result in any given ratio of NMPs.

[0060] In some embodiments, lysis converts 50-98% of endogenous RNA in cells to 5'-NMP (depolymerized). For example, 50-95%, 50-90%, 50-85%, 50-80%, 75-98%, 75-95%, 75-90%, 75-85%, or 75-80% RNA is converted to 5'-NMP (depolymerized). In some embodiments, lysis converts 65-70% of endogenous RNA in cells to 5'-NMP (depolymerized). Lower yields are also acceptable. Elimination of futile circuits

[0061] After the conversion of RNA from biomass (e.g., endogenous RNA) to its monomeric components by endogenous and / or exogenous nucleases, several enzymes, including nucleases and phosphatases, typically remain in the cell lysate, which can have detrimental effects on RNA biogenesis. For example, Escherichia coli has a number of phosphatases, many of which dephosphorylate NTPs, NDPs, and NMPs. Dephosphorylation of NMPs after RNA depolymerization leads to the accumulation of unphosphorylated nucleosides and loss of available NMP substrates, thus reducing the synthetic RNA yield. Dephosphorylation of NMPs, NDPs, or NTPs after RNA depolymerization leads to a futile energy cycle (an energy cycle that results in low yields of synthetic RNA), during which NMPs are phosphorylated to NDPs and NTPs, which in turn are dephosphorylated back to their NMPs or nucleoside starting points. Futile cycles reduce the yield of RNA product per unit of energy input (e.g., polyphosphate, ATP, or other high-energy phosphate sources). In some embodiments, the enzyme activity is eliminated by removal from the host genome. In some embodiments, the enzyme activity is eliminated by heat inactivation. In some embodiments, the enzyme activity is eliminated by protease targeting. In some embodiments, the enzyme activity is eliminated by the use of chemical inhibitors. A combination of any of the aforementioned approaches may also be used.

[0062] Enzymes that are detrimental to the biosynthesis of RNA provided herein can be deleted from the host cell genome during the process of modifying the host cell. However, the enzyme is not essential for host cell (e.g., bacterial cell) survival and / or growth. Deletion of an enzyme or enzyme activity can be achieved, for example, by deleting or modifying a gene encoding an essential enzyme in the host cell genome. An enzyme is "essential for host cell survival" if the enzyme is necessary for host cell survival. That is, an enzyme is considered essential for host cell survival if the host cell cannot survive without the expression and / or activity of a particular enzyme. Similarly, an enzyme is "essential for host cell growth" if the enzyme is necessary for host cell growth. That is, an enzyme is considered essential for host cell growth if the host cell cannot divide and / or grow without the expression and / or activity of a particular enzyme.

[0063] In cases where an enzyme harmful to RNA biosynthesis is essential for host cell survival and / or growth, it may not be possible to delete or modify the gene encoding the enzyme. In such cases, the enzyme may be heat inactivated. "Heat inactivation" refers to the process of heating a cell lysate to a temperature sufficient to inactivate (or at least partially inactivate) endogenous nucleases and phosphatases. Generally, the process of heat inactivation involves denaturation (unfolding) of the harmful enzymes. The temperature at which endogenous proteins of a cell denature varies between organisms. In E. coli, for example, endogenous enzymes of a cell generally denature at temperatures above 41°C. Denaturation temperatures may be higher or lower than 41°C in other organisms. Enzymes of the cell lysates provided herein may be heat inactivated at temperatures between 40°C and 95°C, or higher. In some embodiments, the enzymes of the cell lysate may be heat inactivated at a temperature of 40-90°C, 40-80°C, 40-70°C, 40-60°C, 40-50°C, 50-80°C, 50-70°C, 50-60°C, 60-80°C, 60-70°C, or 70-80°C. For example, the enzymes of the cell lysate may be heat inactivated at a temperature of 40°C, 42°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C. In some embodiments, the enzymes of the cell lysate may be heat inactivated at a temperature of 50-80°C. In some embodiments, the enzymes of the cell lysate may be heat inactivated at a temperature of 70°C. In some embodiments, the enzymes of the cell lysate may be heat inactivated at a temperature of 60°C. It may also be possible to introduce chemical inhibitors of harmful enzymes. Such inhibitors may include, but are not limited to, sodium orthovanadate (an inhibitor of protein phosphotyrosine phosphatases), sodium fluoride (an inhibitor of phosphoserine and phosphothreonine phosphatases), sodium pyrophosphate (a phosphatase inhibitor), sodium phosphate, and / or potassium phosphate.

[0064] The time that the cell lysate is incubated at elevated temperatures to achieve heat inactivation of endogenous enzymes can vary depending, for example, on the volume of the cell lysate and the organism from which the cell lysate was prepared. In some embodiments, the cell lysate is incubated at a temperature of 35° C. to 80° C. for 2 minutes (min) to 48 hours (hr). For example, the cell lysate can be incubated at a temperature of 35° C. to 80° C. for 2 min, 4 min, 5 min, 10 min, 15 min, 30 min, 45 min, or 1 hr. In some embodiments, the cell lysate is incubated at a temperature of 35° C. to 80° C. for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 42, or 48 hr.

[0065] In some embodiments, the enzyme is heat inactivated for 10-20 minutes at a temperature of 60-80° C. In some embodiments, the enzyme is heat inactivated for 15 minutes at a temperature of 70° C.

[0066] In some embodiments, the enzymes that depolymerize endogenous RNA contain one or more modifications (e.g., mutations) that make the enzymes more sensitive to heat. Such enzymes are referred to as "thermosensitive enzymes." Thermosensitive enzymes become denatured and inactivated at lower temperatures than their wild-type counterparts, and / or the time required to reduce the activity of thermosensitive enzymes is shorter than that of their wild-type counterparts.

[0067] It should be understood that in some cases, enzymes that are heat inactivated may retain some degree of activity.For example, the activity level of a heat inactivated enzyme may be less than 50% of the activity level of the same enzyme that is not heat inactivated.In some embodiments, the activity level of a heat inactivated enzyme is less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 1%, or less than 0.1% of the activity level of the same enzyme that is not heat inactivated.

[0068] Therefore, the activity of an enzyme can be completely eliminated or reduced. An enzyme is considered completely inactive when the denatured (heat-inactivated) form of the enzyme no longer catalyzes the reaction catalyzed by the enzyme in its native form. A heat-inactivated denatured enzyme is considered "inactivated" when the activity of the heat-inactivated enzyme is reduced by at least 50% relative to the activity of the enzyme not heated (e.g., in its native environment). In some embodiments, the activity of the heat-inactivated enzyme is reduced by 50-100% relative to the activity of the enzyme not heated. For example, the activity of the heat-inactivated enzyme is reduced by 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, or 50-55% relative to the activity of the enzyme not heated. In some embodiments, the activity of the heat-inactivated enzyme is reduced by 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% relative to the activity of the unheated enzyme.

[0069] Examples of enzymes that can be heat inactivated or deleted from the genome of a host cell include, without limitation, nucleases (e.g., RNase III, RNase I, RNase R, PNPase, RNase II, and RNase T), phosphatases (e.g., nucleoside monophosphatases, nucleoside diphosphatases, nucleoside triphosphatases), and other enzymes that depolymerize RNA or dephosphorylate nucleotides. Enzymes that depolymerize RNA include any enzyme that can cleave, partially hydrolyze, or completely hydrolyze an RNA molecule. Table 2 provides a non-limiting list of examples of nucleases that can be heat inactivated or, in some cases, deleted from a modified host cell. Table 3 provides a non-limiting list of examples of phosphatases that can be heat inactivated or, in some cases, deleted from a modified host cell. Heat inactivation of these and other nucleases and phosphatases is encompassed by the present disclosure. [Table 2] [Table 3]

[0070] E. coli RNase III preferentially cleaves dsRNA and some single-stranded mRNA molecules. The presence of RNase III in cell lysate can limit the accumulation of high concentrations of synthetic RNA (e.g., dsRNA) because synthetic RNA is readily cleaved. Neither RNase III nor the gene rnc that encodes RNase III is essential for cell viability. Therefore, in some embodiments, rnc is deleted or mutated in modified host cells. In other embodiments, RNase III is heat-inactivated after depolymerization of endogenous RNA.

[0071] E. coli RNase I is localized in the periplasmic space in intact cells and catalyzes the depolymerization of a wide range of RNA molecules, including rRNA, mRNA, and tRNA. Under physiological conditions, the periplasmic localization of this enzyme means that it has little effect on RNA stability in cells; however, the mixing of periplasm and cytoplasm in cell lysates allows RNase I access to intracellular RNA. The presence of RNase I in cell lysates can reduce the yield of synthetic RNA by RNA degradation. Neither RNase I nor the gene rna encoding RNase I is essential for cell viability. Therefore, in some embodiments, rna is deleted or mutated in modified host cells. In other embodiments, RNase I is heat inactivated after depolymerization of endogenous RNA.

[0072] E. coli RNase R and RNase T catalyze the depolymerization of dsRNA, rRNA, tRNA, and mRNA, as well as small unstructured RNA molecules. Neither the enzymes nor the genes encoding them, rnr and rnt, respectively, are essential for cell viability. Thus, in some embodiments, rnr and / or rnt are deleted or mutated in engineered host cells (e.g., E. coli host cells). In other embodiments, RNase R and / or RNase T are heat inactivated after depolymerization of endogenous RNA.

[0073] E. coli RNase E and PNPase are components of the degradosome, which is responsible for mRNA turnover in cells. RNase E is believed to function together with PNPase and RNase II to turn over intracellular mRNA pools. Disruption of the gene rne, which encodes RNase E, is lethal in E. coli. Therefore, in some embodiments, RNase E is heat inactivated after depolymerization of endogenous RNA. Neither PNPase nor the gene pnp, which encodes PNPase, is essential for cell viability. Therefore, in some embodiments, pnp is deleted or mutated in engineered host cells (e.g., E. coli host cells). In other embodiments, PNPase is heat inactivated after depolymerization of endogenous RNA.

[0074] E. coli RNase II depolymerizes both mRNA and tRNA in the 3'->5' direction. Neither RNase II nor the gene encoding RNase II, rnb, is essential for cell viability. Therefore, in some embodiments, rnb is deleted or mutated in engineered host cells. In other embodiments, RNase II is heat inactivated after depolymerization of endogenous RNA.

[0075] While neither pnp nor rnb are essential for host cell survival, disruption of both simultaneously can be lethal, therefore, in some embodiments, both PNPase and RNase II are heat inactivated. Phosphorylation of nucleoside monophosphates to nucleoside triphosphates

[0076] After conversion of endogenous RNA to its monomeric form and after heat inactivation of endogenous nuclease and phosphatase, the resulting nucleoside monophosphate (NMP) in cell lysate is phosphorylated and then polymerized to form desired synthetic RNA, such as double-stranded RNA or single-stranded RNA (e.g., mRNA or antisense RNA).This process is highly energy-dependent, and therefore this process requires an energy source.Typically, phosphate is provided from a high-energy phosphate source, such as phosphoenolpyruvate, ATP, or polyphosphate.

[0077] In some embodiments, the energy source is ATP added directly to the cell lysate. In other embodiments, the energy source is provided using an ATP regeneration system. For example, polyphosphate and polyphosphate kinase can be used to produce ATP. Other examples included the use of acetyl phosphate and acetate kinase to produce ATP; creatine phosphate and creatine kinase to produce ATP; and phosphoenolpyruvate and pyruvate kinase to produce ATP. Other ATP (or other energy) regeneration systems can be used. In some embodiments, at least one component of the energy source is added to the cell lysate or cell lysate mixture. The "components" of the energy source include the substrate(s) and enzyme(s) required to produce energy (e.g., ATP). Non-limiting examples of these components include polyphosphate, polyphosphate kinase, acetyl phosphate, acetate kinase, creatine phosphate, creatine kinase, phosphoenolpyruvate, and pyruvate kinase.

[0078] Kinases are enzymes that catalyze the transfer of a phosphate group from a high-energy phosphate donor molecule, such as ATP, to a specific substrate / molecule. This process is called phosphorylation, where the substrate acquires a phosphate group and the high-energy ATP molecule donates a phosphate group. This transesterification produces a phosphorylated substrate and ADP. In some embodiments, the kinases of the present disclosure convert NMP to NDP and NDP to NTP.

[0079] In some embodiments, the kinase is a nucleoside monophosphate kinase, which catalyzes the transfer of high-energy phosphate from ATP to NMP, resulting in ADP and NDP. Non-limiting examples of nucleoside monophosphate kinases are provided in Tables 4 and 5. As discussed below, thermostable variants of the enzymes listed in Tables 4 and 5 are encompassed in the present disclosure. In some embodiments, the cell lysate comprises one or more (or all) of the following four nucleoside monophosphate kinases: thermostable uridylate kinase, thermostable cytidylate kinase, thermostable guanylate kinase, and thermostable adenylate kinase. In some embodiments, the UMP kinase is obtained from Pyrococcus furiosus (e.g., SEQ ID NO: 3, or a variant comprising an amino acid sequence at least 70% identical to the amino acid sequence identified by SEQ ID NO: 3). In some embodiments, the CMP kinase is obtained from Thermus thermophilus (e.g., SEQ ID NO: 4, or a variant comprising an amino acid sequence at least 70% identical to the amino acid sequence identified by SEQ ID NO: 4). In some embodiments, the GMP kinase is obtained from Thermotoga maritima (e.g., SEQ ID NO:5, or a variant comprising an amino acid sequence at least 70% identical to the amino acid sequence identified by SEQ ID NO:5). In some embodiments, the AMP kinase is obtained from Thermus thermophilus (e.g., SEQ ID NO:6, or a variant comprising an amino acid sequence at least 70% identical to the amino acid sequence identified by SEQ ID NO:6).

[0080] Thus, in some embodiments, the NMP kinase has an amino acid sequence identified by the amino acid sequence of any one of SEQ ID NOs: 3-6. In some embodiments, the NMP kinase has an amino acid sequence that is at least 70% identical to the amino acid sequence of any one of SEQ ID NOs: 3-6. For example, the NMP kinase can have an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence identified by any one of SEQ ID NOs: 3-6.

[0081] It should be understood that the disclosure encompasses the use of any one or more of the enzymes and variants of the enzymes (e.g., "PPK2 variants") described herein. Variant enzymes may share a certain degree of sequence identity to the reference enzyme. The term "identity" refers to the relationship between two or more polypeptide or polynucleotide sequences and is determined by comparing the sequences. Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) being handled by a particular mathematical model or computer program (e.g., "algorithm"). Identity between closely related molecules can be readily calculated by known methods. "Percent (%) identity" as applied to amino acid or nucleic acid sequences is defined as the percentage of residues (amino acid or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences to achieve the maximum percent identity and introducing gaps, if necessary. Identity is dependent on the calculation of percent identity, but may vary due to gaps and penalties introduced in the calculation. Variants of a particular sequence can have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that particular reference sequence, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art.

[0082] Comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software for determining the homology between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al. Nucleic Acids Research, 12(1): 387, 1984), the BLAST suite (Altschul, SF et al. Nucleic Acids Res. 25: 3389, 1997), and FASTA (Altschul, SF et al. J. Molec. Biol. 215: 403, 1990). Other techniques include: the Smith-Waterman algorithm (Smith, TF et al. J. Mol. Biol. 147: 195, 1981; the Needleman-Wunsch algorithm (Needleman, SB et al. J. Mol. Biol. 48: 443, 1970; and the Fast Optimal Global Sequence Alignment algorithm (FOGSAA) (Chakraborty, A. et al. Sci Rep.3: 1746, 2013). [Table 4]

[0083] In some embodiments, the kinase is a nucleoside diphosphate kinase, which transfers a phosphoryl group to the NDP, resulting in an NTP. The donor of the phosphoryl group can be, without limitation, ATP, a polyphosphate polymer, or phosphoenolpyruvate. Non-limiting examples of kinases that convert NDP to NTP include nucleoside diphosphate kinase, polyphosphate kinase, and pyruvate kinase. As discussed below, thermostable variants of the aforementioned enzymes are encompassed in the present disclosure. In some embodiments, the NDP kinase(s) are obtained from Aquifex aeolicus (e.g., SEQ ID NO:9, or a variant comprising an amino acid sequence at least 70% identical to the amino acid sequence identified by SEQ ID NO:9). In some embodiments, the NDP kinase has an amino acid sequence at least 70% identical to the amino acid sequence identified by SEQ ID NO:9. For example, an NDP kinase can have an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence identified by SEQ ID NO:9.

[0084] In some embodiments, phosphorylation of NMP to NTP occurs through polyphosphate-dependent kinase pathway (FIGS. 2A and 2B), where high-energy phosphate is transferred from polyphosphate to ADP by polyphosphate kinase (PPK). In some embodiments, polyphosphate kinase belongs to polyphosphate kinase 1 (PPK1) family, which transfers high-energy phosphate from polyphosphate to ADP to form ATP. This ATP is then used by NMP kinase (e.g., AMP kinase, UMP kinase, GMP kinase, and CMP kinase) to convert NMP to their corresponding ribonucleotide diphosphate (NDP). Furthermore, ATP is then used by nucleotide diphosphate kinase to convert NDP to NTP. See, for example, Tables 5 and 6 for exemplary enzymes.

[0085] In some embodiments, the polyphosphate kinase belongs to the polyphosphate kinase 2 (PPK2) family. In some embodiments, the polyphosphate kinase belongs to the class I PPK2 family, which transfers high-energy phosphate from polyphosphate to NDP to form NTP. The ATP produced by the system is used as a high-energy phosphate donor to convert NMP to NDP. In some embodiments, the polyphosphate kinase belongs to the class III PPK2 family, which transfers high-energy phosphate from polyphosphate to NMP and NDP to form NTP. In some embodiments, the class III PPK2 is used alone to produce NTP from NMP. In other embodiments, the class III PPK2 is used in combination with other kinases. The class III PPK2 produces ATP from ADP, AMP, and polyphosphate, which is then used by NMP and NDP kinases to convert NMP to NTP.

[0086] Non-limiting examples of PPK2 enzymes for use provided herein are listed in Table 6 (SEQ ID NOs: 8-18). Thus, in some embodiments, the PPK2 enzyme is thermostable. For example, the PPK2 enzyme can be a thermostable class III PPK2 enzyme, which prefers ATP synthesis over polyphosphate polymerization and converts both ADP and AMP to ATP. In some embodiments, the PPK2 enzyme is used to convert polyphosphate, such as hexametaphosphate, to ATP, for example, at a rate ranging from 10 to 800 mM per hour (e.g., 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, or 800 mM per hour).

[0087] In some embodiments, the RNA biosynthesis methods of the present disclosure utilize a PPK2 enzyme that comprises an amino acid sequence identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18. In some embodiments, the PPK2 enzyme comprises an amino acid sequence that is at least 70% identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18. For example, the PPK2 enzyme can comprise an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18.

