Methods and compositions for the enzymatic production of pseudouridine triphosphate

Abstract

The present invention comprises novel systems, methods and compositions for the enzymatic / chemical production of pseudouridine (Ψ) and its variants, such as N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This international PCT application claims the benefit of and priority to U.S. Provisional Application No. 63 / 323,145, filed March 24, 2022. The specifications, claims and drawings of the above-referenced applications are hereby incorporated by reference in their entireties.

[0002] Sequence Listing This application includes the contents of the electronic sequence listing (90125-00251-Sequence-Listing, size: 27 Kbytes, and creation date: March 22, 2023), the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to the field of chemical and enzymatic production of nucleotide isomers, in particular the enzymatic and chemical production of pseudouridine (Ψ) from uridine, specifically the chemical and / or enzymatic production and modification of N1-methyl-pseudouridine. [Background technology]

[0004] Pseudouridine-5'-triphosphate (ΨTP) is a C5-glycoside isomer of uridine that contains a CC bond between the C1 of the ribose sugar and the C5 of uracil, rather than the usual C1-N1 bond found in uridine. Pseudouridine is found in almost all types of non-coding RNA, such as tRNA, rRNA, small nuclear RNA (snRNA), as well as coding RNA, all generally referred to herein as "RNA" or "RNA oligonucleotides," which refers to oligonucleotides with several RNA nucleosides attached. This base modification can stabilize RNA and improve base stacking by forming an additional hydrogen bond with water through its extra amino group.

[0005] The most common modification for therapeutic RNA is the partial replacement of UTP with ΨTP to stabilize the RNA in the host cell during translation. For this reason, pseudo-UTP is naturally present in tRNAs and other organisms and is naturally produced by enzymes in various organisms called pseudouridine synthases (pUS). These enzymes are responsible for the most abundant post-transcriptional modification of cellular RNA, catalyzing the site-specific isomerization of uridine residues that are already part of RNA oligonucleotides. Therapeutic RNA requires ΨTP as a replacement for uridine-5'-triphosphate (UTP), but the cost of synthetically produced ΨTP is prohibitively high, creating a bottleneck in the production of many RNA-based therapeutics, such as RNA vaccines, and other RNA therapeutics and diagnostics that are increasingly being produced by in vitro transcription systems. Thus, there is a need for an efficient and cost-effective method of ΨTP production that can be applied especially to in vitro RNA production systems. Summary of the Invention

[0006] In one aspect, the invention includes systems, methods, and compositions for producing pseudouridine. In one embodiment, the invention includes systems and methods for enzymatically synthesizing pseudouridine-5'-monophosphate (ΨMP). In this embodiment, the system can include a substrate comprising an amount of an isolated uracil nucleobase and an amount of an isolated ribose-5-phosphate. The substrate can be introduced to an amount of a pseudouridine-5'-phosphate glycosidase (PsuG) enzyme, or a fragment or variant thereof, where the PsuG catalyzes the formation of pseudouridine-5'-monophosphate (ΨMP) from the uracil nucleobase and the ribose-5-phosphate.

[0007] In another embodiment, the present invention can further include a protecting agent that reacts with the hydroxyl group of ΨMP to form a protected ΨMP. The protecting agent of the present invention can preferably include an N-silyl compound such as hexamethyldisilazane (HMDS), which reacts with ΨMP to form a protected ΨMP compound as described herein.

[0008] In another embodiment, the present invention can further include a methylating agent that reacts with the protected ΨMP to form a protected pseudouridine-5'-monophosphate (protected m1ΨMP) compound. In a preferred embodiment, the methylating agent of the present invention includes iodomethane.

[0009] In another embodiment, the present invention can further comprise a deprotecting agent that reacts with the protected m1ΨMP to remove the protecting group(s), thereby forming N1-methyl-pseudouridine-5'-monophosphate (m1ΨMP). In a preferred embodiment, the deprotecting agent of the present invention comprises an amount of ammonia and methanol.

[0010] In another embodiment, the present invention can further comprise a phosphorylating agent that catalyzes the sequential phosphorylation of m1ΨMP to form N1-methyl-pseudouridine-5'-diphosphate (m1ΨDP) and N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP). In a preferred embodiment, the phosphorylating agent of the present invention comprises an amount of polyphosphate kinase (PPK2) or a fragment or variant thereof, and an amount of inorganic polyphosphate, the PPK2 or a fragment or variant thereof catalyzing the sequential phosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP. In a preferred embodiment, the PPK comprises a PPK from a thermophilic bacterium, such as, for example, a Deinococcus or Meiothermus genus, or more preferably, a thermophilic bacterium including Deinococcus geothermalis, Deinococcus radiodurans, or Meiothermus ruber. In a further preferred embodiment, the PPK of the present invention comprises a sequence as set forth in SEQ ID NO: 4, 6, 21, or a sequence having at least 80% homology to SEQ ID NO: 4, 6, or 21.

[0011] In a further aspect of the invention, the concentration of inorganic polyphosphate is in excess and promotes the forward reaction of sequential phosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP. Moreover, in this preferred aspect, the sequential phosphorylation of m1ΨMP by PPK is preferably carried out at a temperature in the presence of excess inorganic polyphosphate generating reaction conditions that drive the forward reaction of sequential phosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP.

[0012] In another aspect, the PsuG of the present invention can comprise a PsuG from an enterobacteria or a thermophilic bacterium, e.g., Deinococcus diothermalis and / or Escherichia coli, respectively. In a further preferred aspect, the PsuG of the present invention can comprise a sequence selected from SEQ ID NO: 1, 3, or a sequence having at least 80% homology to SEQ ID NO: 1 or 3. In another preferred embodiment, the concentrations of uracil nucleobase and ribose-5-phosphate substrate, and reaction temperature, cause reverse catalysis of the substrate by PsuG to form ΨMP.

[0013] In another aspect, the present invention includes systems, methods, and compositions for producing ribose-5-phosphate. In this preferred embodiment, an isolated ribokinase (RbsK) catalyzes the formation of ribose-5-phosphate from ribose in the presence of an adenosine triphosphate (ATP) donor, where the RbsK donates a phosphate group to ribose to form ribose-5-phosphate and adenosine diphosphate (ADP), and an amount of ribose. In another aspect, the RbsK of the present invention can include RbsK from a thermophilic bacterium, such as Deinococcus geothermalis. In another aspect, the RbsK of the present invention can include a sequence as set forth in SEQ ID NO:20, or a sequence having at least 80% homology to SEQ ID NO:20.

[0014] Additional embodiments of the present invention include isolated compounds selected from ΨMP, protected ΨMP, protected m1ΨMP compounds, m1ΨMP, m1ΨDP, and m1ΨTP produced by the methods, systems, and compositions of the present invention.