[0088] The present disclosure also encompasses fusion enzymes. Fusion enzymes may perform multiple activities, each corresponding to the activity of a different enzyme. For example, rather than using an independent nucleoside monophosphate kinase and an independent nucleoside diphosphate kinase, a fusion enzyme (or any other enzyme) may be used that has both nucleoside monophosphate kinase activity and nucleoside diphosphate kinase activity. [Table 5] [Table 6] Polymerization of nucleoside triphosphates into ribonucleic acid

[0089] The final step in the biosynthesis of an RNA of interest is the polymerization of NTPs into an RNA (e.g., dsRNA or ssRNA) end product, for example, using a DNA-dependent RNA polymerase. In this step of the process, a DNA designed to encode the RNA of interest serves as a template for the synthesis of the RNA of interest. In some cases, the DNA template can be modified to have a transcriptional promoter that selectively drives transcription of the RNA of interest. An example DNA template is shown in FIG. 3A. The DNA template encodes three RNA domains: a sense domain (domain 1), a flexible hinge domain (domain 2), and a domain complementary to the sense domain (antisense domain 3). After transcription of the DNA template, the antisense domain binds (hybridizes) to the sense domain to form a double-stranded RNA hairpin stem domain and an adjacent hairpin loop domain. Other examples of DNA templates are shown in FIGS. 3B-3E. The DNA template in FIG. 3B contains convergent promoter sequences on the complementary strands. The RNA sequences transcribed from each template strand anneal after transcription. The DNA template in Figure 3C, encoded as part of a plasmid, contains convergent promoter sequences on the complementary strands and one or more terminator sequences to minimize read-through transcription. The DNA template in Figure 3D, encoded as part of a plasmid, contains independent promoter-terminator cassettes that drive transcription of complementary sequences, which anneal after transcription. The DNA template in Figure 3E encodes a single RNA domain. The use of both DNA-dependent RNA polymerase and RNA-dependent RNA polymerase produces a double-stranded RNA end product.

[0090] Polymerization of RNA requires NTPs, a DNA template containing a transcription promoter, and a polymerase (RNA polymerase) specific to the transcription promoter. Typically, the polymerase for use provided herein is a single subunit polymerase, highly selective for its corresponding transcription promoter, highly accurate, and highly efficient. Examples of polymerases include, without limitation, T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase. Bacteriophage T7 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific to the T7 phage promoter. The 99KD enzyme catalyzes in vitro RNA synthesis from cloned DNA sequences under the control of the T7 promoter. Bacteriophage T3 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific to the T3 phage promoter. The 99KD enzyme catalyzes in vitro RNA synthesis from cloned DNA sequences under the T3 promoter. Bacteriophage SP6 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for the SP6 phage promoter. The 98.5KD polymerase catalyzes in vitro RNA synthesis from cloned DNA templates under the SP6 promoter. T7, T3, and SP6 polymerases are each optimally active at 37-40°C. In some embodiments, thermostable variants of T7, T3, and SP6 polymerases are used. Thermostable variant polymerases are typically optimally active at temperatures above 40°C (or about 50-60°C).

[0091] "Conditions that result in the production of nucleoside triphosphates and polymerization of nucleoside triphosphates" are also referred to as "conditions for RNA biosynthesis", which can be determined by one of skill in the art and take into account optimal conditions for polymerase activity, including, for example, pH, temperature, length of time, and salt concentration of the cell lysate, as well as any exogenous cofactors.

[0092] The pH of the cell lysate during the biosynthesis of RNA may have a value of 3.0 to 8.0. In some embodiments, the pH value of the cell lysate is 3.0 to 8.0, 4.0 to 8.0, 5.0 to 8.0, 6.0 to 8.0, 7.0 to 8.0, 3.0 to 7.0, 4.0 to 7.0, 5.0 to 7.0, 6.0 to 7.0, 3.0 to 6.0, 4.0 to 6.0, 5.0 to 6.0, 3.0 to 5.0, 3.0 to 4.0, or 4.0 to 5.0. In some embodiments, the pH value of the cell lysate is 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0. In some embodiments, the pH value of the cell lysate during the biosynthesis of RNA is 7.0.

[0093] The temperature of the cell lysate during RNA biosynthesis can be from 15° C. to 70° C. In some embodiments, the temperature of the cell lysate during RNA biosynthesis is 15-60° C., 15-50° C., 15-40° C., 15-30° C., 25-70° C., 25-60° C., 25-50° C., 25-40° C., 30-70° C., 30-60° C., 30-50° C., 40-70° C., 40-60° C., 40-50° C., 50-70° C., or 50-60° C. In some embodiments, the temperature of the cell lysate during RNA biosynthesis is 15° C., 25° C., 32° C., 37° C., 42° C., 45° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., or 70° C. In some embodiments, the temperature of the cell lysate during RNA biosynthesis is 50° C.

[0094] The cell lysate during RNA biosynthesis may be incubated for 15 minutes (min) to 72 hours (hr). In some embodiments, the cell lysate during RNA biosynthesis is incubated for 30 min to 48 hr. For example, the cell lysate during RNA biosynthesis may be incubated for 30 min, 45 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, 7 hr, 8 hr, 9 hr, 10 hr, 11 hr, 12 hr, 18 hr, 24 hr, 30 hr, 36 hr, 42 hr, or 48 hr. In some embodiments, the cell lysate during RNA biosynthesis is incubated for 3 hours. In some embodiments, the cell lysate during RNA biosynthesis is incubated for 24 hours at a temperature of 37°C.

[0095] In some embodiments, the cell lysate during RNA biosynthesis is incubated at a pH of 7.0 and a temperature of 50° C. for 2 to 4 hours.

[0096] Some polymerase activities may require the presence of metal ions. Therefore, in some embodiments, metal ions are added to the cell lysate. A non-limiting example of a metal ion is Mg2 + , Li + , Na + , K + , Ni2 + , Ca2 + , Cu2 + , and Mn2 + Other metal ions may be used. In some embodiments, more than one metal ion may be used. The concentration of the metal ion in the cell lysate may be 0.1 mM to 100 mM, or 10 mM to 50 mM. In some embodiments, the concentration of the metal ion in the cell lysate is 0.1, 0.2, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 60.0, 70.0, 80.0, 90.0, or 100.0 mM.

[0097] In some embodiments, salt is added to the cell lysate, for example to prevent enzyme aggregation. For example, sodium chloride, potassium chloride, sodium acetate, potassium acetate, or a combination thereof can be added to the cell lysate. The concentration of salt in the cell lysate during the RNA depolymerization reaction can be 5 mM to 1 M. In some embodiments, the concentration of salt in the cell lysate during the RNA depolymerization reaction is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 500 mM, 750 mM, or 1 M. Heat-resistant enzymes

[0098] One advantage of the cell-free RNA biogenesis methods of the present disclosure is that all of the enzymes required to convert endogenous RNA to synthetic double-stranded RNA can be (but need not be) expressed within, for example, a single engineered cell. For example, a clonal population of engineered cells is cultured to a desired cell density, the cells are lysed, incubated under conditions that result in depolymerization of the endogenous RNA to its monomeric form (e.g., at a temperature of 30-37°C), subjected to a temperature sufficient to inactivate endogenous nucleases and phosphatases (e.g., 40-90°C), and incubated under conditions that result in polymerization of RNA (e.g., dsRNA or ssRNA) (e.g., 30-50°C). To proceed to the final product synthetic RNA, the enzymes required for the conversion of NMP to NDP (e.g., nucleoside monophosphate kinase and / or polyphosphate kinase), NDP to NTP (e.g., nucleoside diphosphate kinase and / or polyphosphate kinase), and NTP to RNA (e.g., polymerase) should be thermostable to avoid denaturation during heat inactivation of endogenous nucleases (and / or exogenous nucleases) and phosphatases. Thermostable refers to the quality of an enzyme to withstand denaturation at relatively high temperatures. For example, if an enzyme is denatured (inactivated) at a temperature of 42°C, an enzyme with a similar activity (e.g., kinase activity) is considered "thermostable" if it does not denature at 42°C.

[0099] An enzyme (e.g., a kinase or polymerase) is considered thermostable if the enzyme (a) retains activity after temporary exposure to high temperatures that would denature an otherwise native enzyme, or (b) functions at a higher rate after temporary exposure to intermediate to high temperatures at which the native enzyme functions at a lower rate.

[0100] In some embodiments, the thermostable enzyme retains more than 50% activity after temporary exposure to relatively high temperatures (e.g., above 41°C for kinases from E. coli and above 37°C for many RNA polymerases) that would otherwise denature a similar (non-thermostable) native enzyme. In some embodiments, the thermostable enzyme retains 50-100% activity after temporary exposure to relatively high temperatures that would otherwise denature a similar (non-thermostable) native enzyme. For example, the thermostable enzyme may retain 50-90%, 50-85%, 50-80%, 50-75%, 50-70%, 50-65%, 50-60%, or 50-55% activity after temporary exposure to relatively high temperatures that would otherwise denature a similar (non-thermostable) native enzyme. In some embodiments, a thermostable enzyme retains 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% activity after temporary exposure to relatively high temperatures that would otherwise denature a similar (non-thermostable) native enzyme.

[0101] In some embodiments, the activity of a thermostable enzyme after temporary exposure to intermediate to high temperatures (e.g., 42-80° C.) exceeds (e.g., 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% greater) the activity of a similar (non-thermostable) enzyme in its native state.

[0102] For example, the activity of a thermostable kinase can be measured by the amount of NMP or NDP that the kinase can phosphorylate.Thus, in some embodiments, a thermostable kinase converts more than 50% of NMP to NDP or more than 50% of NDP to NTP at a relatively high temperature (e.g., 42°C) in the same amount of time required to complete a similar conversion at 37°C.In some embodiments, a thermostable kinase converts more than 60% of NMP to NDP or more than 60% of NDP to NTP at a relatively high temperature (e.g., 42°C) in the same amount of time required to complete a similar conversion at 37°C.In some embodiments, a thermostable kinase converts more than 70% of NMP to NDP or more than 70% of NDP to NTP at a relatively high temperature (e.g., 42°C) in the same amount of time required to complete a similar conversion at 37°C. In some embodiments, the thermostable kinase converts more than 80% of the NMP to NDP, or more than 80% of the NDP to NTP, at a relatively high temperature (e.g., 42° C.) in the same amount of time required to complete a similar conversion at 37° C. In some embodiments, the thermostable kinase converts more than 90% of the NMP to NDP, or more than 90% of the NDP to NTP, at a relatively high temperature (e.g., 42° C.) in the same amount of time required to complete a similar conversion at 37° C.

[0103] For example, the activity of a thermostable polymerase is calculated based on the accuracy and polymerization kinetics (e.g., rate of polymerization). Thus, for example, one unit of thermostable T7 polymerase can incorporate 10 nmoles of NTPs into acid-insoluble material in 30 minutes at temperatures above 37° C. (e.g., at 50° C.).

[0104] A thermostable enzyme (e.g., a kinase or polymerase) can remain active (capable of catalyzing reactions) at temperatures between 42° C. and 80° C. or higher. In some embodiments, the thermostable enzyme remains active at temperatures between 42° C. and 80° C., 42° C. and 70° C., 42° C. and 60° C., 42° C. and 50° C., 50° C. and 80° C., 50° C. and 70° C., 50° C. and 60° C., 60° C. and 70° C. For example, a thermostable enzyme may remain active at temperatures of 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 55° C., 56° C., 57° C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C., 77° C., 78° C., 79° C., or 80° C. A thermostable enzyme may remain active at a relatively high temperature for 15 minutes to 48 hours, or longer. For example, a thermostable enzyme may remain active at a relatively high temperature for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 24, 36, 42, or 48 hours.

[0105] Non-limiting examples of thermostable NMP kinases are listed in Tables 5 and 7. Other thermostable kinases include thermostable nucleoside diphosphate kinases, thermostable pyruvate kinases, and thermostable polyphosphate kinases (see, e.g., Table 6). Other thermostable kinases are encompassed by the present disclosure. [Table 7]

[0106] Non-limiting examples of RNA polymerases are listed in Table 8. Other RNA polymerases, including thermostable RNA polymerases, are encompassed by the disclosure. [Table 8]

[0107] Thermostable RNA polymerases can be prepared by modifying wild-type enzymes. Such modifications (e.g., mutations) are known. For example, variant thermostable T7 RNA polymerases can include one or more of the following point mutations: V426L, A702V, V795I, S430P, F849I, S633I, F880Y, C510R, and S767G (EP2377928 and EP1261696A1, each of which is incorporated herein by reference). In some embodiments, variant thermostable T7 RNA polymerases include V426L, A702V, and V795I mutations. In some embodiments, variant thermostable T7 RNA polymerases include S430P, F849I, S633I, and F880Y mutations. In some embodiments, the variant thermostable T7 RNA polymerase comprises F880Y, S430P, F849I, S633I, C510R, and S767G mutations. In some embodiments, the variant thermostable T7 RNA polymerase comprises Y639V, H784G, E593G, and V685A mutations. In some embodiments, the variant thermostable T7 RNA polymerase comprises S430P, N433T, S633P, F849I, and F880Y mutations. Other variants and recombinant thermostable polymerases are encompassed in the present disclosure.

[0108] In some embodiments, a thermostable T7 polymerase is used to produce the RNA of interest. For example, a thermostable T7 polymerase with a concentration of 1-2% total protein (e.g., incubated at a temperature of 40-60° C.) can be used to synthesize the RNA of interest at a rate of more than 2 g / L / hr (or, for example, 2 g / L / hr to 10 g / L / hr). As another example, a thermostable T7 polymerase with a concentration of 3-5% total protein (e.g., incubated at a temperature of 40-60° C.) can be used to synthesize the RNA of interest at a rate of more than 10 g / L / hr (or, for example, 10 g / L / hr to 20 g / L / hr).

[0109] Although many embodiments of the present disclosure describe the use of a thermostable polymerase / enzyme, it should be understood that other enzymes / polymerases can be used. In some embodiments, a polymerase can be added exogenously to a heat-inactivated cell lysate, for example to compensate for any reduction or loss of activity of the thermostable enzyme(s). RNA of interest

[0110] The method of the present disclosure is used to synthesize an RNA of interest. The RNA can be single-stranded or double-stranded. In some embodiments, the RNA is a double-stranded RNA interference molecule. For example, the RNA of interest can be an siRNA or a hairpin RNA interference molecule. As discussed above, the RNA of interest is encoded by a DNA template, examples of which are shown in Figures 3A-3E. The RNA produced using the template in Figure 3A includes a sense domain (domain 1), a flexible hinge domain (domain 2), and a domain complementary to the sense domain (antisense domain 3). After transcription of the DNA template, the antisense domain binds (hybridizes) to the sense domain to form a double-stranded RNA hairpin stem domain and an adjacent hairpin loop (hinge) domain.

[0111] The double-stranded hairpin stem domain is formed by the binding of two complementary nucleic acid domains (e.g., distinct nucleotide sequences) to each other. Nucleic acid domains are "complementary" when they bind to each other (base pairing, hybridization via Watson-Crick interactions) to form a double-stranded nucleic acid. The complementary domains of the DNA template encoding the RNA of interest can vary depending on the desired end product. The complementary domains can have a length of, for example, 4 to 1000 nucleotides or longer. For example, the complementary domains can have a length of 4 to 10, 4 to 20, 4 to 30, 4 to 50, 4 to 60, 4 to 70, 4 to 80, 4 to 90, 4 to 100, 4 to 200, 4 to 300, 4 to 400, or 4 to 500, or 4 to 1000 nucleotides. In some embodiments, the complementary domains have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some embodiments, the complementary domains have a length of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides.

[0112] Hairpin loop domains are also formed by the binding of two complementary nucleic acid domains. Hairpin loop domains are the intervening sequences between two complementary domains. Typically, hairpin loop domains are non-specific, meaning that they are not designed to bind intramolecularly or to another nucleic acid. Hairpin loop domains form a loop-like structure by the binding of complementary domains to form a double-stranded hairpin stem domain. In some embodiments, hairpin loop domains have a length of 4 to 500 nucleotides or more. For example, hairpin loop domains can have a length of 4 to 10, 4 to 20, 4 to 30, 4 to 50, 4 to 60, 4 to 70, 4 to 80, 4 to 90, 4 to 100, 4 to 200, 4 to 300, 4 to 400, or 4 to 500 nucleotides. In some embodiments, the hairpin loop domain has a length of 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides.

[0113] The "double-stranded RNA" of the present disclosure encompasses fully double-stranded molecules that do not contain single-stranded regions (e.g., loops or overhangs), as well as partially double-stranded molecules that contain double-stranded regions and single-stranded regions (e.g., loops or overhangs). The dsRNA product depicted at the bottom of Figure 3A is considered a partially double-stranded molecule, while the dsRNA product depicted at the bottom of Figure 3B is considered a fully double-stranded molecule.

[0114] Examples of "single-stranded RNA" of interest include messenger RNA (mRNA) and antisense RNA. Thus, provided herein are methods for synthesizing mRNA and other single-stranded RNA molecules.

[0115] These methods include (a) lysing cultured engineered cells containing RNA, an enzyme that depolymerizes RNA, a thermostable kinase, and a thermostable RNA polymerase, thereby producing a cell lysate; (b) incubating the cell lysate produced in step (a) under conditions that result in depolymerization of RNA, thereby producing a cell lysate containing nucleoside monophosphates; and (c) incubating the cell lysate produced in step (b) with endogenous nucleases and nucleases without inactivating the thermostable kinase and the thermostable RNA polymerase. (d) heating to a temperature that inactivates the phosphatase, thereby producing a cell lysate containing heat-inactivated nuclease and phosphatase; and (d) incubating the cell lysate produced in (c) in the presence of an energy source and a modified DNA template containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest under conditions that result in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates, thereby producing a cell lysate containing the mRNA of interest.

[0116] Alternatively, such a method may include (a) combining cell lysates obtained from engineered cells containing endogenous multimeric RNA, an RNA depolymerizing enzyme, a thermostable nucleoside monophosphate (NMP) kinase, a thermostable nucleoside diphosphate (NDP) kinase, a thermostable PPK2 kinase, and / or polyphosphate to produce a cell lysate mixture; (b) incubating the cell lysate mixture produced in step (a) under conditions that result in depolymerization of RNA, thereby producing a cell lysate containing nucleoside monophosphates; and (c) treating the cell lysate produced in step (b) with phosphatases and RNases (as well as R 2 ) without inactivating the thermostable kinases and the thermostable RNA polymerase. (d) heating the cell lysate produced in step (c) to a temperature that inactivates any other activities that may be detrimental to NA stability or polymerization fidelity, such as native RNA polymerase, NMP reductase, and / or nucleosidase, thereby producing a cell lysate containing heat-inactivated phosphatases and RNases (as well as other deleterious cellular activities); and (d) incubating the cell lysate produced in step (c) in the presence of an energy source and a modified DNA template containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest under conditions that result in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates, thereby producing a cell lysate containing mRNA.

[0117] In some embodiments, a DNA template encoding an RNA containing a single target domain is transcribed using a DNA-dependent RNA polymerase, such as T7 RNA polymerase, and the resulting RNA transcript serves as a template for an RNA-dependent RNA polymerase, such as phage φ6 RdRP, to synthesize a complementary RNA molecule and generate dsRNA. See, for example, FIG. 3B. Phage φ6 is a double-stranded RNA virus that infects members of the genus Pseudomonas. This phage encodes an RdRP capable of synthesizing RNA using an RNA template and generating dsRNA molecules. φ6 RdRP is capable of polymerizing RNA without a primer molecule, and therefore the polymerase requires only template RNA (Wright, S. et al, 2012. Journal of Virology. Mar;86(5):2837-49; Van Dijk, AA., et al, 2004. J Gen Virol. May;85(Pt 5). Incorporated herein by reference). Other RNA-dependent RNA polymerases (RdRPs) are encompassed by this disclosure.

[0118] In some embodiments, the modified cell comprises a DNA template encoding the RNA of interest. The DNA template encoding the RNA can be integrated into the genomic DNA of the modified cell, or the DNA template can be introduced into the modified cell on a plasmid. In other embodiments, the DNA template is added to the cell lysate during the biosynthesis of the RNA of interest (e.g., after a heat inactivation step). In some embodiments, the concentration of the DNA template in the cell lysate is 0.05-1 μg / μl. In some embodiments, the concentration of the DNA template in the cell lysate is 0.05 μg / μl, 0.1 μg / μl, 0.5 μg / μl, 1.0 μg / μl.

[0119] As discussed above, other examples of RNA end products of interest include messenger RNA (mRNA) and short / small interfering RNA (siRNA), synthetic RNA duplexes designed to specifically target particular mRNAs for degradation.

[0120] In some embodiments, the concentration of the RNA end product (biosynthesized RNA of interest) is at least 1 g / L to 50 g / L cell lysate. For example, the concentration of the RNA end product can be 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 g / L or more.

[0121] In some embodiments, the RNA of interest is designed to bind to a target nucleic acid of interest and is used, eg, as a therapeutic, prophylactic, or diagnostic agent. Protease targeting

[0122] The modified cells of the present disclosure may express (e.g., endogenously express) enzymes necessary for cell health that may have a negative impact on the production of nucleic acids such as RNA. Such enzymes are referred to herein as "targeted enzymes." For example, the targeted enzymes expressed by the modified cells may compete for substrates or cofactors with enzymes that increase the rate of precursors supplied to an RNA biosynthetic pathway. As another example, the targeted enzymes expressed by the modified cells may compete for substrates or cofactors with enzymes that are key pathway entry enzymes of an RNA biosynthetic pathway. As yet another example, the targeted enzymes expressed by the modified cells may compete for substrates or cofactors with enzymes that supply substrates or cofactors to an RNA biosynthetic pathway.

[0123] To neutralize or reduce this negative effect, targeted enzymes can be modified to include site-specific protease recognition sequences in their protein sequences such that they can be "targeted" and cleaved for inactivation during RNA production (see, e.g., U.S. Publication No. 2012 / 0052547A1, published March 1, 2012; and International Publication No. WO2015 / 021058A2, published February 12, 2015, each of which is incorporated herein by reference).

[0124] Cleavage of the target enzyme containing a site-specific protease recognition sequence results from contact with the corresponding site-specific protease, which is sequestered in the periplasm (away from the target enzyme) of the cell during the cell growth phase (e.g., when the modified cells are cultured) and is contacted with the target enzyme during the RNA production phase (e.g., after cell lysis to produce a cell lysate). Thus, in some embodiments, the modified cells of the present disclosure include (i) a modified nucleic acid encoding a target enzyme that negatively affects the rate of RNA production and includes a site-specific protease recognition sequence in the protein sequence of the target enzyme, and (ii) a modified nucleic acid encoding a site-specific protease that cleaves the site-specific protease recognition sequence of the target enzyme and includes a periplasmic targeting sequence. The periplasmic targeting sequence is responsible for sequestering the site-specific protease in the periplasmic space of the cell until the cell is lysed. Examples of periplasmic targeting sequences are provided below.

[0125] Examples of proteases that may be used in accordance with the present disclosure include, without limitation, alanine carboxypeptidase, protease obtained from Armillaria mellea, astacin, bacterial leucyl aminopeptidase, cancer-derived procoagulant factor, cathepsin B, clostripain, cytoplasmic alanyl aminopeptidase, elastase, endoproteinase Brg-C, enterokinase, gastricin, gelatinase, Gly-X carboxypeptidase, glycyl endopeptidase, human rhinovirus 3C protease, hypodermin C, Iga-specific serine endopeptidase, leucyl aminopeptidase, leucyl endopeptidase, lysC, lysosomal pro-X carboxypeptidase, lysyl aminopeptidase, methionyl aminopeptidase, myxobacter , nardilysin, pancreatic endopeptidase E, picornain 2B, picornain 3C, proendopeptidase, prolyl aminopeptidase, proprotein convertase I, proprotein convertase II, russellysin, saccharopepsin, semenogelase, T-plasminogen activator, thrombin, tissue kallikrein, protease derived from tobacco etch virus (TEV), togavirin, tryptophanyl aminopeptidase, U-plasminogen activator, V8, venombin B, venombin BB, and Xaa-Pro aminopeptidase. Periplasmic targeting

[0126] The enzyme of nucleic acid (e.g., RNA) biosynthetic pathway may include at least one enzyme that has a negative effect on cell health (e.g., viability).To neutralize or reduce this negative effect, the enzyme may be modified to include a translocation sequence, so that the enzyme is translocated to an intracellular or extracellular compartment where it is not naturally located and where the enzyme does not negatively affect cell health (see, for example, Publication No. US-2011-0275116-A1, published November 10, 2011, which is incorporated herein by reference).For example, the enzyme of biosynthetic pathway may be translocated to the periplasmic space of the cell.

[0127] Thus, in some embodiments, the modified cells of the present disclosure comprise at least one enzyme of a nucleic acid (e.g., RNA) biosynthetic pathway linked to a periplasmic targeting sequence. A "periplasmic targeting sequence" is an amino acid sequence that targets a protein to which it is linked to the periplasm of a cell. A protein linked to a periplasmic targeting sequence will be sequestered in the periplasm of the cell in which the protein is expressed.

[0128] For example, periplasmic targeting sequences can be derived from the N-terminus of a bacterial secreted protein. The sequences vary in length from about 15 to about 70 amino acids. Although the primary amino acid sequence of periplasmic targeting sequences varies, they generally share a common structure that includes the following components: (i) an N-terminal portion that is of variable length and generally carries a net positive charge; (ii) followed by a hydrophobic central core of about 6 to about 15 amino acids; (iii) the final component includes 4 to 6 amino acids that define the cleavage site for a signal peptidase.

[0129] In some embodiments, the periplasmic targeting sequences of the present disclosure may be derived from proteins secreted by gram-negative bacteria. The secreted proteins may be encoded by the bacteria or by bacteriophages that infect the bacteria. Examples of gram-negative bacterial sources of secreted proteins include, without limitation, members of the genera Escherichia, Pseudomonas, Klebsiella, Salmonella, Caulobacter, Methylomonas, Acetobacter, Achromobacter, Acinetobacter, Aeromonas, Agrobacterium, Alcaligenes, Azotobacter, Burkholderia, Citrobacter, Comamonas, Enterobacter, Erwinia, Rhizobium, Vibrio, and Xanthomonas.

[0130] Examples of periplasmic targeting sequences for use in accordance with the present disclosure include, without limitation: MKIKTGARILALSALTTMMFSASALA (SEQ ID NO: 19); MKQSTIALALLPLLFTPVTKA (SEQ ID NO:20); MMITLRKLPLAVAVAAGVMSAQAMA (SEQ ID NO:21); MNKKVLTLSAVMASMLFGAAAHA (SEQ ID NO:22); MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO:23); MKKIWLALAGLVLAFSASA (SEQ ID NO:24); MMTKIKLLMLIIFYLIISASAHA (SEQ ID NO:25); MKQALRVAFGFLILWASVLHA (SEQ ID NO:26); MRVLLFLLLSLFMLPAFS (SEQ ID NO:27); and MANNDLFQASRRRFLAQLGGLTVAGMLGPSLLTPRRATA (SEQ ID NO: 28), The invention encompasses a sequence selected from the group consisting of: Engineered Cells

[0131] Typically, the modified cells of the present disclosure contain at least one, most, or all of the enzymatic activities required to biosynthesize RNA. "Modified cells" are cells that contain at least one modified (e.g., recombinant or synthetic) nucleic acid or are otherwise modified to be structurally and / or functionally distinct from their naturally occurring counterparts. Thus, cells that contain modified nucleic acids are considered "modified cells."

[0132] In some embodiments, the modified cells of the present disclosure comprise an RNA, an enzyme that depolymerizes an RNA, a thermostable kinase, and / or a thermostable polymerase. In some embodiments, the modified cells further comprise a DNA template that contains a promoter operably linked to a nucleotide sequence that encodes an RNA of interest.

[0133] In some embodiments, the modified cells express a selectable marker. A selectable marker is typically used to select modified cells that have taken up and expressed the modified nucleic acid after transfection of the cells (or after other procedures used to introduce exogenous nucleic acid into the cells). Thus, the nucleic acid encoding the product may also encode a selectable marker. Examples of selectable markers include, without limitation, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics (e.g., ampicillin resistance genes, kanamycin resistance genes, neomycin resistance genes, tetracycline resistance genes, and chloramphenicol resistance genes) or other compounds. Additional examples of selectable markers include, without limitation, genes encoding proteins that allow cells to grow in media lacking an otherwise essential nutrient (auxotrophic markers). Other selectable markers may be used in accordance with the present disclosure.

[0134] An engineered cell "expresses" a product when the product encoded by a nucleic acid (e.g., an engineered nucleic acid) is produced within the cell. It is known in the art that gene expression refers to the process by which genetic instructions in the form of nucleic acids are used to synthesize a product, such as a protein (e.g., an enzyme).

[0135] The modified cell can be a prokaryotic or eukaryotic cell, hi some embodiments, the modified cell is a bacterial cell, a yeast cell, an insect cell, a mammalian cell, or other type of cell.

[0136] The modified bacterial cells of the present disclosure include, without limitation, modified Escherichia spp., Streptomyces spp., Zymomonas spp., Acetobacter spp., Citrobacter spp., Synechocystis spp., Rhizobium spp., Clostridium spp., Corynebacterium spp., Streptococcus spp., Xanthomonas spp., Lactobacillus spp., Lactococcus spp., Bacillus spp., Alcaligenes spp., Pseudomonas spp., Aeromonas spp., Azotobacter spp., Comamonas spp., Mycobacterium spp., Rhodococcus spp., Gluconobacter spp., Ralstonia spp., Lactobacillus spp., Lactococcus ... spp., Acidithiobacillus spp., Microlunatus spp., Geobacter spp., Geobacillus spp., Arthrobacter spp., Flavobacterium spp., Serratia spp., Saccharopolyspora spp., Thermus spp., Stenotrophomonas spp., Chromobacterium spp., Sinorhizobium spp., Saccharopolyspora spp., Agrobacterium spp., and Pantoea spp.

[0137] The modified yeast cells of the present disclosure include, without limitation, modified Saccharomyces spp., Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, and Pichia.

[0138] In some embodiments, the modified cell of the disclosure is a modified Escherichia coli cell, a Bacillus subtilis cell, a Pseudomonas putida cell, a Saccharomyces cerevisae cell, or a Lactobacillus brevis cell. In some embodiments, the modified cell of the disclosure is a modified Escherichia coli cell. Modified Nucleic Acids

[0139] A "nucleic acid" is at least two nucleotides covalently linked together, and in some cases may contain phosphodiester bonds (e.g., a phosphodiester "backbone"). Nucleic acids (e.g., components or portions of a nucleic acid) may be naturally occurring or modified. A "naturally occurring" nucleic acid is present in a cell in which it occurs in nature in the absence of human intervention. "Modified nucleic acid" includes recombinant and synthetic nucleic acids. "Recombinant nucleic acid" refers to a molecule constructed by joining together nucleic acid molecules (e.g., from the same species or from different species), and typically can be replicated in a living cell. "Synthetic nucleic acid" refers to a molecule that is biologically synthesized, chemically synthesized, or synthesized or amplified by other means. Synthetic nucleic acid includes nucleic acids that have been chemically modified or otherwise modified, but that are capable of base pairing with naturally occurring nucleic acid molecules. Recombinant and synthetic nucleic acids also include molecules that result from replication of either of the foregoing. Modified nucleic acids may contain portions of naturally occurring nucleic acids, but as a whole, modified nucleic acids do not occur in nature and require human intervention. In some embodiments, the nucleic acid encoding the product of the disclosure is a recombinant or synthetic nucleic acid, while in other embodiments, the nucleic acid encoding the product is naturally occurring.

[0140] The modified nucleic acids encoding the RNAs provided herein can be operably linked to a "promoter," a control region of the nucleic acid, which controls the initiation and rate of transcription of the remainder of the nucleic acid. A promoter drives the expression or drives the transcription of the nucleic acid it controls.

[0141] A promoter may be one that is naturally associated with a gene or sequence, or may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter may be said to be "endogenous."

[0142] In some embodiments, the coding nucleic acid sequence can be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coding sequence in its natural environment. Such promoters can include the promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring", such as those that contain mutations that alter expression between different elements of different transcriptional control regions and / or by genetic engineering methods known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences can be produced using recombinant cloning and / or nucleic acid amplification technologies, including polymerase chain reaction (PCR).

[0143] A promoter is considered to be "operably linked" when it is in the correct functional location and orientation with respect to the nucleic acid it controls so as to control ("drive") the transcription initiation and / or expression of that nucleic acid.

[0144] The modified nucleic acid of the present disclosure may contain a constitutive promoter or an inducible promoter. A "constitutive promoter" refers to a promoter that is always active in a cell. An "inducible promoter" refers to a promoter that initiates or enhances transcriptional activity when in the presence, affected by, or in contact with an inducer or agent, or activated in the absence of a factor that causes repression. Inducible promoters for use according to the present disclosure include any inducible promoter described herein or known to those of skill in the art. Examples of inducible promoters include, without limitation, chemically / biochemically regulated and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters, steroid-regulated promoters, metal-regulated promoters, pathogenicity-regulated promoters, temperature / heat-inducible, phosphate-regulated (e.g., PhoA), and light-regulated promoters.

[0145] An inducer or inducer agent can be an endogenous or normally exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein that contacts an inducible promoter in such a way that it is active in controlling transcriptional activity from the inducible promoter. Thus, a "signal that controls transcription" of a nucleic acid refers to an inducer signal that acts on an inducible promoter. A signal that controls transcription can activate or inactivate transcription depending on the control system used. Activating transcription can involve acting directly on the promoter to drive transcription, or indirectly on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, deactivating transcription can involve acting directly on the promoter to prevent transcription, or indirectly on the promoter by activating a repressor that then acts on the promoter.

[0146] The modified nucleic acid can be introduced into the host cell using any means known in the art, including, without limitation, transformation, transfection (e.g., chemical (e.g., calcium phosphate, cationic polymers, or liposomes) or non-chemical (e.g., electroporation, sonoporation, impalefection, optical transfection, hydrodynamic transfection)), and transduction (e.g., viral-based transduction).

[0147] An enzyme or other protein encoded by a naturally occurring intracellular nucleic acid can be referred to as an "endogenous enzyme" or "endogenous protein." Cell cultures and cell lysates

[0148] Typically, the modified cells are cultured. "Culturing" refers to the process in which cells are grown under controlled conditions, typically outside their natural environment. For example, modified cells, such as modified bacterial cells, can be grown as a cell suspension in a liquid nutrient broth, also referred to as a liquid "culture medium."

[0149] Examples of commonly used Escherichia coli bacterial growth media include, without limitation, LB (Lysogeny Broth) Miller Broth (1% NaCl): 1% peptone, 0.5% yeast extract, and 1% NaCl; LB (Lysogeny Broth) Lennox Broth (0.5% NaCl): 1% peptone, 0.5% yeast extract, and 0.5% NaCl; SOB medium (Super Optimal Broth): 2% peptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4; SOC medium (Super Optimal Broth with catabolic repressors): SOB + 20 mM glucose; 2xYT broth (2x yeast extract and tryptone): 1.6% peptone, 1% yeast extract, and 0.5% NaCl; TB (Terrific Broth) medium: 1.2% peptone, 2.4% yeast extract, 72 mM K2HPO4, 17 mM KH2PO4, and 0.4% glycerol; and SB (Super Broth) medium: 3.2% peptone, 2% yeast extract, and 0.5% NaCl, and / or Korz medium (Korz, DJ et al. 1995).

[0150] Examples of high density Escherichia coli bacterial growth media include, but are not limited to, DNAGro™ medium, ProGro™ medium, AutoX™ medium, DetoX™ medium, InduX™ medium, and SecPro™ medium.

[0151] In some embodiments, the engineered cells are cultured under conditions that result in expression of the enzyme or nucleic acid. Such culture conditions can depend on the particular product to be expressed and the desired amount of the product.

[0152] In some embodiments, the modified cells are cultured at a temperature between 30° C. and 40° C. For example, the modified cells can be cultured at a temperature of 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., or 40° C. Typically, the modified cells, e.g., modified E. coli cells, are cultured at a temperature of 37° C.

[0153] In some embodiments, the modified cells are cultured for a period of 12 to 72 hours or more. For example, the modified cells can be cultured for a period of 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, or 72 hours. Typically, modified cells, such as modified bacterial cells, are cultured for a period of 12 to 24 hours. In some embodiments, the modified cells are cultured at a temperature of 37° C. for a period of 12 to 24 hours.

[0154] In some embodiments, the modified cells are cultured (e.g., in liquid cell culture medium) to an optical density measured at a wavelength of 600 nm (OD600) of 5 to 200. In some embodiments, the modified cells are cultured to an OD600 of 5, 10, 15, 20, 25, 50, 75, 100, 150, or 200.

[0155] In some embodiments, the modified cells are at least 1×10 8 (OD<1) to 2 × 10 11 The modified cells are cultured at a density of 1×10 (OD~200) viable cells / ml cell culture medium. 8 , 2×10 8 , 3×10 8 , 4×10 8 , 5×10 8 , 6×10 8 , 7×10 8 , 8×10 8 , 9×10 8 , 1×10 9 , 2×10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 1×10 10 , 2×10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , 9×10 10 , 1×1011 , or 2 × 10 11 Cultured to a density of 1000 viable cells / ml (conversion factor: OD1 = 8 x 10 8 cells / ml).