[0015] In another aspect, the present invention includes novel systems, methods, and compositions for enzymatically producing pseudouridine (Ψ). In a preferred embodiment, Ψ, and preferably pseudouridine-5'-triphosphate (ΨTP), may be enzymatically produced from unpurified RNA samples, preferably extracted from fermentation or cell waste of bacterial cultures routinely grown in the laboratory, or yeast waste from beer and wine fermentation plants. In this embodiment, RNA is isolated from the fermentation waste and enzymatically converted to the Ψ form, which is then enzymatically converted to the monophosphate form (ΨMP) prior to regeneration of triphosphate. In one preferred embodiment, regeneration of triphosphate is accomplished using inorganic polyphosphate (PPi) and adenosine monophosphate (AMP) as part of a dual enzyme system, which may preferably include adenosine kinase (AdK) and polyphosphate kinase (PPK) to regenerate ΨTP from ΨMP.

[0016] In a preferred embodiment of the present invention, N1-methyl-pseudouridine triphosphate (m1Ψ) may be produced from an isolated uridine-stranded RNA oligonucleotide. In this preferred embodiment, the uridine-containing RNA may be isolated, for example, from fermentation waste. The uridine residues of the RNA oligonucleotide may be converted to pseudouridine-5'-triphosphate (Ψ) by pseudouridine synthase (pUS). The Ψ residues may then be methylated by N1-pseudouridine methyltransferase to form m1Ψ residues, and further preferably digested to nucleotide monophosphate (m1ΨMP) by a nuclease such as P1 or 5'-phosphodiesterase (5'-PDase). The resulting m1ΨMP may be purified and enzymatically converted to m1Ψ triphosphate by a nucleoside diphosphate kinase (NdK), such as adenosyl kinase, in the presence of an adenosine triphosphate (ATP) donor.

[0017] In another preferred embodiment, the present invention includes modified N1-pseudouridine methyltransferases or fragments or variants thereof that can further catalyze the methylation of newly formed Ψ residues in RNA oligonucleotides to form a series of N1-methyl-pseudouridine (m1Ψ) residues. In one preferred embodiment, the 129R and 132R residues of the exemplary N1-pseudouridine methyltransferase Nep1 are converted to 129A and 132A to form modified N1-pseudouridine methyltransferase (mNep1). These engineered mutations can reduce the cooperation of mNep1 with nucleotides adjacent to pUTP residues in RNA oligonucleotides, thereby allowing the formation of individual N1-methyl-pseudouridine (m1Ψ) residues from Ψ residues of RNA oligonucleotides.

[0018] In another aspect, the present invention includes alternative novel systems, methods and compositions utilizing pseudouridine-5'-phosphate glycosidase (PsuG) enzyme to enzymatically produce ΨTP. In this preferred aspect, PsuG may be used to catalyze the formation of pseudouridine-5'-monophosphate (ΨMP) from uracil nucleobase and ribose-5-phosphate substrates. The temperature and substrate concentration of this reaction cause the reverse action of the PsuG enzyme, which under normal conditions catalytically cleaves the ΨMP substrate to form uracil and ribose-5-phosphate. The resulting ΨMP can be enzymatically converted to Ψtriphosphate by a nucleoside diphosphate kinase (NdK), such as adenosyl kinase (Adk), in the presence of an adenosine triphosphate (ATP) donor. Alternatively, the resulting ΨMP can be enzymatically converted to Ψtriphosphate (ΨTP) by a deoxynucleoside kinase, such as PPK2, in the presence of a phosphate donor, which may preferably include sodium hexametaphosphate. ΨTP may be further optionally methylated to form m1ΨTP.

[0019] Additional aspects of the present invention may become apparent from the specification, claims, and drawings provided below. [Brief description of the drawings]

[0020] [Figure 1] 1 shows a schematic scheme for the enzymatic production of ΨTP, specifically m1-pseudouridine-5'-triphosphate (m1ΨTP) in one embodiment. [Diagram 2] 1 shows the expression and characteristics of a recombinantly expressed pseudouridine glycosidase (PsuG) enzyme in one embodiment. [Figure 3A] 1 shows a schematic scheme of the enzymatic production of uridine-5′-monophosphate (ΨMP) from uracil and ribose-5-phosphate catalyzed by the reverse reaction of PsuG in one embodiment. [Figure 3B] 1 shows the time course production of ΨMP catalyzed by PsuG in one embodiment. [Figure 4A] 1 shows a schematic scheme of the enzymatic regeneration of ΨTP from ΨMP using sodium hexametaphosphate catalyzed by deoxynucleoside kinase (PPK2) in one embodiment. [Figure 4B] FIG. 1 shows the enzymatic regeneration of ΨMP to ΨTP using an ATP donor to regenerate ΨMP to ΨTP, thereby producing AMP that is recycled back to ATP in one embodiment. [Diagram 5] 1 shows a stepwise scheme 1 and m1ΨTP of the enzymatic and chemical production in one embodiment. [Figure 6] Enzymatic production of ΨMP is shown using the scheme in FIG. 5, where ribose and polyP were dissolved in about 500 ml of H2O and the solution shown in the figure was fed to the reaction over about 20 hours and adjusted to pH 7. [Figure 7] Stepwise chemical methylation of ΨMP to form 1mΨMP is shown. As shown in the figure, a small aliquot (200 mL) of the chemical methylation reaction was removed after 36 hours, the solvent was removed with a stream of nitrogen, deprotected using 300 mL of 7 M NH3 in MeOH, the solvent was removed again, dissolved in an aqueous solution of 10 mM DBAA (500 mL), and 5 mL of the solution was subjected to LC-MS analysis. [Figure 8] Enzymatic production of 1mΨTP from 1mΨMP. Reaction conditions include 25 mM 1mΨTP, 10 mM polyP, 0.25 mM PPK2, buffer: 50 mM HEPES, 25 mM Mg2+, 1 mM Mn2+ (pH 7). [Figure 9A] 1 shows HPLC analysis of purified 1mΨTP synthesized according to Scheme 1 described herein. [Figure 9B] 1 shows MS analysis of purified 1mΨTP synthesized according to Scheme 1 described herein. [Figure 10] FIG. 1 shows in vitro transcription of RNA using 1mΨTP synthesized according to Scheme 1 described herein in comparison to commercially available transcription products. [Figure 11A] HPLC analysis of mlΨ versus standards is shown. [Figure 11B] HPLC analysis of the amount and conversion of 1mΨTP, 1mΨDP and 1mΨMP from in vitro RNA transcription mixtures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] The present invention comprises novel systems, methods and compositions for the scalable enzymatic production of pUTP from uridine-contiguous RNA oligonucleotides, preferably RNA oligonucleotides isolated from fermentation waste and other natural or synthetic sources. In a preferred embodiment, the present invention may comprise generating a sample comprising a plurality of RNA oligonucleotides, preferably isolated from fermentation waste such as bacterial cultures grown in a laboratory or polymer production system, or yeast waste from beer and wine fermentation plants, and yeast-based polymer production systems.

[0022] Preferably, in an in vitro reaction chamber such as a biochamber, an isolated pseudouridine synthase enzyme, or a fragment or variant thereof, may catalytically convert a uridine nucleotide residue in an RNA oligonucleotide to a Ψ residue. In a preferred embodiment, the pseudouridine synthase of the present invention may be selected from the group consisting of SEQ ID NOs: PUS1-PUS9 (SEQ ID NOs: 11-19) or a fragment or variant thereof. In the same or a separate in vitro reaction chamber, an N1-pseudouridine methyltransferase, or a fragment or variant thereof, may further catalyze the methylation of the newly formed Ψ residue in the RNA oligonucleotide to form a series of N1-methyl-pseudouridine (m1Ψ) residues. In a preferred embodiment, the N1-pseudouridine methyltransferase may comprise Nep1, more preferably the mNep1 enzyme as set forth in SEQ ID NO: 8, or a fragment or variant thereof.

[0023] In another preferred embodiment, in the same or separate in vitro reaction chamber, a modified N1-pseudouridine methyltransferase (mNep1), or a fragment or variant thereof, may catalyze the methylation of newly formed Ψ residues in an RNA oligonucleotide to form a series of N1-methyl-pseudouridine (m1Ψ) residues. In this embodiment, the 129R and 132R residues of Nep1 are converted to 129A and 132A to form mNep1 (SEQ ID NO: 9). These engineered mutations may reduce the cooperation of mNep1 with the nucleotide(s) adjacent to the Ψ residue in the RNA oligonucleotide, thereby allowing the formation of individual m1Ψ residues from the Ψ residues of the RNA oligonucleotide.

[0024] Here, an RNA oligonucleotide containing a series of m1Ψ residues may be digested to form multiple nucleotide monophosphates, including m1Ψ-monophosphate (m1ΨMP). In a preferred embodiment, the RNA oligonucleotide may be digested with nuclease P1, or 5'-phosphodiesterase (5'-PDase), or a fragment or variant thereof. The resulting m1ΨMP may be further isolated and / or purified, for example, via a weak anion exchange column or other similar purification device.

[0025] The enzymatically generated m1ΨMP may be further regenerated to their N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP) form. In this preferred embodiment, a nucleoside diphosphate kinase (NdK), in the presence of an adenosine triphosphate (ATP) donor, catalyzes the transfer of a phosphate group to m1ΨMP to form adenosine monophosphate (AMP) while sequentially forming m1Ψ-diphosphate (m1ΨDP) and finally pseudouridine-5'-triphosphate (m1ΨTP) which can be further isolated and / or purified. In one embodiment, the NdK of the present invention may comprise an adenosyl kinase (AdK), preferably an Adk as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0026] AMP obtained from the ATP donor of the present invention may be further regenerated through the action of polyphosphate kinase (PPK). In this embodiment, AMP may be regenerated back to ATP by the transfer of inorganic polyphosphate to AMP by the PPK enzyme. In a preferred embodiment, the PPK enzyme of the present invention may comprise a PPK2 enzyme selected from the group consisting of SEQ ID NO: 4, 6, or a fragment or variant thereof. The regenerated ATP may be recycled back to the reaction cycle that regenerates m1ΨTP from m1ΨMP.

[0027] As mentioned above, the reactions may occur in vitro either individually or in the same or consecutive in vitro reaction chambers. In a preferred embodiment, the reactions may be carried out in an in vitro transcription system, such as a cell-free expression system, or an in vitro RNA production system.

[0028] The present invention includes novel systems, methods and compositions for the enzymatic production of Ψ from uracil nucleobase and ribose-5-phosphate. In a preferred embodiment, the pseudouridine-5'-phosphate glycosidase (PsuG) enzyme may catalyze the formation of pseudouridine-5'-monophosphate (ΨMP) from the reverse reaction of two substrates, i.e., uracil nucleobase and ribose-5-phosphate, preferably in an in vitro system such as in vitro transcription, or in an RNA production system. The substrate and temperature conditions may be adjusted to induce the reverse reaction of PsuG, thereby catalyzing the formation of ΨMP from uracil nucleobase and ribose-5-phosphate substrates. Here, the normal forward reaction proceeds in the reverse direction to catalyze the cleavage of the ΨMP substrate to form uracil nucleobase and ribose-5-phosphate products. In this embodiment, the reverse reaction of PsuG may be promoted by increasing the reaction temperature to about 37°C to 50°C and increasing the concentration of both substrates, i.e., uracil and ribose-5-phosphate. In one embodiment, the concentration of both uracil and ribose-5-phosphate may be at least 1 millimolar each. In this preferred embodiment, PsuG may be selected from a thermophilic bacterium, such as D. geothermalis (SEQ ID NO: 1 or 2), allowing the reaction to run at higher temperatures, preferably at least 50° C.

[0029] As described above, the invention may include producing ΨTP from ΨMP by contacting ΨMP with PPK2 in the presence of an inorganic polyphosphate source such as sodium hexametaphosphate. In an alternative embodiment, enzymatically produced ΨMP may be regenerated to form ΨTP via nucleoside diphosphate kinase (NdK) catalysis in the presence of an adenosine triphosphate (ATP) donor, where the resulting ATP donor, here AMP, is regenerated as described above.

[0030] The enzymatically produced ΨTP may be further methylated by an N1-pseudouridine methyltransferase, or a fragment or variant thereof, to form N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP). In a preferred embodiment, the N1-pseudouridine methyltransferase comprises Nep1 (SEQ ID NO:8).

[0031] In one embodiment, the invention includes systems, methods and compositions for enzymatically and chemically producing m1ΨTP, and preferably its use in an in vitro transcription reaction to generate RNA incorporating it. With general reference to the scheme provided in FIG. 5, the invention can include the enzymatic synthesis of Ψ from uracil nucleobase and ribose-5-phosphate. In one embodiment, a ribokinase (RbsK) enzyme can convert ribose to ribose-5-phosphate, where a phosphate group is donated from a molecule of ATP, which converts ATP to ADP. As shown in FIG. 5, ADP can be regenerated by reacting with a PPK enzyme in the presence of polyphosphate, which forms ATP, which can subsequently be combined with ribose by RbsK to form ribose-5-phosphate. In a preferred embodiment, a ribokinase (RbsK) enzyme set forth in the amino acid sequence of SEQ ID NO: 20, or a fragment or variant thereof, can convert ribose to ribose-5-phosphate, where a phosphate group is donated from a molecule of ATP, which converts ATP to ADP. Again, the resulting ADP can be regenerated by reaction with the PPK enzyme set forth in SEQ ID NO: 4, 6, 21, or a fragment or variant thereof, in the presence of polyphosphate, thereby forming ATP, which is subsequently bound to ribose by RbsK set forth in the amino acid sequence of SEQ ID NO: 20, or a fragment or variant thereof, to form ribose-5-phosphate.