[0156] In some embodiments, the modified cells are cultured in a bioreactor. A bioreactor simply refers to a container in which cells are cultured, such as a culture flask, dish, or bag, which may be single-use (disposable), autoclavable, or sterilizable. Bioreactors may be made of glass, or may be based on polymers, or may be made of other materials.

[0157] Examples of bioreactors include, without limitation, stirred tank (e.g., complete mixing) and tubular (e.g., extrusion flow) bioreactors, airlift bioreactors, membrane stirred tanks, spin filter stirred tanks, vibromixers, fluidized bed reactors, and membrane bioreactors. The mode of operation of the bioreactor can be a batch or continuous process and will depend on the engineered cells to be cultured. A bioreactor is continuous when feed and product streams are continuously fed and removed from the system. Batch bioreactors may have a continuous recirculation flow, but there is no continuous feed of nutrients or product harvest. In intermittent harvest and fed-batch (or batch fed) cultures, cells are inoculated at a lower viable cell density in a medium whose composition is similar to the batch medium. The cells are allowed to grow exponentially essentially without external manipulation until nutrients are somewhat depleted and the cells approach a stationary growth phase. At this point, in an intermittent harvest batch fed process, a portion of the cells and product may be harvested and the removed culture medium is replenished with new medium. This process may be repeated several times. For the production of recombinant proteins and antibodies, a fed-batch process may be used. While the cells are growing exponentially but becoming depleted of nutrients, concentrated feed medium (e.g., 10-15 times concentrated basal medium) is added either continuously or intermittently to provide additional nutrients and allow a further increase in cell concentration and the length of the production phase. New medium may be added in proportion to the cell concentration without the removal of culture medium (broth). To accommodate the addition of medium, a fed-batch culture is started at a volume much lower than the full capacity of the bioreactor (e.g., approximately 40% to 50% of the maximum volume).

[0158] Some methods of the present disclosure are directed to large-scale production of RNA (e.g., ssRNA or dsRNA). In large-scale production methods, modified cells can be grown in liquid culture medium of 5 liters (L) to 250,000 L or more. In some embodiments, modified cells can be grown in liquid culture medium of more than (or equal to) 10 L, 100 L, 1000 L, 10000 L, or 100000 L. In some embodiments, modified cells are grown in liquid culture medium of 5 L, 10 L, 15 L, 20 L, 25 L, 30 L, 35 L, 40 L, 45 L, 50 L, 100 L, 500 L, 1000 L, 5000 L, 10000 L, 100000 L, 150000 L, 200000 L, 250000 L, or more. In some embodiments, the modified cells may be grown in a volume of liquid culture medium from 5L to 10L, 5L to 15L, 5L to 20L, 5L to 25L, 5L to 30L, 5L to 35L, 5L to 40L, 5L to 45L, 10L to 15L, 10L to 20L, 10L to 25L, 20L to 30L, 10L to 35L, 10L to 40L, 10L to 45L, 10L to 50L, 15L to 20L, 15L to 25L, 15L to 30L, 15L to 35L, 15L to 40L, 15L to 45L, or 15 to 50L. In some embodiments, the modified cells may be grown in volumes of liquid culture medium from 100 L to 300,000 L, 100 L to 200,000 L, or 100 L to 100,000 L.

[0159] Typically, culturing the modified cells is followed by lysing the cells. "Lysing" refers to a process in which cells are fragmented, for example, by viral, enzymatic, mechanical, or osmotic mechanisms. "Cell lysate" refers to a liquid containing the contents of a lysed cell (e.g., a lysed modified cell), including, for example, organelles, membrane lipids, proteins, nucleic acids, and inverted membrane vesicles. The cell lysates of the present disclosure can be produced by lysing any population of modified cells provided herein.

[0160] Methods of cell lysis, referred to as "lysing," are known in the art, any of which may be used in accordance with the present disclosure. Such cell lysis methods include, without limitation, physical lysis, such as homogenization.

[0161] Cell lysis can disrupt the tightly controlled intracellular environment, leading to unregulated protein degradation and modification by endogenous proteases and phosphatases. Therefore, in some embodiments, protease and / or phosphatase inhibitors can be added to cell lysates or cells prior to lysis, or their activity can be removed by heat inactivation, genetic inactivation, or protease targeting.

[0162] In some embodiments, cell lysate can be combined with at least one nutrient.For example, cell lysate can be combined with Na2HPO4, KH2PO4, NH4Cl, NaCl, MgSO4, CaCl2.Other examples of nutrients include, without limitation, magnesium sulfate, magnesium chloride, magnesium orotate, magnesium citrate, monobasic potassium phosphate, dibasic potassium phosphate, tribasic potassium phosphate, monobasic sodium phosphate, dibasic sodium phosphate, tribasic sodium phosphate, monobasic ammonium phosphate, dibasic ammonium phosphate, ammonium sulfate, ammonium chloride, and ammonium hydroxide.

[0163] In some embodiments, the cell lysate can be combined with at least one cofactor. For example, the cell lysate can be combined with adenosine diphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD+), or other non-protein chemical compounds required for the activity of the enzyme (e.g., inorganic ions and coenzymes).

[0164] In some embodiments, the cell lysate is incubated under conditions that result in RNA depolymerization, hi some embodiments, the cell lysate is incubated under conditions that result in the production of ssRNA or dsRNA.

[0165] The volume of cell lysate used in a single reaction can vary. In some embodiments, the volume of cell lysate ranges from 0.001 to 250 ml. 3 For example, the volume of a cell lysate is 0.001 m 3 , 0.01m 3 , 0.1m 3 , 1m 3 , 5m 3 , 10m 3 , 15m 3 , 20m 3 , 25m 3 , 30m 3 , 35m 3 , 40m 3 , 45m 3 , 50m 3 , 55m 3 , 60m 3 , 65m 3 , 70m 3 , 75m 3 , 80m 3 , 85m 3 , 90m 3 , 95m 3 , 100m 3 , 105m 3 , 110m 3 , 115m 3 , 120m 3 , 125m 3 , 130m 3 , 135m 3 , 140m 3 , 145m 3 , 150m 3 , 155m 3 , 160m 3 , 165m 3 , 170m 3 , 175m 3 , 180m 3 , 185m 3 , 190m 3 , 195m 3 , 200m3 , 205m 3 , 210m 3 , 215m 3 , 220m 3 , 225m 3 , 230m 3 , 235m 3 , 240m 3 , 245m 3 , or 250m 3 In some embodiments, the volume of the cell lysate is 25 ml. 3 250m from 3 , 50m 3 250m from 3 , or 100m 3 250m from 3 It is. Downstream Processing

[0166] The methods and systems provided herein, in some embodiments, produce an RNA (e.g., dsRNA, ssRNA) product at a concentration of 1-50 g / L (e.g., 30, 35, 40, 45, or 50 g / L). Downstream processing increases the purity to as much as 99% (e.g., 75, 80, 85, 90, 95, 96, 97, 98, or 99%) dsRNA by weight. An example of downstream processing is shown in FIG. 4, starting with the addition of a protein precipitant (e.g., ammonium acetate), followed by disk centrifugation (DSC) to remove proteins, lipids, and some DNA from the product stream. Ultrafiltration is then performed to remove salts and volume. Addition of lithium chloride to the product stream leads to precipitation of the RNA product, which is then separated from the bulk liquid using a disk centrifuge, yielding an RNA product stream of, for example, ∼80% purity. Further chromatographic polishing produces a ∼99% pure product. Additional Aspects

[0167] Additional aspects of the present disclosure are contained in the following numbered paragraphs 1-46: 1. A cell-free method for biosynthesizing ribonucleic acid (RNA), the method comprising: (a) lysing a culture of engineered cells containing RNA, an enzyme that depolymerizes RNA, a thermostable kinase, and a thermostable RNA polymerase, thereby producing a cell lysate; (b) incubating the cell lysate produced in step (a) under conditions that result in depolymerization of RNA, thereby producing a cell lysate that includes nucleoside monophosphates; (c) heating the cell lysate produced in step (b) to a temperature that inactivates endogenous nucleases and phosphatases without inactivating thermostable kinases and thermostable RNA polymerases, thereby producing a cell lysate containing heat-inactivated nucleases and phosphatases; (d) incubating the cell lysate produced in (c) in the presence of an energy source and a modified DNA template containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest under conditions conducive to the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates, thereby producing a cell lysate containing the RNA of interest; Includes. 2. The method of paragraph 1, wherein the energy source is polyphosphate, polyphosphate kinase, or both polyphosphate and polyphosphate kinase. 3. The method of paragraph 1 or 2, wherein the cultured modified cells contain a modified DNA template. 4. The method of paragraph 1 or 2, wherein a modified DNA template is added to the cell lysate in step (d). 5. The method of any one of paragraphs 1-4, wherein an ATP regenerating system is added to the cell lysate of step (d). 6. The method of paragraph 1, wherein the cultured modified cells further comprise a thermostable polyphosphate kinase. 7. The method of any one of paragraphs 1 to 6, wherein the RNA of the modified cell in step (a) is endogenous RNA. 8. The method of any one of paragraphs 1-7, wherein the RNA comprises ribosomal RNA, messenger RNA, transfer RNA, or a combination thereof. 9. The method of any one of paragraphs 1-8, wherein the cultured modified cells comprise at least two enzymes that depolymerize RNA. 10. The method of any one of paragraphs 1-9, wherein the enzyme that depolymerizes RNA is selected from the group consisting of: S1 nuclease, nuclease P1, RNase II, RNase III, RNase R, RNase JI, NucA, PNPase, RNase T, RNase E, RNaseG, and combinations thereof. 11. The method of paragraph 10, wherein the RNA depolymerizing enzyme is nuclease P1. 12. The method of any one of paragraphs 1 to 11, wherein the cell lysate of step (b) is 2+ Contains a chelating agent. 13. The method of paragraph 12, further comprising: 2+ The chelating agent is ethylenediaminetetraacetic acid (EDTA). 14. The method of paragraph 13, wherein the concentration of EDTA is from 0.1 mM to 25 mM. 15. The method of paragraph 14, wherein the concentration of EDTA is 8 mM. 16. The method of any one of paragraphs 1-15, wherein the thermostable kinase comprises a thermostable nucleoside monophosphate kinase. 17. The method of paragraph 16, wherein the thermostable nucleoside monophosphate kinase is selected from the group consisting of thermostable uridylate kinase, thermostable cytidylate kinase, thermostable guanylate kinase, and thermostable adenylate kinase. 18. The method of paragraph 17, wherein the stable nucleoside monophosphate kinase is selected from the group consisting of a thermostable Pyrococcus furiosus uridylate kinase (PfPyrH) encoded by the pyrH gene, a thermostable Thermus thermophilus adenylate kinase (TthAdk) encoded by the adk gene, a thermostable Thermus thermophilus cytidylate kinase (TthCmk) encoded by the cmk gene, and a thermostable Pyrococcus furiosus guanylate kinase (PfGmk) encoded by the gmk gene. 19. The method of any one of paragraphs 1-18, wherein the thermostable kinase comprises a thermostable nucleoside diphosphate kinase. 20. The method of paragraph 19, wherein the thermostable nucleoside diphosphate kinase is selected from the group consisting of a thermostable nucleoside phosphate kinase, a thermostable pyruvate kinase, and a thermostable polyphosphate kinase. 21. The method of paragraph 20, wherein at least one of the thermostable nucleoside diphosphate kinases is a thermostable Aquifex aeolicus enzyme encoded by an ndk gene. 22. The method of any one of paragraphs 1-21, wherein the cell contains a thermostable nucleoside monophosphate kinase and a thermostable nucleoside diphosphate kinase. 23. The method of any one of paragraphs 1-22, wherein the cultured modified cells comprise a thermostable uridylate kinase, a thermostable cytidylate kinase, a thermostable guanylate kinase, a thermostable adenylate kinase, and a thermostable polyphosphate kinase. 24. The method of any one of paragraphs 1 to 23, wherein the thermostable RNA polymerase is a thermostable DNA-dependent RNA polymerase. 25. The method of paragraph 24, wherein the DNA-dependent RNA polymerase is selected from the group consisting of thermostable T7 RNA polymerase, thermostable SP6 RNA polymerase, and thermostable T3 RNA polymerase. 26. The method of paragraph 25, wherein the DNA-dependent RNA polymerase is a thermostable T7 RNA polymerase. 27. The method of any one of paragraphs 1 to 26, wherein the temperature in step (c) is at least 50°C. 28. The method of paragraph 27, wherein the temperature in step (c) is 50°C to 80°C. 29. The method of any one of paragraphs 1-28, wherein step (c) comprises heating the cell lysate for at least 15 minutes. 30. The method of any one of paragraphs 1-29, wherein step (c) comprises heating the cell lysate to a temperature of at least 65°C for 15 minutes. 31. The method of any one of paragraphs 1-30, wherein the nucleoside triphosphate in step (d) is produced at a rate of 15-30 mM / hour. 32. The method of any one of paragraphs 1 to 31, wherein the RNA of interest produced in step (d) is double-stranded RNA. 33. The method of any one of paragraphs 1 to 32, wherein the RNA of interest produced in step (d) is an RNA interference molecule. 34. The method of any one of paragraphs 1 to 33, wherein the RNA of interest produced in step (d) is an mRNA that contains complementary domains linked by a hinge domain. 35. The method of any one of paragraphs 1-34, wherein the RNA of interest produced in step (d) is produced at a concentration of at least 4 g / L, at least 6 g / L, at least 6 g / L, or at least 10 g / L. 36. The method of paragraph 35, further comprising purifying the double-stranded RNA. 37. The method of paragraph 36, wherein the purification step comprises combining the cell lysate of step (d) with a protein precipitant and removing precipitated proteins, lipids, and DNA. 38. A cell lysate produced by the method of any one of paragraphs 1 to 37. 39. A modified cell comprising RNA, an enzyme that depolymerizes RNA, a thermostable kinase, and a thermostable RNA polymerase. 40. The modified cell of paragraph 39, further comprising a modified DNA template containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest. 41. A population of modified cells of paragraph 39 or 40. 42. A method comprising: maintaining the modified cells of paragraph 39 in a cell culture medium. 43. The method of paragraph 42, further comprising lysing the cultured modified cells to produce a cell lysate. 44. The method of paragraph 43, further comprising incubating the cell lysate under conditions that result in depolymerization of RNA to produce a cell lysate that includes nucleoside monophosphates. 45. The method of paragraph 44, further comprising heating the cell lysate to a temperature that inactivates endogenous nucleases and phosphatases without inactivating the thermostable kinase and the thermostable RNA polymerase to produce a cell lysate containing heat-inactivated nucleases and phosphatases. 46. ​​The method of paragraph 45, further comprising incubating the cell lysate containing the heat-inactivated nuclease and phosphatase in the presence of an energy source and a modified DNA template containing a promoter operably linked to a nucleotide sequence encoding an RNA of interest under conditions resulting in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates to produce a cell lysate containing the RNA of interest. EXAMPLES

[0168] Example 1 Nuclease selection To identify the optimal nuclease(s) for digesting lysate RNA, a series of screening experiments was performed with commercially available enzymes selected based on their ability to generate 5'-NMPs or oligonucleotides. First, the activity of the enzymes was determined using purified E. coli RNA and reaction conditions recommended by the manufacturer, where RNA depolymerization was monitored by the release of acid-soluble nucleotides. Under these conditions, four nucleases demonstrated depolymerization activity above background. The endonucleases benzonase and RNase A, which served as positive controls, produced rapid conversion of RNA to acid-soluble nucleotides (Figure 5A). Treatment of RNA with exonuclease P1 and RNase R produced a time-dependent conversion of RNA to acid-soluble nucleotides, with RNase R reaching nearly 100% depolymerization in 2 h. The remaining nucleases (terminator exonuclease, RNase III, and RNase T) did not produce detectable depolymerization in this assay. Subsequent analysis by LC-MS revealed NMP release in samples treated with RNase R and Nuclease P1, but not with Benzonase or RNase A (Figure 5B). These results suggest that RNase R and Nuclease P1 may be suitable for depolymerizing lysate RNA into 5'-NMPs. RNase R was chosen for further study for several reasons, including its lack of DNAse activity, its ability to degrade dsRNA and structured RNA, and its processive 3'->5' exonuclease activity. RNA depolymerization in lysates

[0169] RNase R was then tested for its ability to depolymerize endogenous RNA in bacterial lysates. In these experiments, purified RNase R (0.5 mg / mL final concentration) was added to the lysates (50% final concentration) and free nucleotides were quantified by UPLC. A representative experiment is shown in Figure 6. Addition of purified RNase R to the lysates resulted in rapid release of 5'-NMP from the lysate RNA, with maximum NMP release after 5-10 min. After this initial period of rapid depolymerization, NMP concentrations stabilized and then began to decline slowly. Endogenous RNase activity also resulted in 5'-NMP release, albeit at a much slower rate. Importantly, consistent with its known mechanism of action, RNase R addition did not increase the rate of 2' or 3' NMP release from RNA. In multiple independent experiments, addition of RNase R to lysates resulted in the conversion of 68% of lysate RNA to 5'-NMP in 5-10 minutes at a rate exceeding 200 mM / hr.

[0170] To assess the toxicity of RNase R expression, two bacterial strains were constructed. One strain contained the parent strain (GL16-170) transformed with an empty protein expression vector, and the other contained GL16-170 transformed with the same protein expression vector encoding RNase R. Both strains were grown in 1 L bioreactors under batch conditions and the OD 600 Strains were induced at 0.5 C for 1 h and harvested prior to glucose exhaustion. Induction produced robust expression of RNase R (Figure 7A) without detectable changes in growth rate (Figure 7B). Upon lysis and 50% dilution, the RNase R-expressing strain exerted rapid depolymerization of RNA to acid-soluble nucleotides (Figure 7C), indicating that the overexpressed RNase R was functional. Notably, additional RNase R activity supplied by adding purified enzyme to the reaction did not increase the rate of depolymerization or the yield of acid-soluble nucleotides, suggesting that the overexpressed RNase R was fully active upon lysis and dilution.