[0032] Referring again to the synthetic scheme of Figure 5, the present invention further includes novel systems, methods and compositions for enzymatically producing Ψ from uracil nucleobase and ribose-5-phosphate, preferably produced by the methods described above. In a preferred embodiment, a pseudouridine-5'-phosphate glycosidase (PsuG) enzyme may catalyze the formation of pseudouridine-5'-monophosphate (ΨMP) from the reverse reaction of two substrates, i.e., uracil nucleobase and ribose-5-phosphate, preferably in an in vitro system such as an in vitro transcription or RNA production system. The substrate and temperature conditions may be adjusted to cause the reverse reaction of PsuG, thereby catalyzing the formation of ΨMP from the uracil nucleobase and ribose-5-phosphate substrate. Here, the normal forward reaction proceeds in the reverse direction to catalyze the cleavage of the ΨMP substrate to form the uracil nucleobase and ribose-5-phosphate product. In this embodiment, the reverse reaction of PsuG may be facilitated by increasing the reaction temperature to about 37° C. to 50° C. and increasing the concentration of both substrates, i.e., uracil and ribose-5-phosphate. In one embodiment, the concentrations of both uracil and ribose-5-phosphate may each be at least 1 millimolar. In this preferred embodiment, PsuG may be selected from a thermophilic bacterium, such as D. geothermalis, and may comprise the amino acid sequence set forth in SEQ ID NO: 1 or 2, or a fragment or variant thereof. Thus, the use of an enzyme from a thermophilic organism allows the reaction to be carried out at higher temperatures, preferably at least 50° C.

[0033] Referring again to the synthetic scheme of Figure 5, the hydroxyl group of the ΨMP substrate formed by uracil nucleobase and ribose-5-phosphate is methylated to form m1ΨMP. In one embodiment, the hydroxyl group of ΨMP can be reacted with a protecting agent, preferably an N-silyl compound such as an amount of hexamethyldisilazane (HMDS), to generate a trimethylsilyl ether (-OTMS) protecting group at the original hydroxyl position of ΨMP to form what is referred to herein as protected ΨMP according to the formula provided below. [ka]

[0034] The resulting protected ΨMP compound can be methylated at the 1-position to form a protected m1ΨMP compound by reacting with a methylating agent. In a preferred embodiment, the protected m1ΨM is reacted with an amount of a methylating agent, preferably iodomethane, which methylates the 1-position of the protected ΨMP to form the protected m1ΨM according to the formula provided below. [ka]

[0035] The protected m1ΨMP compound of the present invention can be further deprotected. In this embodiment, a deprotecting agent can react with the protected m1ΨMP to form m1ΨMP. In one embodiment, for example, m1ΨMP with a deprotecting agent containing an amount of ammonia in methanol. Removal of the protecting group forms m1ΨMP.

[0036] In a preferred embodiment, the m1ΨMP synthesized by the above steps can be enzymatically converted to m1ΨTP by diphosphorylation mediated by a phosphorylating agent, in a preferred embodiment, the phosphorylating agent can include a PPK enzyme, preferably a PPK2 enzyme from a thermophilic bacterium. Referring again to Figure 5, a phosphate group from ATP is transferred to m1ΨMP to form m1ΨDP, and then another phosphate group is transferred from ATP to m1ΨDP to form m1ΨTP, respectively. In this preferred embodiment, the diphosphorylation is mediated by a PPK enzyme, preferably a PPK2 enzyme, as set forth in SEQ ID NO: 4, 6, 21, or a fragment or variant thereof.

[0037] In certain embodiments, the present invention further includes systems, methods and compositions for producing heterologous proteins used in the production of m1ΨTP. For example, in one embodiment, a cell, preferably a yeast, bacterial or other prokaryotic cell, can be transformed with an expression vector expressing one or more of the enzymes of the present invention. In this embodiment, the cell can express a heterologous nucleotide operably linked to a promoter that encodes the PsuG and / or PPK2 protein of the present invention, or a fragment or variant thereof. In another preferred embodiment, the cell, such as a yeast cell, can express a heterologous nucleotide operably linked to a promoter that encodes the PsuG enzyme set forth in SEQ ID NO: 1 or 2, or a fragment or variant thereof, and / or the PPK2 enzyme set forth in SEQ ID NO: 4, 6, 21, or a fragment or variant thereof. In this preferred embodiment, the transformed yeast cell can be cultured in a suitable medium. Expression of the PsuG and / or PPK2 enzyme can occur and the resulting proteins can be actively or passively induced from the yeast cells into the supernatant from which they can be isolated.

[0038] Unless otherwise defined, all technical terms, notations and other scientific terms used herein shall have the meanings commonly understood by those skilled in the art. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a substantial difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly used by those skilled in the art using conventional techniques, such as the widely used molecular cloning techniques described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, and Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001). Where appropriate, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and / or parameters, unless otherwise noted.

[0039] Unless otherwise required by context, the use of the singular herein shall be construed to include the plural and vice versa. The terms "a" or "an" when used in reference to an entity shall be construed to refer to one or more of that entity. Thus, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0040] As used herein, the term "comprise" or variations thereof, such as "comprises" or "comprising," should be interpreted to indicate the inclusion of any recited component (e.g., feature, element, property, property, method / process step, or limitation) or group of components (e.g., feature, element, property, property, method / process step, or limitation) and not the exclusion of other components or groups of components. Thus, as used herein, the term "comprising" is inclusive or open-ended and does not exclude additional unrecited components or method / process steps.

[0041] The term "polynucleotide" or "nucleotide" as used herein refers to an organic polymer composed of two or more monomers, including nucleotides, nucleosides or analogs thereof. The term "nucleotide" refers to any of several compounds consisting of a ribose or deoxyribose sugar linked to a purine or pyrimidine base and a phosphate group, and is the basic structural unit of nucleic acids. The term "nucleoside" refers to compounds consisting of a purine or pyrimidine base combined with deoxyribose or ribose, particularly found in nucleic acids (such as guanosine or adenosine). The terms "nucleotide analog" or "nucleoside analog" refer to nucleotides or nucleosides, respectively, in which one or more individual atoms have been replaced with different atoms or different functional groups. Thus, the term "polynucleotide" includes nucleic acids of any length, particularly DNA, RNA, analogs and fragments thereof. Polynucleotides of three or more bases are also called "oligomers" or "oligonucleotides".