[0171] Next, the activity of overexpressed RNase R was assessed in high-density lysates. Mg, which is known to stabilize ribosomal structure and protect rRNA from nucleases, was also detected. 2+ is also required (in low amounts) for RNase R activity. Thus, the depolymerization rate was measured in the presence of various concentrations of EDTA (Figure 8). Lysates from empty vector strains exhibited relatively slow depolymerization rates and were insensitive to EDTA, whereas lysates with overexpressed RNase R exhibited higher rates of depolymerization with increasing EDTA concentrations, with a maximum rate at 8 mM EDTA. 2+ Above 8 mM the depolymerization rate decreased due to inactivation of RNase R. Taken together, these results suggest that overexpressed RNase R is non-toxic and can be activated by lysis. NMP stability in lysate

[0172] After RNA depolymerization, the resulting NMP pool is progressively phosphorylated to NTPs before polymerization to dsRNA. Detrimental enzymatic activities, such as NMP degradation to nucleosides and subsequent hydrolysis to sugars and bases, negatively affect dsRNA yield. Therefore, the stability of individual NMPs was assessed in the lysate. Stability assessments were performed by adding isotopically labeled "heavy" NMPs (hAMP, hCMP, hUMP, and hGMP) to the lysate and quantifying their abundance over time using LC-MS (Figures 9A-9D, solid lines). In contrast to hAMP, which is relatively stable, hCMP, hUMP, and hGMP are actively degraded by the lysate with approximate half-lives (t 1 / 2 ).

[0173] One pathway for the metabolism of NMP is dephosphorylation to nucleosides. To assess whether dephosphorylation contributes to NMP degradation, stability assessments were repeated with the addition of inexpensive phosphatase inhibitors. Increasing concentrations of phosphate (PO4, 150 mM) and the structural mimetic orthovanadate (VO4, 10 mM) were preincubated with lysates prior to the addition of hNMP. Increasing the phosphate concentration stabilized hUMP (t 1 / 2 ≈60 min), whereas it had minimal effect on hCMP and hGMP (Figures 9A-9D, dotted lines). In contrast, orthovanadate inhibited hCMP (t 1 / 2 >>60 minutes), hUMP(t 1 / 2 ≒60 min), and hGMP (t 1 / 2 ≈45 min) (Figures 9A-9D, dotted lines). Taken together, these stability calculations define an overall NMP degradation rate of 13 mM / hr in the lysate, with 70% of the exogenously added NMP present after 15 min. Even in the absence of phosphatase inhibitors, the relatively low rate of hNMP consumption (13 mM / hr) compared with the rate of RNase R-dependent depolymerization (>200 mM / hr) suggested that NMP released from RNA should be available for polymerization into dsRNA. Development of heat inactivation

[0174] A heat inactivation protocol was developed to stabilize NMPs, as well as NDPs, NTPs, and dsRNA. The objective was to identify the lowest temperature and shortest incubation time that would eliminate nucleotide and RNA degradation activity in the lysate. To assess the efficacy of heat inactivation, the NTP consumption rate (at 37°C) was compared by LC-MS between heat-inactivated lysates and varying heat inactivation time and temperature. Before heat inactivation, the lysate consumed NTPs at approximately 120 mM / hr (Figure 10). Temperatures below 70°C did not affect NTPase activity, while incubation at 70°C caused a time-dependent decrease in NTPase activity. Complete inhibition of NTPase activity occurred after 15 min incubation at 70°C (Figure 10).

[0175] These conditions were then evaluated for their ability to stabilize NMPs and dsRNA in the lysate. To evaluate the effect of heat inactivation on NMP degradation, the lysate was treated with exogenous RNase R to release RNA and then subjected to heat inactivation at 70°C. After inactivation, the temperature was reduced to 37°C and the reaction was incubated for an additional 60 minutes. As shown in Figure 11A, treatment with RNase R for 5 minutes rapidly depolymerized the RNA. After heat inactivation and incubation at 37°C, the NMP concentration was unchanged, suggesting that the selected heat inactivation conditions stabilized NMPs.

[0176] Finally, these conditions were evaluated for their ability to stabilize reactants and products (including NTPs and dsRNA) of in vitro transcription reactions in lysates. First, lysates were preincubated at elevated temperatures for 15 minutes. The temperature was then lowered to 37°C, and transcription reactants (including exogenous NTPs, DNA template, and purified T7 RNA polymerase) were added. As shown in FIG. 11B, transcription reactions in lysates heat-inactivated at ≧70°C yielded RNA products qualitatively similar to the positive control reactions (performed in buffer). No RNA products were evident in reactions performed at 60°C.

[0177] Taken together, these results suggest that a 15 minute 70° C. incubation is sufficient to stabilize NMPs, NTPs, and dsRNA in cell-free reactions. Selection and evaluation of thermostable kinases

[0178] After heat inactivation, a series of kinase activities are required to sequentially phosphorylate the 5'-NMPs released from RNA to NTPs that can be polymerized to form dsRNA. Those kinases that phosphorylate NMPs and NDPs with high-energy phosphate groups from ATP must be sufficiently thermostable to remain active after high-temperature incubation and active enough to produce NTPs at high rates (21 mM / hr NTPs at 1 g dsRNA / L / hr). Enzymes from thermophilic organisms were chosen for evaluation based on literature reports of successful expression in E. coli and biochemical characterization of the recombinant enzymes (Table 9). [Table 9]

[0179] To evaluate the suitability of these enzymes for cell-free production of dsRNA, the enzymes were cloned into an E. coli protein expression vector with an N-terminal hexahistidine tag, overexpressed, and purified using immobilized metal affinity chromatography (IMAC). The activity of the purified enzymes was first quantified using a luciferase assay, in which ATP consumption served as a proxy for NMP and NDP phosphorylation. The assays were performed at various incubation temperatures to determine the optimal reaction temperature for each enzyme.

[0180] Expression of UMP kinase (encoded by the pyrH gene) from T. thermophilus and E. coli yielded insoluble protein under the induction and purification conditions tested. Purified PyrH from P. furiosus exerted a specific activity of approximately 2 μmol / min / mg protein, which was largely temperature independent (FIG. 12). Based on this specific activity, high-density cell lysates (90 g dcw / L) expressing PfPyrH at 1% total protein would phosphorylate UMP at a rate in excess of 50 mM / hr in the presence of excess ATP (Table 10). Because dsRNA production at 1 g / L / hr requires approximately 5.25 mM / hr of UMP kinase activity, PfPyrH was chosen for further evaluation. Table 10 shows the predicted PyrH rates in high-density (90 g dcw / L) lysates, assuming that PfPyrH constitutes 1% of the total lysate protein. [Table 10]

[0181] Expression of AMP kinase (encoded by the adk gene) from E. coli and T. thermophilus yielded soluble recombinant protein, whereas the Thermosynechococcus enzyme was insoluble under the expression and purification conditions tested. The purified E. coli enzyme was active at 37°C and 50°C, but did not exert detectable activity at higher temperatures (Figure 13). The T. thermophilus enzyme exerted higher specific activity than the E. coli enzyme at all temperatures tested, with an optimum activity of nearly 1 mM / min per mg enzyme at 70°C. This activity results in a predicted rate of AMP phosphorylation in lysates of over 250 mM / hr when the enzyme is expressed at 0.01% of total protein in high-density lysates (Table 11). TthAdk was chosen for further studies because approximately 5.25 mM / hr of AMP kinase activity is required to synthesize dsRNA at 1 g / L / hr. Table 11 shows the predicted Adk reaction rates in high-density (90 g dcw / L) lysates, assuming that TthAdk constitutes 0.01% of the total lysate protein. [Table 11]

[0182] Expression of CMP kinase (encoded by the cmk gene) from E. coli and P. furiosus produced insoluble protein, whereas expression of the T. thermophilus enzyme produced soluble protein under the conditions tested. T. thermophilus CMP kinase showed largely temperature-independent activity, although enzyme activity decreased slightly at temperatures above 60°C (Figure 14). Based on these results, expression of TthCmk in high-density lysate at 0.02% of total protein would produce CMP kinase activity of 30-45 mM / hr (temperature dependent, Table 12), well in excess of the target of 5.25 mM / hr. Thus, TthCmk was selected for further evaluation. Table 12 shows the predicted Cmk reaction rates in high-density (90 g dcw / L) lysate, assuming that TthCmk constitutes 0.02% of total lysate protein. [Table 12]

[0183] In contrast to the CMP kinases tested, expression of GMP kinases from E. coli, T. thermophilus, and T. maritima yielded soluble recombinant proteins. The E. coli enzyme had the highest specific activity tested at 37°C, but was less active at higher temperatures (Figure 14). Both the T. thermophilus and T. maritima enzymes exerted optimal activity at higher temperatures, while the T. maritima enzyme was more active at all temperatures tested. Based on the measured specific activity of TmGmk, expression in high density cell lysates at 0.1% of total protein would yield a predicted rate of over 60 mM / hr at 70°C in the presence of excess ATP, compared to a target rate of 5.25 mM / hr (Table 13). Thus, TmGmk was selected for further evaluation. Table 13 shows the predicted Gmk reaction rates in high density (90 g dcw / L) lysates, assuming that TmGmk constitutes 0.1% of the total lysate protein. [Table 13-1] [Table 13-2]

[0184] Unlike NMP kinases, which are largely specific for a single substrate, NDP kinases phosphorylate ADP, CDP, UDP, and GDP. To compare the NDP kinases, enzymes from the thermophiles T. thermophilus and A. aeolicus were cloned and expressed in E. coli. The T. thermophilus enzyme was insoluble under the conditions tested, whereas expression of the A. aeolicus enzyme yielded a soluble protein. Activity measurements in a luciferase assay using ATP and GDP as substrates revealed that AaNdk is highly active over a wide range of temperatures, with an optimum temperature of 50°C (Figure 16). Comparison between substrates revealed specific activities exceeding 100 μmol / min / mg at 50°C for each nucleotide, confirming that AaNdk can phosphorylate multiple NDP substrates (Table 14). Based on these measurements, expression of AaNdk in high density lysates at 0.01% total protein results in a UDP kinase rate well in excess of 60 mM / hr. Given that 21 mM / hr of NDP kinase activity is required to support 1 g / L / hr of dsRNA synthesis, AaNdk was chosen for further evaluation. Table 14 shows that at 50°C, AaNdk is highly active with CDP, UDP, and GDP substrates. [Table 14]

[0185] Polyphosphate kinase (Ppk) reversibly transfers high-energy phosphate groups between polymeric phosphate chains and adenosine nucleotides. Several Ppk enzymes belonging to the type I family of polyphosphate kinases, including enzymes from E. coli and the thermophiles T. thermophilus and Thermosynechococcus elongatus, were evaluated for activity. These enzymes were selected for testing because they belong to the well-characterized type I family or have been previously shown to be active. Expression and purification of the Ppk enzymes yielded soluble proteins, which were then tested for activity by a luciferase assay system using ADP and sodium hexametaphosphate as substrates. As shown in Figure 17, the specific activities of all tested Ppk enzymes were relatively low compared to the activities of other kinases. E. coli Ppk had the highest specific activity at lower temperatures (up to 60°C), while the Thermosynechococcus enzyme had the highest activity at 70°C. In addition, incubation of the E. coli enzyme under heat inactivation conditions (70°C for 15 min) resulted in irreversible inactivation (not shown). Based on the specific activity of the Thermosynechococcus enzyme, expression in high-density lysate at 2% of total protein would result in a predicted rate of 42 mM / hr at 70°C, matching the required ATP production rate to support 1 g / L / hr dsRNA synthesis (Table 15). However, cell-free dsRNA synthesis at lower temperatures (e.g., 50°C) would require higher expression of TePpk (greater than 4% total protein). Table 15 shows the predicted Ppk reaction rates in high-density (90 g dcw / L) lysate, assuming that TePpk constitutes 2% of total lysate protein. [Table 15]

[0186] After assessing each enzyme activity in the purified system, the enzymes were tested for activity in heat-inactivated lysates. Lysates expressing individual kinases were pre-incubated at 70°C for 15 minutes before adding substrate. As in the purified reactions, ATP consumption (for NMP and NDP kinases) or ATP production (for polyphosphate kinase) was quantified using a luciferase assay kit. As shown in Table 16 below, the rates of NMP and NDP phosphorylation significantly exceed the targets. [Table 16]

[0187] After confirming that the individual kinases were fully active in the lysate (with the exception of Ppk), the kinases were evaluated by the multienzyme system for their ability to convert NMPs to NTPs. In these studies, equal volumes of lysates expressing individual kinases (5) were combined, heat inactivated, and then assayed for ATP-dependent production of NTPs from an equimolar mix of NMPs by LC-MS. As shown in Table 17, the overall NTP production rates exceeded 24 mM / hr for UTP, CTP, and GTP, suggesting that a simple mixture of lysates can provide NTPs at a rate sufficient to support the synthesis of 1 g / L / hr dsRNA in the presence of sufficient ATP without any optimization of reaction conditions. [Table 17] RNA polymerase selection

[0188] Following depolymerization of RNA to NMPs and phosphorylation of NMPs to their corresponding NTPs, an RNA polymerase is required to convert the NTPs to dsRNA products. RNA polymerase from bacteriophage T7 is an attractive enzyme for use in recombinant systems for several reasons. T7 RNA polymerase (unlike many RNA polymerases from bacteria and eukaryotes) contains a single subunit and has been extensively characterized by biochemical and molecular biology studies. In addition, several T7 RNA polymerase mutations that confer improved thermostability have been described (see Table 18). [Table 18]

[0189] First, the activity of commercially available T7 RNA polymerase was assessed using a double-stranded DNA template (e.g., Figure 3B) and a reaction temperature of 37°C. RNA production was quantified over time using the Quant-iT RNA Kit (Broad Range) (Thermo Fisher Scientific). Under the conditions recommended by each manufacturer, ThermoT7 and MegaScript enzymes were highly active, while the NEB enzyme displayed significantly lower activity (Figure 18).

[0190] Next, the LVI mutant of T7 RNA polymerase was cloned with an N-terminal hexahistidine tag, expressed in E. coli, and purified using IMAC.

[0191] Next, the activity of MegaScript and ThermoT7 polymerases was tested side-by-side with the LVI mutant polymerase in diluted heat-inactivated lysate (35% lysate final concentration) under standardized reaction conditions (Figure 19). As in the purified system, ThermoT7 showed higher specific activity than the MegaScript enzyme at 37°C (3.1 nmol RNA / min / mg protein). The LVI mutant had the lowest specific activity (0.33 nmol / min / mg) of the three enzymes tested. When tested at 50°C under otherwise identical conditions, neither the MegaScript enzyme nor the LVI mutant exerted any detectable activity. In contrast, ThermoT7 activity was higher at 37°C. With ThermoT7 polymerase making up approximately 4% of the total protein in the assay, the rate of RNA synthesis exceeded 11 g / L / hr at 50°C with a double-stranded DNA template and ThermoT7 polymerase (Figure 19). Therefore, ThermoT7 was selected for further characterization.

[0192] After confirming the activity of ThermoT7 at 50°C in diluted heat-inactivated lysate, the activity of ThermoT7 was examined under conditions representative of large-scale cell-free dsRNA production. In addition to increased lysate concentration, large-scale reactions may contain precipitated lysate components (e.g., proteins) resulting from the heat inactivation process. To assess the performance of ThermoT7 under these conditions, RNA polymerization was quantified in heat-inactivated high-density lysate (68% lysate final concentration) with and without clarification to remove precipitated proteins after heat inactivation (Figure 20). RNA synthesis rates were significantly higher in matrix than in buffer, with the highest rates occurring in heat-inactivated matrix clarified by centrifugation. In unclarified reactions, the overall RNA synthesis rate (from a 2-hour reaction) exceeded 2 g / L / hr with ThermoT7 polymerase representing 1.4% of the total protein in the assay.

[0193] Finally, the thermostability of ThermoT7 was tested at higher temperatures to assess its compatibility with the heat inactivation conditions previously established in this program. In these studies, ThermoT7 enzyme was preincubated at elevated temperatures (50-70°C) for various lengths of time (0-15 min) and the remaining activity was quantified at 37°C. As shown in Figure 21, incubation at 50°C is well tolerated by the enzyme, while higher temperatures result in rapid irreversible inactivation of the polymerase activity. Thus, in these experiments, this particular Thermo T7 was not compatible with heat inactivation at 50-70°C. Therefore, in some cases, purified ThermoT7 enzyme can be added to the cell-free reaction after heat inactivation, or an alternative T7 RNA polymerase variant with sufficient half-life at, for example, 70°C can be used. material and method Nucleotide analysis

[0194] Analysis of nucleotide monophosphates (AMP, CMP, UMP, and GMP) was performed by liquid chromatography-mass spectrometry (LC-MS). Samples were separated using an Agilent 1100 series HPLC equipped with a ZIC-cHILIC column (2.1 × 20 mm, 3 μm id) (Merck) at room temperature with a flow rate of 0.5 mL / min and a 2 μL injection volume. The mobile phase consisted of 20 mM ammonium acetate (A) and 20 mM ammonium acetate (B) in 90% acetonitrile. The separation method consisted of a gradient of 15-50% (B) for 3.5 min, followed by 50% (B) for 1.5 min, then 15% B for 3 min. Quantification was performed by an ABSciex API 3200 mass spectrometer in multiple reaction monitoring (MRM) mode with electrospray ionization (capillary voltage: -3000 V, temperature: 600 °C, desolvation gas: 20 psi). Analysis of nucleotide monophosphate, diphosphate, and triphosphate species (NMPs, NDPs, and NTPs) used the method described above with the following separation gradient: 15% B to 50% B in 3.5 min, followed by 50% B for 2.5 min, then 15% B for 4 min. Peak areas were compared to a standard curve consisting of purified compounds (Sigma-Aldrich). For analysis of samples in lysate, a standard curve was generated in an acid quenched, clarified, pH neutralized, and filtered lysate background as per the sample preparation steps described below.

[0195] Analysis of 2', 3', and 5'-NMP was performed by liquid chromatography using an ACQUITY H-class UPLC (Waters) equipped with an ACQUITY CSH Fluorophenyl column (2.1 × 150 mm, 1.7 μm id) (Waters) at 40 °C with a flow rate of 0.5 mL / min and 0.5 μL injection volume. The mobile phase consisted of 10 mM ammonium acetate in 0.2% formic acid (A) and 95% acetonitrile, 10 mM ammonium acetate in 0.2% formic acid (B). The separation method consisted of 1% B for 2.8 min, followed by a gradient from 1% to 30% B in 2.2 min, 100% B for 7 min, then 1% B for 3 min. Quantification was performed at 260, 254, and 210 nm using an ACQUITY UPLC PDA (Waters). Peak areas were compared to standard curves consisting of purified compounds (purchased from Sigma-Aldrich, with the exception of 2' and 3' CMP, UMP, and GMP, which were purchased from Biolog Life Science Institute). For analysis of samples in lysate, standard curves were generated in acid quenched, clarified, pH neutralized, and filtered lysate background as per the sample preparation steps described below. E. coli RNA extraction and purification

[0196] RNA was extracted and purified from high-density E. coli lysates (protein concentration: 40–50 mg / mL) according to established protocols (Mohanty, BK, Giladi, H., Maples, VF, & Kushner, SR (2008). Analysis of RNA decay, processing, and polyadenylation in Escherichia coli and other prokaryotes. Methods in enzymology, 447, 3-29). For every 400 μL of frozen E. coli lysate, 67 μL of 20 mM acetic acid was added to reduce RNase activity. Samples were thawed at 37°C in a bead bath. Immediately after thawing, 400 μL of a 10% (w / v) solution of trimethyl(tetradecyl)ammonium bromide (Sigma-Aldrich) was added. The resulting suspension was then clarified by centrifugation at 10,000 × g in a microcentrifuge at 4°C, and the supernatant was removed. The pellet was resuspended in 1 mL of a 2 M solution of lithium chloride (Sigma-Aldrich) in 35% ethanol. The suspension was incubated for 5 min at room temperature and then clarified by centrifugation at 15,000×g for 6 min at 4°C and the supernatant was removed. The pellet was then resuspended in 1 mL of a 2 M solution of lithium chloride in water and incubated for 2 min at room temperature before clarification at 15,000×g for 6 min. The supernatant was then removed and the remaining pellet was washed by resuspension in 70% ethanol and centrifugation at maximum speed (21,000×g) for 5 min at 4°C. The supernatant was then removed and the pellet was air-dried for 15 min at room temperature. The pellet was then resuspended in 200 μL nuclease-free water and incubated overnight at 4°C to solubilize the RNA. The RNA solution was clarified by centrifugation (maximum speed for 5 min at 4°C) and the supernatant containing the soluble RNA was transferred to a sterile RNase-free tube and stored at -20°C. Nuclease selection

[0197] Nucleases were obtained from the following commercial sources: Benzonase and Nuclease P1 were obtained from Sigma-Aldrich, RNase R, Terminator Exonuclease, and RNase III were obtained from Epicentre, RNase A was obtained from Thermo Fisher, and Exonuclease T was obtained from New England BioLabs. For screening assays, 1 μL of each enzyme solution was added to 100 μL of 2× assay buffer (100 mM potassium phosphate pH 7.4, 10 mM magnesium chloride, 1 mM zinc chloride) and then combined with an equal volume of 1 mM RNA solution (~340 ng / μL) at time t=0 and mixed well. Reactions were incubated at 37°C and sampled periodically by transferring 20 μL to acid quench solution (180 μL of 0.2 M sulfuric acid) on ice. After completion of the time series, quenched samples were clarified by centrifugation at 3,000×g for 5 min at 4°C. Then, 170 μL of the supernatant from each sample was transferred to a UV-transparent 96-well half-area plate (Corning) and the acid-soluble nucleotides were analyzed using a microplate reader and a 10665 M NMR spectroscopy estimated by averaging the individual extinction coefficients of each mononucleotide. -1 cm -1 Quantification was by absorbance at 260 nm with an extinction coefficient of 0.01 μL. For subsequent analysis by LC-MS, 45 μL of the clarified supernatant was pH neutralized with 5 μL of 2.5 M potassium hydroxide. Total nucleotide pool (i.e., 100% depolymerization) was determined by alkaline hydrolysis of RNA: RNA was combined with an equal volume of 0.2 M potassium hydroxide and then heated to 99° C. for 20 min. The alkaline hydrolyzed samples were then quenched and analyzed as described above. Protein expression and purification

[0198] Recombinant proteins were cloned into pETDuet-1 (Novagen) from synthetic DNA encoding the gene with a hexahistidine tag. Plasmids were transformed into E. coli T7Express (New England Biolabs) and then grown in 1 L cultures in ZYM-505 medium (Studier, FW (2005). Protein production by auto-induction in high-density shaking cultures. Protein expression and purification, 41(1), 207-234) supplemented with 50 μg / mL carbenicillin. Expression was confirmed by A 600= 0.6. For RNase R and kinase, expression was induced with 0.1 mM IPTG, the temperature was reduced to 16°C, and the cultures were grown at 16°C for 24 h. For T7 RNA polymerase, expression was induced with 0.8 mM IPTG, and the cultures were grown at 37°C for 3 h. Biomass was harvested by centrifugation, the supernatant was decanted, and the cell pellet was stored at -80°C. Cell pellets were thawed and lysed by resuspension in 4 volumes of B-PER Complete (Thermo Fisher Scientific) supplemented with benzonase (0.04 μL / mL) and incubation at room temperature for 15 min with gentle shaking. The lysate was then clarified by centrifugation at 16,000 × g for 1 h at 4°C. Proteins were purified by immobilized metal affinity chromatography using His GraviTrap columns (GE Healthcare) or HisTrap HP columns connected to an AKTAPrime Plus FPLC system (GE Healthcare). For both purification methods, the column was equilibrated with equilibration / wash buffer (50 mM phosphate buffer pH 7.4, 500 mM NaCl, 20 mM imidazole), the lysate was loaded, and then washed with 30 column volumes of equilibration / wash buffer. Proteins were eluted with elution buffer (50 mM phosphate buffer pH 7.4, 500 mM NaCl, 500 mM imidazole). For purification of kinases, the equilibration / wash and elution buffers were 50 mM Tris-HCl pH 7.5 instead of phosphate buffer. Elution fractions were analyzed by SDS-PAGE, and protein content was quantified by BCA (Thermo Fisher Scientific). Fractions were then combined and buffer exchanged by dialysis against 1000 volumes of 2× storage buffer. For RNase R, the 2x storage buffer consisted of 2x PBS with an additional 500 mM NaCl. For T7 RNA polymerase, the 2x storage buffer consisted of 2x PBS. For kinase, the 2x storage buffer consisted of 100 mM Tris-HCl pH 7.0 with 100 mM NaCl.After dialysis, the protein was mixed with an equal volume of 100% glycerol (50% final concentration) and stored at -20°C. Cell lysate preparation

[0199] E. coli strain GL16-170(BL21(DE3).t526pgi.Δedd.ΔtktB.ΔtolC_wt-7-E1.ΔmgsA*-F3.ΔappA*.Δamn*-F1.nag D(keio)::zeoR-1.ΔphoA*.t352BAA1644.ΔushA*-C4.rna::tolC-B04) and GL14-322(BL21(DE3).t526p gi.Δedd.ΔtktB.ΔtolC_wt-7-E1.ΔmgsA*-F3.ΔappA*.Δamn*-F1.nagD(keio)::zeoR-1.ΔphoA*.t352BAA1644.ΔushA::tolC-A01) was grown in a 10 L bioreactor in Korz medium to the end of the batch phase, then harvested by centrifugation and frozen at -80°C. The pellet was resuspended in 58.8 mM potassium phosphate dibasic to 10% dry cell weight and lysed using two passes through a PandaPLUS homogenizer (GEA Niro Soavi) cooled to 4°C at 15,000 psi. The lysate was clarified by centrifugation at 16,000 × g for 1 h at 4°C, and protein content was analyzed by BCA assay (Thermo Fisher) before storage at -80°C. Depolymerization of lysate RNA by exogenous RNase R

[0200] Reactions were initiated after pre-equilibration of GL16-170 lysate (protein content 34.5 mg / mL) and RNase R solution (1 mg / mL in 300 mM potassium phosphate buffer pH 7.4, 200 mM KCl, 2 mM MgCl) at 2°C. At time t=0, reactions were initiated by mixing 50 μL of E. coli lysate and 50 μL of RNase R solution and transferring to a pre-heated 37°C block. Reactions involving deoxycholate were assembled as described above, except that the lysate was pre-mixed with 0.2 volumes of 5× sodium deoxycholate solution in water and incubated at 2°C for 15 min before initiation. After initiation, reactions were incubated at 37°C and sampled periodically by transferring 10 μL to acid quench solution (90 μL of 0.2 M sulfuric acid) on ice. After completion of the time series, the quenched samples were clarified by centrifugation at 3,200×g for 10 min at 2° C. Depolymerization was first quantified by absorbance of acid-soluble nucleotides: 10 μL of the quenched and clarified reaction was added to 160 μL of 0.2 M sulfuric acid on a UV-transparent 96-well half-area plate (Corning). Acid-soluble nucleotides were quantified by absorbance at 260 nm using a microplate reader (see above). Depolymerization was also quantified by UPLC analysis of 5′, 2′, and 3′ NMPs: 30 μL of each acid-quenched sample was pH-neutralized by adding 10 μL of 1 M KOH and then passed through a 0.2 μm filter before UPLC analysis. Total nucleotide pools (i.e., 100% depolymerized) were determined by alkaline hydrolysis of lysate RNA: 50 μL lysate was combined with 150 μL of 0.2 M potassium hydroxide and then heated at 99° C. for 20 min. The alkaline hydrolyzed samples were then quenched and analyzed as described above. Depolymerization of RNA in lysates by overexpressed RNase R

[0201] E. coli strain GL16-170 was transformed with pETDuet-1, encoding the E. coli rnr gene with a C-terminal hexahistidine tag. This strain was grown in batch mode in Korz medium supplemented with 50 mg / L carbenicillin alongside GL16-170 transformed with empty pETDuet-1. The cultures were grown in A 600 The cultures were induced with 0.8 mM IPTG at 0.5 °C = 20 °C and an additional 10 g / L glucose was added at the time of induction. One hour after induction, biomass was harvested by centrifugation and frozen. Lysates were prepared from frozen biomass as described above (protein concentrations: 36.6 mg / mL for GL16-170 biomass with empty pETDuet-1; 53.2 mg / mL for GL16-170 with pETDuet-1 with cloned RNase R). Depolymerization in diluted lysates was calculated as described above for a 50% lysate final concentration in the reaction. Depolymerization in concentrated lysates was calculated by preincubating 9 volumes of lysate with 1 volume of 10x EDTA solution for 5 min at 2 °C. Reactions were then started by transferring to a preheated 37 °C block and sampling as described above. NMP stability calculation

[0202] Four volumes of GL14-322 lysate (protein concentration: 50.5 mg / mL) were combined with one volume of phosphatase inhibitor solution (final concentrations of 50 mM potassium phosphate pH 7.4, 150 mM potassium phosphate pH 7.4, or 10 mM sodium orthovanadate) on ice. 13 C 10 , 15 N55'-monophosphate, cytidine- 15 N3-5'-monophosphate, uridine- 15 N2-5'-monophosphate, and guanosine- 15An equimolar solution of N5-5'-monophosphate [Sigma-Aldrich], 25 mM each) was prepared in water. The lysate and NMP were equilibrated to 37°C for 10 min before initiating the reaction. To initiate the reaction, 90 μL lysate solution was added to 10 μL NMP solution and the reaction was mixed well. The reaction was monitored by sampling at the designated time points. Between samples, 12 μL of the reaction mixture was transferred to 108 μL of 0.2 M sulfuric acid on ice. The quenched reaction was then clarified by centrifugation, pH neutralized, and filtered for LC-MS analysis as described above. Development of heat inactivation

[0203] The GL14-322 lysate was aliquoted into five microcentrifuge tubes on ice and then transferred to a heat block equilibrated at the desired heat inactivation temperature. At the indicated times, the tubes were cooled on ice and then clarified by centrifugation (21,000 x g for 5 min) and the supernatant harvested. The supernatant from the heat inactivated lysate was equilibrated with an equimolar mixture of NTPs (Sigma-Aldrich, 25 mM each) for 10 min at 37°C. At time t = 0, 9 volumes of heat inactivated lysate supernatant were combined with 1 volume of NTP solution and the reaction was monitored by sampling into acid quench solution, pH neutralized, filtered, and analyzed by LC-MS as described above. For transcription reactions in lysate, 10 μL reactions were performed using the MegaScript T7 Transcription Kit (Thermo Fisher) according to the kit instructions, with the exception of a reduced amount of enzyme mix (5% of the final reaction volume) and inclusion of heat-inactivated lysate supernatant (40% of the final reaction volume). Positive control reactions were performed in MegaScript reaction buffer alone, while negative control reactions included lysate but omitted the enzyme mix. Reactions were analyzed by agarose gel electrophoresis stained with SYBR Safe (Invitrogen) alongside a 1 kb DNA ladder (New England Biolabs). Nucleotide kinase activity assay

[0204] Nucleotide kinases were assayed at various temperatures (37°C, 50°C, 60°C, 70°C, and 80°C) in a buffer consisting of 50 mM Tris-HCl pH 7.0, 4 mM MgSO4, 4 mM ATP, and 4 mM of the corresponding NMP or NDP. The reaction was started after pre-equilibration of the reaction buffer (1.2x concentrated) and enzyme solution (0.5 mg / mL) at the reaction temperature for 1 min. At time t=0, the reaction was started by mixing 80 μL reaction buffer with 20 μL enzyme. The reaction was monitored by sampling at the designated time points. Between samplings, 15 μL of the reaction mixture was transferred to 135 μL of 0.2 M sulfuric acid on ice. After completion of the reaction, the samples were pH neutralized with 1 M KOH as described above and then diluted 1:10 with ice-cold water. ATP in each sample was quantified using an ATP bioluminescence assay kit (Sigma-Aldrich) following the kit instructions. For reactions in lysate, the above protocol was modified as follows: lysate was aliquoted into individual reaction tubes and then heat inactivated by incubation at 70° C. for 15 minutes. Reaction buffer (2× concentrated) and heat inactivated lysate were pre-equilibrated to the reaction temperature, and reactions were initiated by combining equal volumes of lysate and reaction buffer. Reactions were sampled by quenching individual reaction tubes with 9 volumes of acid quench solution and then analyzed as described above.

[0205] To assay the combined (i.e., NMP to NTP) activity of the kinases in the lysates, lysates expressing each kinase individually were mixed in a 1:1 ratio, divided into 10 μL aliquots, and then heat inactivated as described above. Kinase activity was analyzed in a buffer consisting of 50 mM Tris-HCl pH 7.0, 16 mM MgSO4, 2 mM each nucleotide monophosphate (AMP, CMP, UMP, and GMP), and 16 mM ATP. Reactions were initiated by combining an equal volume of lysate with reaction buffer (2× concentrated), pre-equilibrated at the reaction temperature. Reactions were performed at 70° C. and sampled by quenching individual reaction tubes with 9 volumes of acid quench solution, then analyzed as described above. Polyphosphate kinase activity assay

[0206] Polyphosphate kinase was assayed at various temperatures (37°C, 50°C, 60°C, 70°C, and 80°C) in a buffer consisting of 50 mM Tris-HCl pH 7.0, 4 mM MgSO4, 25 mM (NH4)2SO4, 1 mM ADP, and 1 mM sodium hexametaphosphate. The reaction was started after pre-equilibration of the reaction buffer (1.2x concentrated) and enzyme solution (0.25 mg / mL) at the reaction temperature for 1 min. At time t=0, the reaction was started by mixing 80 μL reaction buffer with 20 μL enzyme. The reaction was monitored by sampling at the indicated time points. Between samplings, 15 μL of the reaction mixture was transferred to 135 μL of 0.2 M sulfuric acid on ice. After completion of the reaction, the samples were pH neutralized with 1 M KOH as described above and then diluted 1:10 with ice-cold water. ATP was quantified in each sample using an ATP bioluminescence assay kit (Sigma-Aldrich) following the kit instructions. For reactions in lysates, the above protocol was modified as follows: lysates were aliquoted into individual reaction tubes and then heat-inactivated by incubation at 70°C for 15 min. Reaction buffer (2x concentrated) and heat-inactivated lysate were pre-equilibrated to the reaction temperature, and reactions were initiated by combining equal volumes of lysate and reaction buffer. Reactions were sampled by quenching individual reaction tubes with 9 volumes of acid quench solution and then analyzed as described above. Reaction rates in lysates were calculated by subtracting the signal from a control lysate (without overexpressed polyphosphate kinase) under the same reaction conditions. Generation of transcription templates

[0207] Double-stranded DNA templates were prepared by PCR amplification of synthetic gBlock DNA (Integrated DNA Technologies). Reactions were purified and concentrated by isopropanol precipitation. RNA polymerase selection

[0208] Commercially available RNA polymerases were compared using conditions recommended by each manufacturer. Each 50 μL reaction consisted of 10× reaction buffer (supplied by the manufacturer), NTPs, DNA template (0.5 μg), and enzyme. For NEB T7 RNA polymerase, reactions contained 0.5 mM each NTP, 5 mM DTT, and 100 U enzyme. Reactions with ThermoT7 polymerase were identical except that DTT was omitted. Reactions with MegaScript T7 contained 7.5 mM each NTP and 5 μL enzyme mix. Enzyme concentrations were determined by BCA assay (Thermo Fisher). Reactions were monitored by sampling at the indicated time points. Between samples, 10 μL of the reaction mixture was transferred to 90 μL of RNA quenching solution (10 mM Tris-HCl pH 8.0, 5 mM EDTA) and stored on ice. RNA samples in the quench solution were quantified using the Quant-iT RNA Broad Range Assay Kit (Thermo Fisher) according to the kit instructions. A standard curve for quantification was constructed using serial dilutions of purified dsRNA, prepared and purified using the MegaScript kit according to the kit instructions. Reactions were qualitatively analyzed by agarose gel electrophoresis. RNA polymerase assay in lysates

[0209] GL14-322 lysates were heat inactivated as described previously and clarified by centrifugation. Each 20 μL reaction consisted of clarified lysate (7 μL), 10× cofactor solution (300 mM MgCl2, 20 mM spermidine), NTPs (7.5 mM each, prepared from pH-neutralized stock solutions), DNA template (0.6 μg), and enzyme (1 μL). Reactions were incubated at 37° C. or 50° C. for 1 hour and then quenched by adding 9 volumes of RNA quenching solution. The quenched reactions were further diluted 10-fold with quenching solution (final dilution: 100-fold). The diluted reactions were then quantified using the Quant-iT kit (see above). RNA produced by the reaction was calculated by subtracting the RNA quantified in the control reaction (RNA polymerase was omitted).