[0042] The term "messenger ribonucleic acid" (mRNA) refers to a ribonucleic acid (RNA) molecule that mediates the transfer of genetic information to ribosomes in the cytoplasm, where it serves as a template for protein synthesis. It is synthesized from a DNA template during the process of transcription. "Ribonucleic acid" (RNA) is a polymer of nucleotides linked by phosphodiester bonds, each containing ribose or modified versions thereof as a sugar moiety. Each nucleotide contains adenine (A), guanine (G), cytosine (C), uracil (U) or modified versions thereof as a base. The genetic information in an mRNA molecule is encoded in the sequence of nucleotide bases of the mRNA molecule, which are arranged in codons, each of which consists of three nucleotide bases. Each codon codes for a specific amino acid of a polypeptide, except for the stop codon, which ends translation (protein synthesis). In living cells, the mRNA is transported to the ribosomes, the site of protein synthesis, where it provides the genetic information for protein synthesis (translation). For a more detailed description, see Alberts B et al. (2007) Molecular Biology of the Cell, Fifth Edition, Garland Science.

[0043] As used herein, "in vitro transcription" (IVT) or "RNA production system" refers to a cell-free reaction in which a double-stranded DNA (dsDNA) template is copied by a DNA-directed RNA polymerase (typically a bacteriophage polymerase) to produce a product that includes an RNA molecule copied from the template. Examples of in vitro transcription may include cell-free expression systems that produce RNA transcripts or other macromolecules such as peptides. In certain embodiments, the invention may encompass the in vitro production of artificial mRNA as well as wild-type mRNA. An artificial mRNA (sequence) may typically be understood to be an mRNA molecule that does not occur in nature. In other words, an artificial mRNA molecule may be understood as a non-natural mRNA molecule. Such an mRNA molecule may be non-natural due to its individual sequence (not occurring in nature) and / or other modifications, such as structural modifications of non-naturally occurring nucleotides. Typically, an artificial mRNA molecule may be designed and / or generated by genetic engineering methods to correspond to a desired artificial nucleotide sequence (heterologous sequence). In this context, an artificial sequence is usually a sequence that may not occur in nature, i.e. differs from the wild-type sequence by at least one nucleotide. The term "wild-type" may be understood as a sequence that occurs in nature. Furthermore, the term "artificial nucleic acid molecule" is not limited to meaning "one single molecule" but is typically understood to include a collection of identical molecules. It may therefore relate to a plurality of identical molecules contained in an aliquot.

[0044] In certain embodiments, the invention may encompass the in vitro production of bicistronic / multicistronic mRNA, which is typically an mRNA that may have two (bicistronic) or multiple (multicistronic) open reading frames (ORFs) (coding regions or coding sequences). An open reading frame in this context is a sequence of several nucleotide triplets (codons) that can be translated into a peptide or protein. Translation of such an mRNA results in two (bicistronic) or multiple (multicistronic) separate translation products (if the ORFs are not identical). For expression in eukaryotes, such an mRNA may, for example, include an internal ribosome entry site (IRES) sequence.

[0045] In one embodiment, an in vitro mRNA is generated, preferably in a host organism such as a mammalian or human subject in need thereof, that is configured to be translated to form a peptide. A peptide is a polymer of amino acid monomers. Typically, the monomers are linked by peptide bonds. The term "peptide" does not limit the length of the polymeric chain of amino acids. In some embodiments of the invention, a peptide may, for example, comprise less than 50 monomer units. Longer peptides, also called polypeptides, typically have 50-600 monomer units, more specifically 50-300 monomer units.

[0046] Further examples of IVT systems include in vitro recombinant cell-free expression systems, which refer to cell-free synthesis of polypeptides in a reaction mixture or solution containing biological extracts and / or defined cell-free reaction components, such as the exemplary systems described by Koglin et al., in PCT / US2020 / 028005 and PCT / US2021 / 027774, which are incorporated herein by reference. The reaction mixture may optionally include a template or genetic template for producing a polymer, such as DNA, mRNA, and monomers of the polymer to be synthesized, such as amino acids, nucleotides, and such cofactors, enzymes and other reagents required for synthesis, such as ribosomes, tRNAs, polymerases, transcription factors, etc. The components of the recombinant cell-free synthesis reaction and / or the cellular adenosine triphosphate (ATP) energy regeneration system, which are incorporated herein by reference, may be performed / added as a batch, continuous flow, or semi-continuous flow.

[0047] Examples of in vitro production systems have also been previously described in the art, including U.S. Patent No. 11,136,586 and PCT Application No. PCT / US2020 / 028005. The methods and conditions for producing synthetic RNA disclosed in each of the aforementioned references, including the methods, systems and compositions outlined in the claims, examples, and materials and methods, are incorporated herein by reference in their entirety. For example, synthetic RNA oligonucleotides may be generated and capped in a cell-free (CF) expression system. In one embodiment, the CF expression system may comprise a fully recombinant, stable, reliable, and functional in vitro transcription system for continuous flow production of RNA. As described above, the exemplary CF system generally described by A. Koglin and M. Humbert et al. in PCT / US2018 / 0121121 and PCT / US2021 / 027774 (already identified as incorporated by reference) may be used as an in vitro platform for producing synthetic mRNA. As pointed out in the field, lysate-based in vitro systems suffer from the limited stability of typical E. coli enzymes, the activity of most metabolic processes (nucleotide recycling), the presence of nucleases and proteases, and insufficient ATP regeneration. Utilizing the CF system described by Koglin and Humbert, and using only the following components: a linear DNA template, an affinity-tagged RNA polymerase, nucleotides in a defined buffer system, and the capping enzyme of the present invention, in vitro synthesis of mRNA may be carried out in a hollow fiber reactor using a continuous flow system and other conventional bioreactors known in the field. Using this in vitro system, the inner chamber (hollow fiber) of the bioreactor provides additional nucleotides to the flow, and the outer chamber holds the RNA polymerase and each linear DNA template. With this setup, we demonstrate that the total turnover of RNAP is at least 50 times higher than in a batch reaction, and coupled with the selected enzyme modifications, produces cleaner mRNA without smearing.All enzymes are designed with affinity tags, allowing the entire reaction to be washed through a bed of affinity resin that is partially loaded with DNase to remove the template and capture the RNA polymerase. In this way, the process avoids the need to deal with phenol precipitation and spin column purification (which remains an issue in traditional vaccine processes). In this system, leaching or carryover of components from the RNA biosynthesis may be in the moderate ppb range. After a scalable and simple precipitation and drying process, the resulting mRNA is stable as a powder and does not contain any traces of any components from the manufacturing process. This does not require costly shipping conditions that are difficult to monitor and can be shipped in reduced volumes.