[0210] RNA polymerase assays in high density lysates were performed as described above with the following modifications. Each 100 μL reaction consisted of lysate (67.5 μL), 10× cofactor solution (300 mM MgCl2, 20 mM spermidine), NTPs (7.5 mM each, prepared from pH-neutralized stock solutions), DNA template (3 μg), and enzyme (10 μL). For reactions performed in unclarified reaction matrix, GL14-322 lysate (67.5 μL) was dispensed into individual reaction tubes and then heat inactivated as previously described. Additional reactants were added to the heat inactivated matrix and then mixed well by vortexing until homogeneous. For reactions performed in buffer, the 10× cofactor solution consisted of 300 mM MgCl2, 20 mM spermidine, and 400 mM DTT. Additionally, buffer-only reactions included 50 mM potassium phosphate pH 7.4. All reactions were incubated at 50°C for 2 hours. Samples from each reaction were quenched by adding 9 volumes of RNA Quench Solution and then clarified by centrifugation at maximum speed (21,000 x g) for 1 minute. Supernatants from these reactions were further diluted 40-fold with Quench Solution (final dilution: 400-fold) and then quantified using the Quant-iT kit (see above). Example 2

[0211] The thermostable PPK2 enzyme was codon-optimized for expression in E. coli, synthesized, and cloned into pETDuet-1 (Novagen). The plasmid was then transformed into GL16-170. To generate the control strain, the empty pETDuet-1 plasmid was transformed into GL16-170. After an overnight preculture in 5 mL lysogeny broth (LB), the cell density was increased to approximately OD 0.5. 600 The strain was grown in 1 L of LB at 37°C until it reached a final concentration of 0.25 mM. The culture was then briefly chilled on ice and PPK2 expression was induced by adding isopropylthiogalactopyranoside (IPTG) to a final concentration of 0.25 mM. After induction, the culture was grown at 20°C for approximately 16 hours. The culture was harvested by centrifugation and the cell pellet was stored at -80°C. Lysates were produced by thawing the frozen pellets and resuspending in two pellet volumes of 150 mM MOPS-NaOH pH 7 and homogenizing with four passes at 15,000 psi with an EmulsiFlex C3 homogenizer (Avestin). The lysates were then clarified by centrifugation at 15,000 x g for 1 hour at 4°C. Aliquots of the lysates were frozen at -80°C prior to use.

[0212] Then, the thermostable PPK2 activity in the heat-inactivated lysates was measured. The melted lysates expressing the PPK2 enzyme were first diluted 1:100 with the lysates prepared from the control strains, with the exception of the D. geothermalis PPK2 lysate, which was diluted 1:10. Pre-chilled solutions of manganese chloride (MnCl2) and sodium hexametaphosphate (HMP) were added to final concentrations of 10 mM and 1 mM, respectively. The lysates were then heat-inactivated by incubation in a pre-heated 70°C thermocycler for 15 min. The reaction was then initiated by mixing the heat-inactivated lysates with an equal volume of 2× reaction buffer consisting of 10 mM MnCl2, 2 mM adenosine diphosphate (ADP) or adenosine monophosphate (AMP), and 9 mM HMP. Reactions were incubated at 70°C and time points were taken by removing an aliquot of the reaction mixture and diluting with 9 parts quenching solution (200 mM H2SO4) on ice. An initial time point (t0) was taken by directly mixing the lysate with the quenching solution, storing the quenched lysate on ice for 15 min, and then adding 2x reaction buffer. At assay termination, the quenched time point solutions were clarified by centrifugation at 3,200 x g for 10 min. The supernatants from the quenched reactions were then pH neutralized by mixing 3 parts quenched reaction solution with 1 part neutralization solution (1 M KOH). The quenched and neutralized samples were then diluted 1:10 with water before quantification using an Adenosine 5'-triphosphate (ATP) Bioluminescence Assay Kit (Sigma-Aldrich cat#:FLAA) following the kit instructions. Initial reaction rates were calculated based on ATP accumulation in PPK2-containing reactions and by subtracting ATP concentrations from control lysates.

[0213] Five class III PPK2 enzymes demonstrated soluble expression, thermostability, and high reaction rates in lysates at 70° C. Representative expression and activity data are shown in Figures 22A-22B.

[0214] A summary of the expression and kinetic data for each enzyme tested is shown in Table 17. The C. aerophila, Roseiflexus, A. thermophila, and R. castenholzii enzymes rapidly converted AMP and ADP to ATP using HMP as a substrate. The D. geothermalis enzyme showed roughly 20× slower conversion rates for both AMP and ADP substrates than the other tested PPK2s. [Table 19-1] [Table 19-2] Example 3

[0215] Thermostable C. aerophila PPK2 was then used to provide ATP for cell-free production of dsRNA from NMP, ADP, and HMP. Cell-free dsRNA synthesis reactions were carried out with a mixture of six E. coli lysates individually overexpressing the kinases listed in Table 18. [Table 20]

[0216] First, the lysates listed in Table 18 were combined in equal volumes on ice. Pre-chilled solutions of manganese chloride (MnCl2), magnesium sulfate (MgSO4), and sodium hexametaphosphate (HMP) were added to final concentrations of 0-2.5 mM, 30 mM, and 1 mM, respectively. The lysate mixture was then heat inactivated by incubation in a pre-heated 70°C thermocycler for 15 minutes. To initiate the reaction, heat-inactivated lysate was combined with the following components: an equimolar mixture of nucleotide monophosphates (adenosine 5'-monophosphate, cytidine 5'-monophosphate, uridine 5'-monophosphate, and guanosine 5'-monophosphate; 2 mM each), 50 mM Tris pH 7.0, 30 mM MgSO4, 0-2.5 mM MnCl2; 1 mM adenosine 5'-diphosphate, 2 mM spermidine, 1.5 µg plasmid DNA template, and 3 µg thermostable T7 RNA polymerase (S430P, F849I, F880Y) in a total volume of 20 µL. As a control, dsRNA was synthesized from an equimolar mixture of 2 mM NTPs (lysate, ADP, and HMP were omitted). As an additional control, dsRNA was synthesized from an equimolar mixture of 2 mM NMP (PPK2 expression lysate, ADP, and HMP were omitted, but 8 mM ATP was included as an energy source). As a negative control, a duplicate reaction was performed in which the polymerase was omitted. All reactions were incubated at 50°C for 2 hours and then stopped by the addition of 9 volumes of TE+ buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA). Samples were mixed with an equal volume of 2x RNA loading dye (New England Biolabs) and heated to 70°C for 10 minutes, followed by agarose / TAE gel electrophoresis.

[0217] As shown in Figure 23, the desired dsRNA products were produced using NTPs in buffer (left lane), from NMP and ATP in a nucleotide kinase-expressing lysate (middle lane), and from NMP and HMP in a nucleotide kinase- and polyphosphate kinase-expressing lysate (right lane). Manganese chloride was not required for either reaction, and the C. aerophila enzyme used Mn as a cofactor. 2+ and Mg 2+ Therefore, it was demonstrated that Mn 2+ is not a priori required for acellular reactions containing C. aerophila PPK2.

[0218] As shown in Figure 24, the desired dsRNA products were produced using NTPs in buffer (left lane), from NMPs and ATP in nucleotide kinase-expressing lysate (middle lane), and from NMPs and HMPs in nucleotide kinase- and polyphosphate kinase-expressing lysate (right lane). dsRNA production from NMPs and HMPs did not require exogenous ADP or T. thermophilus AMP kinase. Thus, C. aerophila PPK2 can be used as part of a 5-kinase system to produce dsRNA from NMPs and HMPs by cell-free reactions. Other Aspects

[0219] In the claims, articles such as "a," "an," and "the" can mean one or more than one, unless indicated to the contrary or otherwise clear from the context. A claim or description including "or" between one or more members of a group is considered to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise involved in a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present in, used in, or otherwise involved in a given product or process. The invention includes embodiments in which more than one or all of the members of a group are present in, used in, or otherwise involved in a given product or process.

[0220] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim may be modified to include one or more limitations found in any other claim that is dependent on the same original claim. Where elements are submitted as lists, e.g., in Markush group format, each subgroup of elements is also disclosed, and any element(s) may be removed from the group. In general, where the invention or aspects of the invention are said to include certain elements and / or features, it should be understood that certain inventive embodiments or aspects of the invention consist of or consist essentially of such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set out herein one by one. It is also noted that the terms "comprise" and "contain" are intended to be open-ended, permitting the inclusion of additional elements or steps. Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can, in different embodiments of the invention, take any specific value or subrange within the stated range, down to 1 / 10 of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0221] This specification refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. In addition, any particular aspect of the present invention that is in the prior art may be expressly excluded from any one or more of the claims. Because such aspects are deemed to be known to those of skill in the art, they may be excluded even if the exclusion is not expressly set forth herein. Any particular aspect of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

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Nucleic Acids Res. 15:2653-2664. array E. coli RNase R MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGLRRRLR AMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYLSSEQMKTCIH GDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFVVPDDSRLSFDILIPP DQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGTGMAVDIALRTHEIPYIWPQA VEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGEDARDFDDAVYCEKKRGGGWRLWVAIA DVSYYVRPSTPLDREARNRGTSVYFPSQVIPMLPEVLSNGLCSLNPQVDRLCMVCEMTVS SKGRLTGYKFYEAVMSSHARLTYTKVWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKA REERGGISFESEEAKFIFNAERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEP ALFRIHDKPSTEAITSFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLL RSMKQAIYDPENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETG GYHYSMEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNMDERKI DFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFRGEKKTKPKAA KKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE (SEQ ID NO: 1) T. elongatus Ppk (PPK1) MPSAKSPRRKAPEPIDLDNPQYYFNRSLSWLEFNKRVLHEAYDPRTPLLERLKFMAIFSS NLDEFFMVRVAGLKQQVESGILQVGADGMPPAEQLQAVRQYLLPIVTEQHRYFDQELRAL LAKESIFLTRFNELTPEQQAYLNDYFQAQVFPVLTPLAVDPAHPFPYISSLSLNLAVLIR DPESGQERLARVKVPNQFPRFVALPQHLHSPQGVHWLGVPLEEIIAHNLSALFPGMEIEA YFAFRITRSADLELETDKADDLLIAIEQEIRKRRFGSVVRLEVQRGIPPLLRQTLMEEMD LEEIDVYELEGLLCLNDLFAFMGLPLPQFKDPEWQPQVPPSFQRVEERESMFDTSSEITT LGTDYWEAVANELFSLIREGDIIVHHPYHSFAATVQRFITLAAHDPQVLAIKITLYRTSG DSPIVSALIKAAENGKQVAVLVELKARFDEENNILWARKLEKVGVHVVYGVPGLKTHTKT VLVVRQEAGQIRRYVHIGTGNYNPKTASLYEDLGLFSCREELGADLSELFNVLTGYARQR DYRKLLVAPVTMRDRTLQLIYREIEHARNGQPARIIAKMNAITDTQVIRALYEASQAGVD IDLIIRGMCCLRPGVPGVSDRIRVISIIGRFLEHSRIFYFGNNGDPEYYIGSADWRSRNL DRRVEAITPIEDPAIQLELKERLEIMLADNRQAWELQPDGTYRQRQPAPGEAERGTHSVL MARTLKDVQGSH (SEQ ID NO: 2) P. furiosus Umk MRIVFDIGGSVLVPENPDIDFIKEIAYQLTKVSEDHEVAVVVGGGKLARKYIEVAEKFNSSE TFKDFIGIQITRANAMLLIAALREKAYPVVVEDFWEAWKAVQLKKIPVMGGTHPGHTTDAVA ALLAEFLKADLLVVITNVDGVYTADPKKDPTAKKIKKMKPEELLEIVGKGIEKAGSSSVIDP LAAKIIARSGIKTIVIGKEDAKDLFRVIKGDHNGTTIEP (SEQ ID NO: 3) T. thermophilius Cmk MRGIVTIDGPSASGKSSVARRVAAALGVPYLSSGLLYRAAAFLALRAGVDPGDEEGLLAL LEGLGVRLLAQAEGNRVLADGEDLTSFLHTPEVDRVVSAVARLPGVRAWVNRRLKEVPPP FVAEGRDMGTAVFPEAAHKFYLTASPEVRAWRRARERPQAYEEVLRDLLRRDERDKAQSA PAPDALVLDTGGMTLDEVVAWVLAHIRR (SEQ ID NO: 4) T. maritima Gmk MKGQLFVICGPSGAGKTSIIKEVLKRLDNVVFSVSCTTRPKRPHEEDGKDYFFITEEEFL KRVERGEFLEWARVHGHLYGTLRSFVESHINEGKDVVLDIDVQGALSVKKKYSNTVFIYV APPSYADLRERILKRGTEKEADVLVRLENAKWELMFMDEFDYIVVNENLEDAVEMVVSIV RSERAKVTRNQDKIERFKMEVKGWKKL (SEQ ID NO: 5) T. thermophilus Adk MDVGQAVIFLGPPGAGKGTQASRLAQELGFKKLSTGDILRDHVARGTPLGERVRPIMERG DLVPDDLILELIREELAERVIFDGFPRTLAQAEALDRLLSETGTRLLGVVLVEVPEEELV RRILRRAELEGRSDDNEETVRRRLEVYREKTEPLVGYYEARGVLKRVDGLGTPDEVYARI RAALGI (SEQ ID NO: 6) A. aeolicus Ndk MAVERTLIIVKPDAMEKGALGKILDRFIQEGFQIKALKMFRFTPEKAGEFYYVHRERPFF QELVEFMSSGPVVAAVLEGEDAIKRVREIIGPTDSEEARKVAPNSIRAQFGTDKGKNAIH ASDSPESAQYEICFIFSGLEIV (SEQ ID NO: 7) Meiothermus ruber DM 1279 PPK2 MGFCSIEFLMGAQMKKYRVQPDGRFELKRFDPDDTSAFEGGKQAALEALAVLNRRLEKLQEL LYAEGQHKVLVVLQAMDAGGKDGTIRVVFDGVNPSGVRVASFGVPTEQELARDYLWRVHQQV PRKGELVIFNRSHYEDVLVVRVKNLVPQQVWQKRYRHIREFERMLADEGTTILKFFLHISKD EQRQRLQERLDNPEKRWKFRMGDLEDRRLWDRYQEAYEAAIRETSTEYAPWYVIPANKNWYR NWLVSHILVETLEGLAMQYPQPETASEKIVIE (SEQ ID NO: 8) Meiothermus silvanus DSM 9946 PPK2 MAKTIGATLNLQDIDPRSTPGFNGDKEKALALLEKLTARLDELQEQLYAEHQHRVLVILQGM DTSGKDGTIRHVFKNVDPLGVRVVAFKAPTPPELERDYLWRVHQHVPANGELVIFNRSHYED VLVARVHNLVPPAIWSRRYDHINAFEKMLVDEGTTVLKFFLHISKEEQKKRLLERLVEADKH WKFDPQDLVERGYWEDYMEAYQDVLDKTHTQYAPWHVIPADRKWYRNLQVSRLLVEALEGLR MKYPRPKLNIPRLKSELEKM (SEQ ID NO: 9) Deinococcus geothermalis DSM 11300 PPK2 MQLDRYRVPPGQRVRLSNWPTDDDGGLSKAEGEALLPDLQQRLANLQERLYAESQQALLIVL QARDAGGKDGTVKHVIGAFNPSGVQVSNFKVPTEEERAHDFLWRIHRQTPRLGMIGVFNRSQ YEDVLVTRVHHLIDDQTAQRRLKHICAFESLLTDSGTRIVKFYLHISPEEQKKRLEARLADP SKHWKFNPGDLQERAHWDAYTAVYEDVLTTSTPAAPWYVVPADRKWFRNLLVSQILVQTLEE MNPQFPAPAFNAADLRIV (SEQ ID NO: 10) Thermosynechococcus elongatus BP-1 PPK2 MIPQDFLDEINPDRYIVPAGGNFHWKDYDPGDTAGLKSKVEAQELLAAGIKKLAAYQDVLYA QNIYGLLIIFQAMDAAGKDSTIKHVMSGLNPQACRVYSFKAPSAEELDHDFLWRANRALPER GCIGIFNRSYYEEVLVVRVHPDLLNRQQLPPETKTKHIWKERFEDINHYERYLTRNGILILK FFLHISKAEQKKRFLERISRPEKNWKFSIEDVRDRAHWDDYQQAYADVFRHTSTKWAPWHII PANHKWFARLMVAHFIYQKLASLNLHYPMLSEAHREQLLEAKALLENEPDED (SEQ ID NO: 11) Anaerolinea thermophila UNI-1 PPK2 MGEAMERYFIKPGEKVRLKDWSPDPPKDFEGDKESTRAAVAELNRKLEVLQERLYAERKHKV LVILQGMDTSGKDGVIRSVFEGVNPQGVKVANFKVPTQEELDHDYLWRVHKVVPGKGEIVIF NRSHYEDVLVVRVHNLVPPEVWKKRYEQINQFERLLHETGTTILKFFLFISREEQKQRLLER LADPAKHWKFNPGDLKERALWEEYEKAYEDVLSRTSTEYAPWILVPADKKWYRDWVISRVLV ETLEGLEIQLPPPLADAETYRRQLLEEDAPESR (SEQ ID NO: 12) Caldilinea aerophila OSM 14535 PPK2 MDVDRYRVPPGSTIHLSQWPPDDRSLYEGDKKQGKQDLSALNRRLETLQELLYAEGKHKVLI ILQGMDTSGKDGVIRHVFNGVNPQGVKVASFKVPTAVELAHDFLWRIHRQTPGSGEIVIFNR SHYEDVLVVRVHGLVPPEVWARRYEHINAFEKLLVDEGTTILKFFLHISKEEQRQRLLERLE MPEKRWKFSVGDLAERKRWDEYMAAYEAVLSKTSTEYAPWYIVPSDRKWYRNLVISHVIINA LEGLNMRYPQPEDIAFDTIVIE (SEQ ID NO: 13) Chlorobaculum tepidum TLS PPK2 MKLDLDAFRIQPGKKPNLAKRPTRIDPVYRSKGEYHELLANHVAELSKLQNVLYADNRYAIL LIFQAMDAAGKDSAIKHVMSGVNPQGCQVYSFKHPSATELEHDFLWRTNCVLPERGRIGIFN RSYYEEVLVVRVHPEILEMQNIPHNLAHNGKVWDHRYRSIVSHEQHLHCNGTRIVKFYLHLS KEEQRKRFLERIDDPNKNWKFSTADLEERKFWDQYMEAYESCLQETSTKDSPWFAVPADDKK NARLIVSRIVLDTLESLNLKYPEPSPERRKELLDIRKRLENPENGK (SEQ ID NO: 14) Oceanithermus profundus DSM 14977 PPK2 MDVSRYRVPPGSGFDPEAWPTREDDDFAGGKKEAKKELARLAVRLGELQARLYAEGRQALLI VLQGMDTAGKDGTIRHVFRAVNPQGVRVTSFKKPTALELAHDYLWRVHRHAPARGEIGIFNR SHYEDVLVVRVHELVPPEVWGRRYDHINAFERLLADEGTRIVKFFLHISKDEQKRRLEARLE NPRKHWKFNPADLSERARWGDYAAAYAEALSRTSSDRAPWYAVPADRKWQRNRIVAQVLVDA LEAMDPRFPRVDFDPASVRVE (SEQ ID NO: 15) Roseiflexus castenholzii DSM 13941 PPK2 MYAQRVVPGMRVRLHDIDPDANGGLNKDEGRARFAELNAELDVMQEELYAAGIHALLLILQG MDTAGKDGAIRNVMLNLNPQGCRVESFKVPTEEELAHDFLWRVHRVVPRKGMVGVFNRSHYE DVLVVRVHSLVPESVWRARYDQINAFERLLADTGTIIVKCFLHISKEEQEQRLLARERDVSK AWKLSAGDWRERAFWDDYMAAYEEALTRCSTDYAPWYIIPANRKWYRDLAISEALVETLRPY RDDWRRALDAMSRARRAELEAFRAEQHAMEGRPQGAGGVSRR (SEQ ID NO: 16) Roseiflexus sp. RS-1 PPK2 MHYAHTVIPGTQVRLRDIDPDASGGLTKDEGRERFASFNATLDAMQEELYAAGVHALLLILQ GMDTAGKDGAIRNVMHNLNPQGCRVESFKVPTEEELAHDFLWRVHKVVPRKGMVGVFNRSHY EDVLVVRVHSLVPEHVWRARYDQINAFERLLTDTGTIIVKCFLHISKDEQEKRLLAREQDVT KAWKLSAGDWRERERWDEYMAAYEEALTRCSTEYAPWYIIPANRKWYRDLAISEVLVETLRP YRDDWQRALDAMSQARLAELKAFRHQQTAGATRL (SEQ ID NO: 17) Truepera radiovictrix DSM 17093 PPK2 MSQGSAKGLGKLDKKVYARELALLQLELVKLQGWIKAQGLKVVVLFEGRDAAGKGSTITRIT QPLNPRVCRVVALGAPTERERTQWYFQRYVHHLPAAGEMVLFDRSWYNRAGVERVMGFCTEA EYREFLHACPTFERLLLDAGIILIKYWFSVSAAEQERRMRRRNENPAKRWKLSPMDLEARAR WVAYSKAKDAMFYHTDTKASPWYVVNAEDKRRAHLSCIAHLLSLIPYEDLTPPPLEMPPRDL AGADEGYERPDKAHQTWVPDYVPPTR (SEQ ID NO: 18)