[0048] In some embodiments, the IVT of the present invention may include a "bioreactor," which is any type of enclosed apparatus configured to maintain an environment conducive to the production of macromolecules in vitro. The bioreactor may be configured to operate in batch, continuous, or semi-continuous mode, for example, with a feeder reaction solution. Examples of bioreactors and conditions for synthesizing RNA or other macromolecules are previously described in Koglin, et al. PCT / US2021 / 027774.

[0049] As used herein, the terms "isolated," "purified," or "biologically pure" refer to a material that is substantially or essentially free of components that normally accompany the material in its natural state or when it is produced. In exemplary embodiments, purity and homogeneity are determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A nucleic acid or a particular bacterium that is the predominant species present in a preparation is substantially purified. In exemplary embodiments, the term "purified" refers to a nucleic acid or protein that results in essentially one elevated band in an electrophoretic gel. Typically, an isolated nucleic acid or protein has a level of purity expressed as a range. The lower limit of the range of purity of a component is about 60%, about 70%, or about 80%, and the upper limit of the range of purity is about 70%, about 80%, about 90%, or greater than about 90%.

[0050] It may be convenient or desirable to prepare, purify and / or process active compounds in a chemically protected form. The term "chemically protected form" as used herein refers to a compound in which one or more reactive functional groups are protected from undesired chemical reactions, i.e., in the form of a protected or protective group (also called a masked or masking group, or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be carried out without adversely affecting the protected group. Protecting groups can usually be removed in a subsequent step without substantially adversely affecting the remainder of the molecule. As used herein, "protecting group" refers to a group of atoms that, when attached to a reactive functional group in a molecule, masks, reduces or prevents the reactivity of the functional group. Non-limiting examples of protecting groups can be found in "Protective Groups in Organic Chemistry", TW Greene, PG M Huts, ISBN 0-471-62301-6, John Wiley & Sons, Inc, New York. A "deprotecting agent" is any compound or mixture of compounds that removes a protecting group.

[0051] In a preferred embodiment, the protecting group may include a hydroxy "protecting group" which may be any suitable protecting group for a hydroxy functional group. Representative hydroxy protecting groups include, but are not limited to, silane, ether, ester, and the like. Representative hydroxy protecting groups include, but are not limited to, hexamethyldisilazane (HMDS), trimethylsilane (TMS), t-butyldimethylsilane (TBDMS), t-butyldiphenylsilane (TBDPS), methoxymethyl (MOM), tetrahydropyran (THP), t-butyl, allyl, benzyl, acetyl, pivaloyl, or benzoyl. In some embodiments, the hydroxy protecting group may be trimethylsilane (TMS), t-butyldimethylsilane (TBDMS), t-butyldiphenylsilane (TBDPS), methylmethoxy (MOM), tetrahydropyran (THP), t-butyl, allyl, benzyl, acetyl, pivaloyl, or benzoyl. In some embodiments, the hydroxy protecting group may be benzyl. In some embodiments, the hydroxy protecting group can be TBS.

[0052] "Methylating agent" refers to a reactive species having electrophilicity that can introduce a "methyl group" to the nitrogen atom of naltrexone to form a covalent bond therebetween. An exemplary methylating agent can be represented by the formula CH3Z, where "Z" is a leaving group that, when eliminated, allows CH3 to form a covalent bond with the nitrogen atom of naltrexone to form MNTX. Methylating agents in general, and leaving groups in general, are known to those skilled in the art and are widely described in both patent literature and chemistry textbooks. Suitable Z groups include, but are not limited to, fluoro, chloro, bromo, iodo, iodomethane, -OSO2CF3, CH3OSO2O-, -OSO2CH3, -OSO2C6H4-p-CH3, -OSO2C6H4-p-Br.

[0053] Phosphorylating agent refers to a reagent or enzyme generally used for phosphorylating hydroxyl groups. Examples of such phosphorylating agent compounds include diesters of phosphoric acid such as dibenzyl phosphate, dithioesters of phosphoric acid such as monocyclohexylammonium S,S'-diphenylphosphorodithioate, and phosphoric acid chlorides such as phosphoryl chloride, diallyl chlorophosphonate. Additives may include, for example, azo compounds such as diethyl azodicarboxylate and diisopropyl azodicarboxylate, phosphines such as triphenylphosphine, arenesulfonic acid chlorides such as 2,4,6-triisopropylbenzenesulfonic acid chloride, pyridine, tert-butylmagnesium chloride, and other bases. Examples of such phosphorylating agents may also include enzymes such as PPK2.

[0054] In a preferred embodiment, the output of the cell-free expression system may be a product, RNA, or other macromolecule such as a peptide or fragment thereof that may be isolated or purified. In this embodiment, the isolation or purification of a target protein, which is at least partially separated from at least one other component in the reaction mixture, for example, by organic solvent precipitation such as methanol, ethanol or acetone precipitation, organic or inorganic salt precipitation such as trichloroacetic acid (TCA) or ammonium sulfate precipitation, non-ionic polymer precipitation such as polyethylene glycol (PEG) precipitation, pH precipitation, temperature precipitation, immunoprecipitation, chromatographic separation such as adsorption, ion exchange, affinity and gel exclusion chromatography, chromatofocusing, isoelectric focusing, high performance liquid chromatography (HPLC), gel electrophoresis, dialysis, microfiltration, etc.

[0055] The term "nucleic acid" as used herein refers to a polymer of ribonucleotides or deoxyribonucleotides. Typically, "nucleic acid" polymers exist in single-stranded or double-stranded form, but are also known to form structures containing three or more strands. The term "nucleic acid" includes naturally occurring nucleic acid polymers as well as nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, have similar binding properties as the reference nucleic acid, and are metabolized in a similar manner as the reference nucleotide. Exemplary analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, and peptide nucleic acids (PNAs). "DNA", "RNA", "polynucleotide", "polynucleotide sequence", "oligonucleotide", "nucleotide", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", "nucleic acid fragment", and "isolated nucleic acid fragment" are used interchangeably herein. For nucleic acids, sizes are given in kilobases (kb) or base pairs (bp). Estimates are typically obtained from agarose or acrylamide gel electrophoresis, sequenced nucleic acids, or published DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or amino acid residue numbers. Protein sizes are estimated from gel electrophoresis, sequenced proteins, derived amino acid sequences, or published protein sequences.

[0056] As is known in the art, different organisms preferentially utilize different codons to generate polypeptides. Such "codon usage" selection may be used in the design of nucleic acid molecules encoding the proteins and chimeras of the present invention to optimize expression in a particular host cell system. All nucleotide sequences described in the present invention may be codon optimized for expression in a particular organism or to increase production yield. Codon optimization generally improves protein expression by increasing the translation efficiency of the gene of interest. Gene functionality may also be increased by optimizing codon usage within a custom-designed gene. In an embodiment of codon optimization, a low-frequency codon in a species may be replaced by a high-frequency codon, e.g., for leucine, the low-frequency codon UUA may be replaced by the high-frequency codon CUG. Codon optimization may improve mRNA stability and therefore alter protein translation rate or protein folding rate. Additionally, codon optimization may customize transcriptional and translational control, modify ribosome binding sites, or stabilize mRNA degradation sites.

[0057] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), complementary (or complementary) and reverse complementary sequences, as well as sequences explicitly set forth. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (see, e.g., Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). In addition to the degeneracy of nucleotide codons encoding amino acids, modifications of polynucleotides that produce chemically equivalent amino acids at a given site but do not adversely affect the functionality of the encoded polypeptide are well known in the art. A "conservative amino acid substitution" is a substitution that is predicted to cause the least interference with the properties of the reference polypeptide. In other words, a conservative amino acid substitution substantially preserves the structure and function of the reference protein. Thus, a codon for the amino acid alanine, which is a hydrophobic amino acid, may be replaced by a codon that codes for another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes that result in the substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine or histidine, can also be expected to produce a functionally equivalent protein or polypeptide. Exemplary conservative amino acid substitutions are known to those of skill in the art. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., a beta-sheet or alpha-helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and / or (c) the bulk of the side chain. Further disclosure of a nucleotide sequence specifically includes the resulting amino acid sequence which it encodes, and vice versa.

[0058] As used herein, the term "transformation" or "genetic modification" refers to the transfer of one or more nucleic acid molecules into a cell, preferably via an expression vector. A microorganism is "transformed" or "genetically modified" by a nucleic acid molecule that is transduced into a bacterium or cell or organism if the nucleic acid molecule is stably replicated. As used herein, the term "transformation" or "genetic modification" encompasses all techniques by which a nucleic acid molecule can be introduced into a cell or organism, such as a bacterium.

[0059] As used herein, the term "promoter" refers to a region of DNA that may be upstream of the start of transcription and may be involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter may be operably linked to a coding sequence for expression in a cell, or a promoter may be operably linked to a nucleotide sequence encoding a signal sequence that may be operably linked to a coding sequence for expression in a cell.

[0060] The term "operably linked" when used in reference to a regulatory sequence and a coding sequence means that the regulatory sequence influences the expression of the linked coding sequence. "Regulatory sequence" or "control element" refers to a nucleotide sequence that influences the timing and level / amount of transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, enhancers, stem-loop structures, repressors or binding sequences, termination sequences, polyadenylation recognition sequences, and the like. A particular regulatory sequence may be located upstream and / or downstream of the coding sequence to which it is operably linked. Also, a particular regulatory sequence operably linked to a coding sequence may be located on the associated complementary strand of a double-stranded nucleic acid molecule.

[0061] As used herein, the term "expression," or "expression of a coding sequence" (e.g., gene or transgene), refers to the process by which the encoded information of a nucleic acid transcription unit (e.g., including genomic DNA or cDNA) is converted into operative, non-operative, or structural parts of a cell, often including the synthesis of a protein. Gene expression may be influenced by external signals, such as exposure of a cell, tissue, or organism to an agent that increases or decreases gene expression. Expression of a gene may also be controlled anywhere along the pathway from DNA to RNA to protein. Control of gene expression occurs, for example, through controls acting on transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of a particular protein molecule once it has been made, or a combination thereof. Gene expression may be measured at the RNA or protein level by any method known in the art, including, but not limited to, Northern blot, RT-PCR, Western blot, or in vitro, in situ, or in vivo protein activity assay(s).

[0062] An "expression vector" is a nucleic acid that can replicate in a selected host cell or organism, or in an in vitro environment, such as a cell-free expression system or other IVT system. An expression vector can replicate as an autonomous structure, or alternatively, can be integrated in whole or in part into the nucleic acid of a host cell's chromosome or organelle, or can be used as a shuttle to deliver foreign DNA to a cell, and thus replicated together with the host cell's genome. Thus, an expression vector is a polynucleotide, such as a plasmid, a virus, an artificial chromosome, a nucleic acid fragment, or any suitable construct known in the art, that can replicate in a selected host cell, organelle, or organism, and that allows a certain gene (including a gene of interest) on the expression vector to be transcribed and translated into a polypeptide or protein in the cell, organelle, or organism. In contrast, as described in the examples herein, a "cassette" is a polynucleotide that comprises a portion of an expression vector of the present invention. The use of a cassette is useful in constructing an expression vector. An expression vector is a replicon, such as a plasmid, a phage, a virus, a chimeric virus, or a cosmid, that comprises a desired polynucleotide sequence operably linked to an expression control sequence(s).

[0063] As generally described herein, the term "expression product" with respect to proteins expressed in cell-free expression systems is used interchangeably and generally refers to any peptide or protein having more than about five amino acids. Polypeptides may be homologous to the organism from which the cell-free extract is derived, such as human proteins, plant proteins, viral proteins, yeast proteins, etc., produced in the cell-free extract, or may be foreign, meaning that they are heterologous, i.e., foreign.

[0064] In some embodiments, the term nucleic acid or peptide may be from a source such as a virus. In this context, a "derived" nucleic acid, such as RNA, or peptide means extracted or expressed and isolated from other sources such as bacteria, eukaryotic cells, or fermentation waste. For example, in one embodiment, a capping protein may be derived from an expression vector that is expressed in bacteria or eukaryotic cells.

[0065] As used herein, "RNA sample" or "sample" refers to a composition comprising one or more oligonucleotides that contain uridine residues. RNA samples may include naturally occurring RNA (e.g., extracted from a cell, tissue, or organism), RNA produced by in vitro transcription, and / or chemically synthesized RNA, or RNA samples taken from fermentation waste cultures and / or cells.

[0066] As used herein, "variant" refers to a protein that has an amino acid sequence that differs from a naturally occurring amino acid sequence (i.e., has less than 100% sequence identity to the amino acid sequence of a naturally occurring protein), but is at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identical to the naturally occurring amino acid sequence.

[0067] As used herein, a "fragment" refers to a portion of a peptide or nucleotide sequence that still retains the activity of the whole.

[0068] Unless otherwise indicated, the disclosure of a DNA sequence also includes the corresponding RNA and amino acid sequences, including all redundant codons and conservative amino acid substitutions, the disclosure of an RNA sequence also includes the corresponding DNA and amino acid sequences, including all redundant codons and conservative amino acid substitutions, and finally, the disclosure of an amino acid sequence also includes the corresponding RNA and DNA sequences, including all redundant codons and conservative amino acid substitutions, and vice versa.