Claims

1. 1. A cell-free method for biosynthesizing ribonucleic acid (RNA), the method comprising: (a) incubating a cell lysate mixture comprising RNA and at least one enzymatic activity selected from the group consisting of an enzymatic activity that depolymerizes RNA, a thermostable kinase activity, and a thermostable RNA polymerase activity under conditions that result in depolymerization of the RNA to produce a cell lysate mixture comprising nucleoside monophosphates; (b) heating the cell lysate mixture produced in step (a) to a temperature that inactivates or partially inactivates endogenous nucleases and phosphatases without completely inactivating the thermostable kinase activity and the thermostable RNA polymerase activity to produce a cell lysate mixture containing heat-inactivated nucleases and phosphatases; and (c) incubating the cell lysate mixture produced in (b) in the presence of an energy source and a deoxyribonucleic acid (DNA) template encoding the RNA of interest under conditions conducive to the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates to produce a cell lysate mixture containing the RNA of interest; Including, The method.

2. The method of claim 1, wherein the cell lysate mixture comprises a single cell lysate obtained from a cell that contains RNA and expresses at least one enzyme or fusion enzyme that acts as a ribonuclease, as a kinase, and / or as an RNA polymerase.

3. 2. The method of claim 1, wherein the cell lysate mixture comprises at least two cell lysates, at least one cell lysate being obtained from a cell containing RNA and at least one cell lysate being obtained from a cell expressing at least one enzyme or fusion enzyme acting as a nuclease, as a kinase and / or as an RNA polymerase.

4. The method of any one of claims 1 to 3, wherein the RNA in step (a) is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA).

5. The method according to any one of claims 1 to 4, wherein the cell lysate mixture comprises at least one ribonuclease, at least one thermostable kinase, and / or at least one thermostable RNA polymerase.

6. 6. The method of claim 5, wherein the at least one ribonuclease is selected from the group consisting of S1 nuclease, nuclease P1, RNase II, RNase III, RNase R, RNase JI, NucA, PNPase, RNase T, RNase E, and RNase G.

7. The method of claim 6 , wherein the at least one ribonuclease is Escherichia coli RNase R.

8. The method according to any one of claims 5 to 7, wherein the at least one thermostable kinase is selected from the group consisting of a thermostable nucleoside monophosphate kinase, a thermostable nucleoside diphosphate kinase, and a thermostable polyphosphate kinase.

9. 9. The method of claim 8, wherein the heat-stable nucleoside monophosphate kinase is selected from the group consisting of heat-stable uridylate kinase, heat-stable cytidylate kinase, heat-stable guanylate kinase, and heat-stable adenylate kinase.

10. 10. The method of claim 9, wherein the thermostable nucleoside monophosphate kinase is selected from the group consisting of thermostable Pyrococcus furiosus uridylate kinase (PfPyrH) encoded by the pyrH gene, thermostable Thermus thermophilus adenylate kinase (TthAdk) encoded by the adk gene, thermostable Thermus thermophilus cytidylate kinase (TthCmk) encoded by the cmk gene, and thermostable Thermotoga maritima guanylate kinase (TmGmk) encoded by the gmk gene.

11. The method according to any one of claims 8 to 10, wherein the thermostable nucleoside diphosphate kinase is selected from the group consisting of thermostable nucleoside diphosphate kinases encoded by the Aquifex aeolicus ndk gene.

12. 11. The method according to any one of claims 8 to 10, wherein the thermostable polyphosphate kinase is selected from the group consisting of thermostable polyphosphate kinase 1 (PPK1) enzyme and thermostable polyphosphate kinase 2 (PPK2) enzyme.

13. 13. The method of claim 12, wherein the thermostable PPK1 enzyme is selected from the group consisting of thermostable Thermosynechococcus elongatus PPK1 enzymes.

14. 14. The method of claim 12 or 13, wherein the thermostable PPK2 enzyme is selected from the group consisting of thermostable Class III PPK2 enzymes.

15. 15. The method of claim 14, wherein the thermostable Class III PPK2 enzyme is selected from the group consisting of Meiothermus ruber, Meiothermus silvanus, Deinococcus geothermalis, Thermosynechococcus elongates, Anaerolinea thermophile, Caldilinea aerophila, Chlorobaculum tepidum, Oceanithermus profundus, Roseiflexus castenholzii, Roseiflexus sp., and Truepera radiovctrix PPK2 enzymes.

16. 16. The method of claim 15, wherein the thermostable Class III PPK2 enzyme is selected from the group consisting of thermostable Class III PPK2 enzymes comprising an amino acid sequence that is at least 70% identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18.

17. 17. The method of claim 16, wherein the thermostable Class III PPK2 enzyme is selected from the group consisting of thermostable Class III PPK2 enzymes comprising an amino acid sequence identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18.

18. 18. The method of any one of claims 1 to 17, wherein the cell lysate mixture comprises at least one ribonuclease, at least one thermostable nucleoside monophosphate kinase, at least one thermostable nucleoside diphosphate kinase, and at least one polyphosphate kinase.

19. The method according to any one of claims 5 to 18, wherein the at least one thermostable RNA polymerase is selected from the group consisting of thermostable DNA-dependent RNA polymerases.

20. 20. The method of claim 19, wherein the thermostable DNA-dependent RNA polymerase is selected from the group consisting of thermostable T7 RNA polymerase, thermostable SP6 RNA polymerase, and thermostable T3 RNA polymerase.

21. 21. The method of claim 20, wherein the at least one thermostable RNA polymerase is a thermostable T7 RNA polymerase.

22. 22. The method of any one of claims 1 to 21, wherein the energy source is adenosine triphosphate (ATP).

23. The method according to any one of claims 1 to 21, wherein the energy source is an ATP regeneration system.

24. 24. The method of claim 23, wherein the ATP regeneration system comprises polyphosphate, optionally hexametaphosphate, nucleoside monophosphate, and polyphosphate kinase.

25. 25. The method of any one of claims 1 to 24, wherein an energy source, or at least one component of an energy source, is added to the cell lysate mixture of step (c).

26. 26. The method of any one of claims 1 to 25, wherein at least one purified enzyme or fusion enzyme is added to the cell lysate mixture of step (a), the at least one purified enzyme or fusion enzyme having an enzymatic activity selected from the group consisting of an RNA depolymerizing enzyme activity, a thermostable kinase activity, and a thermostable RNA polymerase activity under conditions that result in depolymerization of RNA to produce a cell lysate mixture comprising nucleoside monophosphates.

27. 27. The method of any one of claims 1 to 26, wherein at least one purified enzyme or fusion enzyme is added to the cell lysate mixture of step (c), the at least one purified enzyme or fusion enzyme having an enzymatic activity selected from the group consisting of an RNA depolymerizing enzymatic activity, a thermostable kinase activity, and a thermostable RNA polymerase activity under conditions that result in depolymerization of RNA to produce a cell lysate mixture comprising nucleoside monophosphates.

28. The method of any one of claims 1 to 27, wherein the cell lysate mixture of step (a) comprises a DNA template encoding the RNA of interest.

29. The method of any one of claims 1 to 27, wherein a DNA template encoding the RNA of interest is added to the cell lysate mixture of step (c).

30. The method of any one of claims 1 to 29, wherein the RNA of interest is single-stranded RNA.

31. 31. The method of claim 30, wherein the single-stranded RNA is messenger RNA (mRNA).

32. 31. The method of claim 30, wherein the single-stranded RNA is an antisense RNA.

33. The method of any one of claims 1 to 29, wherein the RNA of interest is double-stranded RNA.

34. 34. The method of claim 33, wherein the double-stranded RNA is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA).

35. The method of any one of claims 1 to 29, wherein the RNA of interest is a single-stranded RNA containing complementary domains linked to each other by a hinge domain.

36. The cell lysate mixture of step (a) further comprises Mg ２＋ The method of any one of claims 1 to 35, comprising a chelating agent.

37. Mg ２＋ 37. The method of claim 36, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA).

38. The cell lysate mixture of step (a) may further comprise manganese chloride (MnCl ２ ) and / or magnesium sulfate (MgSO ４ 38. The method of claim 1, comprising:

39. The method of any one of claims 1 to 38, wherein the temperature in step (b) is from 50°C to 80°C.

40. The method of any one of claims 1 to 39, wherein the RNA of interest is produced at a concentration of at least 1 g / L.

41. 41. The method of claim 40, wherein the RNA of interest is produced at a concentration of at least 5 g / L.

42. 41. The method of claim 40, wherein the RNA of interest is produced at a concentration of at least 10 g / L.

43. 43. The method of any one of claims 1 to 42, optionally further comprising purifying the RNA of interest by combining the heat inactivated cell lysate mixture with a protein precipitant and removing precipitated proteins, lipids, and DNA.

44. The method of any one of claims 1 to 43, wherein the cell is a bacterial cell.

45. 45. The method of claim 44, wherein the bacterial cell is an Escherichia coli cell.

46. The method of any one of claims 1 to 43, wherein the cell is a yeast cell.

47. A method comprising culturing in a cell culture medium: (a) cells comprising RNA; and (b) cells comprising at least one ribonuclease, at least one thermostable kinase, and optionally at least one thermostable RNA polymerase.

48. 48. The method of claim 47, further comprising lysing the cells of (a) and (b) to produce a cell lysate and combining the cell lysates to produce a mixture containing the plurality of enzymatic activities; or combining the cells of (a) and (b) and lysing the combined cells to produce a mixture containing the plurality of enzymatic activities.

49. 49. The method of claim 48, further comprising heating the cell lysate to a temperature that inactivates or partially inactivates endogenous nucleases and phosphatases without completely inactivating thermostable kinases to produce a heat-inactivated cell lysate.

50. 50. The method of claim 49, further comprising incubating the heat-inactivated cell lysate in the presence of an energy source and a deoxyribonucleic acid (DNA) template encoding the RNA of interest under conditions that result in the production of nucleoside triphosphates and polymerization of the nucleoside triphosphates to produce a cell lysate mixture containing the RNA of interest.

51. An engineered cell comprising at least one thermostable nucleoside monophosphate kinase, at least one thermostable nucleoside diphosphate kinase, and at least one thermostable polyphosphate kinase.

52. 52. The cell of claim 51, further comprising at least one ribonuclease and / or at least one thermostable RNA polymerase.

53. A cell lysate containing nucleoside triphosphates, deoxyribonucleic acid (DNA) encoding a ribonucleic acid (RNA) of interest, and RNA polymerase activity.

54. 54. The cell lysate of claim 53, further comprising an energy source, a nucleoside monophosphate, and at least one kinase activity that directly or indirectly catalyzes the conversion of a 5'-nucleoside monophosphate to a 5'-nucleoside triphosphate.

55. 55. The cell lysate of claim 53 or 54, further comprising multimeric RNA and an enzymatic activity that depolymerizes RNA.

56. 56. The cell lysate of any one of claims 53 to 55, wherein the cell lysate and / or at least one component of the cell lysate is obtained from a modified cell.

57. 57. The cell lysate according to any one of claims 53 to 56, wherein the cell lysate and / or at least one component of the cell lysate is obtained from bacterial cells.

58. 58. The cell lysate of claim 57, wherein the bacterial cells are Escherichia coli cells.

59. 59. A cell lysate according to any one of claims 53 to 58, wherein the cell lysate comprises RNA and is obtained from a cell expressing at least one enzyme or fusion enzyme acting as a ribonuclease, as a kinase and / or as an RNA polymerase.

60. 59. A cell lysate according to any one of claims 53 to 58, comprising a mixture of at least two cell lysates, at least one cell lysate being obtained from a cell comprising RNA and at least one cell lysate being obtained from a cell expressing at least one enzyme or fusion enzyme acting as a nuclease, acting as a kinase and / or acting as an RNA polymerase.

61. 61. The cell lysate of any one of claims 55 to 60, wherein the multimeric RNA is messenger RNA (mRNA), transfer RNA (tRNA), or ribosomal RNA (rRNA).

62. 62. The cell lysate of any one of claims 53 to 61, wherein the cell lysate comprises at least one ribonuclease, at least one kinase, and / or at least one RNA polymerase.

63. 63. The cell lysate of claim 62, wherein the at least one ribonuclease is selected from the group consisting of S1 nuclease, nuclease P1, RNase II, RNase III, RNase R, RNase JI, NucA, PNPase, RNase T, RNase E, and RNase G.

64. 64. The cell lysate of claim 63, wherein at least one ribonuclease is Escherichia coli RNase R.

65. 65. The cell lysate of any one of claims 62 to 64, wherein the at least one kinase is at least one thermostable kinase and / or the at least one RNA polymerase is at least one thermostable RNA polymerase.

66. 66. The cell lysate of any one of claims 62 to 65, wherein the at least one kinase is selected from the group consisting of nucleoside monophosphate kinase, nucleoside diphosphate kinase, and polyphosphate kinase.

67. 66. The cell lysate of claim 65, wherein the nucleoside monophosphate kinase is selected from the group consisting of uridylate kinase, cytidylate kinase, thermostable guanylate kinase, and adenylate kinase.

68. 68. The cell lysate of claim 67, wherein the nucleoside monophosphate kinase is selected from the group consisting of Pyrococcus furiosus uridylate kinase (PfPyrH) encoded by the pyrH gene, Thermus thermophilus adenylate kinase (TthAdk) encoded by the adk gene, Thermus thermophilus cytidylate kinase (TthCmk) encoded by the cmk gene, and Thermotoga maritima guanylate kinase (TmGmk) encoded by the gmk gene.

69. 69. The cell lysate of any one of claims 66 to 68, wherein the nucleoside diphosphate kinase is selected from the group consisting of nucleoside diphosphate kinases encoded by the Aquifex aeolicus ndk gene.

70. 70. The cell lysate of any one of claims 66 to 69, wherein the polyphosphate kinase is selected from the group consisting of polyphosphate kinase 1 (PPK1) enzyme and polyphosphate kinase 2 (PPK2) enzyme.

71. 71. The cell lysate of claim 70, wherein the PPK1 enzyme is selected from the group consisting of Thermosynechococcus elongatus PPK1 enzymes.

72. 72. The cell lysate of claim 70 or 71, wherein the PPK2 enzyme is selected from the group consisting of class III PPK2 enzymes.

73. 73. The cell lysate of claim 72, wherein the Class III PPK2 enzyme is selected from the group consisting of Meiothermus ruber, Meiothermus silvanus, Deinococcus geothermalis, Thermosynechococcus elongates, Anaerolinea thermophile, Caldilinea aerophila, Chlorobaculum tepidum, Oceanithermus profundus, Roseiflexus castenholzii, Roseiflexus sp., and Truepera radiovctrix PPK2 enzymes.

74. 74. The cell lysate of claim 72 or 73, wherein the class III PPK2 enzyme is selected from the group consisting of class III PPK2 enzymes comprising an amino acid sequence that is at least 70% identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18.

75. 75. The cell lysate of claim 74, wherein the class III PPK2 enzyme is selected from the group consisting of class III PPK2 enzymes comprising an amino acid sequence identical to an amino acid sequence identified by any one of SEQ ID NOs:8-18.

76. 76. The cell lysate of any one of claims 53 to 75, wherein the cell lysate mixture comprises at least one ribonuclease, at least one thermostable nucleoside monophosphate kinase, at least one thermostable nucleoside diphosphate kinase, and at least one polyphosphate kinase.

77. 77. The cell lysate of any one of claims 62 to 76, wherein at least one RNA polymerase is selected from the group consisting of DNA-dependent RNA polymerases.

78. 78. The cell lysate of claim 77, wherein the DNA-dependent RNA polymerase is selected from the group consisting of T7 RNA polymerase, SP6 RNA polymerase, and T3 RNA polymerase.

79. 79. The cell lysate of claim 78, wherein at least one RNA polymerase is T7 RNA polymerase.

80. 80. The cell lysate of any one of claims 54 to 79, wherein the energy source is adenosine triphosphate (ATP).

81. A cell lysate described in any one of claims 54 to 79, wherein the energy source is an ATP regeneration system.

82. 82. The cell lysate of claim 81, wherein the ATP regeneration system comprises polyphosphate, optionally hexametaphosphate, nucleoside monophosphate, and polyphosphate kinase.

83. 83. The cell lysate of any one of claims 53 to 82, wherein the RNA of interest is single-stranded RNA.

84. The cell lysate of claim 83, wherein the single-stranded RNA is messenger RNA (mRNA).

85. The cell lysate of claim 84, wherein the single-stranded RNA is an antisense RNA.

86. 83. The cell lysate of any one of claims 53 to 82, wherein the RNA of interest is double-stranded RNA.

87. 87. The cell lysate of claim 86, wherein the double-stranded RNA is a small interfering RNA (siRNA) or a short hairpin RNA (shRNA).

88. 83. The cell lysate of any one of claims 53 to 82, wherein the RNA of interest is a single-stranded RNA containing complementary domains linked to each other by a hinge domain.

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