[0069] Additional Embodiments In one preferred embodiment, the present invention includes a method for producing pseudouridine, the method comprising: - establishing a sample containing an RNA oligonucleotide; - contacting the RNA oligonucleotide with a pseudouridine synthase, or a fragment or variant thereof, to convert one or more uridine nucleotide residues in the RNA oligonucleotide to one or more pseudouridine residues; - contacting the pseudouridine residue with an N1-pseudouridine methyltransferase, or a fragment or variant thereof, to form an N1-methyl-pseudouridine (m1Ψ) residue; - digesting said RNA oligonucleotide containing m1Ψ residues to form nucleotide monophosphates containing m1Ψ-monophosphate (m1ΨMP); - isolating m1ΨMP from said sample; - optionally regenerating pseudouridine-5'-triphosphate (m1ΨTP) from said m1ΨMP.

[0070] In another preferred embodiment, the invention includes a method for producing pseudouridine, wherein said establishing step comprises isolating said RNA oligonucleotide.

[0071] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said isolating step comprises isolating a quantity of RNA oligonucleotides from a fermentation waste stream.

[0072] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said fermentation waste is selected from the group consisting of bacterial culture waste, yeast culture waste, and food and / or beverage fermentation waste.

[0073] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said pseudouridine synthase is selected from the group consisting of SEQ ID NOs: 11-19, or fragments or variants thereof.

[0074] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said N1-pseudouridine methyltransferase comprises Nep1 or a fragment or variant thereof.

[0075] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said Nep1 comprises the peptide set forth in SEQ ID NO: 8, or a fragment or variant thereof.

[0076] In another preferred embodiment, the present invention includes a method for producing pseudouridine, wherein the N1-pseudouridine methyltransferase comprises a modified N1-pseudouridine methyltransferase (mNep1).

[0077] In another preferred embodiment, the invention comprises a method for producing pseudouridine, wherein said modified mNep1 comprises modified mNep1 in which 129R and 132R are converted to 129A and 132A.

[0078] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein the modified N1-pseudouridine methyltransferase comprises the peptide set forth in SEQ ID NO: 9, or a fragment or variant thereof.

[0079] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein the digesting step comprises digesting the RNA oligonucleotide with nuclease P1, or 5'-phosphodiesterase (5'-PDase), or a fragment or variant thereof.

[0080] In another preferred embodiment, the invention comprises a method of producing pseudouridine, wherein said isolating step comprises purifying mlΨMP.

[0081] In another preferred embodiment, the invention includes a method for producing pseudouridine, wherein the step of purifying m1ΨMP comprises purifying m1ΨMP on a weak anion exchange column.

[0082] In another preferred embodiment, the present invention includes a method for producing pseudouridine, wherein the regenerating step comprises contacting the m1ΨMP with nucleoside diphosphate kinase (NdK) in the presence of an adenosine triphosphate (ATP) donor.

[0083] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said NdK is adenosyl kinase (AdK) as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0084] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, further comprising the step of isolating said m1ΨTP.

[0085] In another preferred embodiment, the present invention includes a method for producing pseudouridine further comprising the step of regenerating the ATP donor by contacting adenosine monophosphate (AMP) with a polyphosphate kinase (PPK) or enzyme in the presence of inorganic polyphosphate.

[0086] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said PPK2 is selected from the group consisting of SEQ ID NO: 4, 6, 21, or a fragment or variant thereof.

[0087] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein one or more of the steps of the present invention are carried out in an in vitro transcription system.

[0088] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said regenerating step is carried out in an in vitro transcription system.

[0089] In another preferred embodiment, the invention comprises a method for producing pseudouridine, wherein the in vitro transcription system comprises an in vitro RNA production system.

[0090] In a preferred embodiment, the present invention includes a system for producing pseudouridine, said system comprising: a sample comprising a quantity of RNA oligonucleotides; - a pseudouridine synthase enzyme, or a fragment or variant thereof, which converts one or more uridine nucleotide residues in said RNA oligonucleotide into one or more pseudouridine residues; - an N1-pseudouridine methyltransferase enzyme, or a fragment or variant thereof, which methylates the pseudouridine residues to form N1-methyl-pseudouridine (m1Ψ) residues; - a nuclease enzyme that digests said RNA oligonucleotide containing an m1Ψ residue to form a nucleotide monophosphate containing m1Ψ-monophosphate (m1ΨMP); - a nucleoside diphosphate kinase (NdK) and an adenosine triphosphate (ATP) donor, which regenerates pseudouridine-5'-triphosphate (m1ΨTP) from said m1ΨMP.

[0091] In another preferred embodiment, the invention comprises a system for producing pseudouridine, wherein said sample comprising a quantity of RNA oligonucleotide comprises an isolated sample comprising a quantity of RNA oligonucleotide.

[0092] In another preferred embodiment, the invention includes a system for producing pseudouridine, wherein the isolated sample includes an isolated sample from a fermentation waste stream.

[0093] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the fermentation waste is selected from the group consisting of bacterial culture waste, yeast culture waste, and food and / or beverage fermentation waste.

[0094] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the pseudouridine synthase is selected from the group consisting of SEQ ID NOs: 11-19, or fragments or variants thereof.

[0095] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the N1-pseudouridine methyltransferase comprises Nep1 or a fragment or variant thereof.

[0096] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said Nep1 comprises the peptide set forth in SEQ ID NO: 8, or a fragment or variant thereof.

[0097] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the N1-pseudouridine methyltransferase comprises a modified N1-pseudouridine methyltransferase (mNep1).

[0098] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the modified mNep1 comprises mNep1 in which 129R and 132R are converted to 129A and 132A.

[0099] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the modified N1-pseudouridine methyltransferase comprises the peptide set forth in SEQ ID NO: 9, or a fragment or variant thereof.

[0100] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said nuclease is selected from nuclease P1, or 5'-phosphodiesterase (5'-PDase), or a fragment or variant thereof.

[0101] In another preferred embodiment, the present invention includes a system for producing pseudouridine, further comprising an m1ΨMP purification apparatus.

[0102] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the mlΨMP purification apparatus step comprises a weak anion exchange column.

[0103] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said NdK is adenosyl kinase (AdK) set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0104] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, further comprising an ATP regeneration system, the ATP regeneration system comprising: - Polyphosphate kinase (PPK2), - adenosyl kinase (AdK), - inorganic polyphosphates, - adenosine monophosphate (AMP).

[0105] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said PPK2 is selected from the group consisting of SEQ ID NO: 4, 6, or a fragment or variant thereof.

[0106] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the AdK comprises the peptide set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0107] In another preferred embodiment, the present invention includes a system for producing pseudouridine, further comprising an in vitro transcription system.

[0108] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said in vitro transcription system comprises an in vitro RNA production system.

[0109] In one preferred embodiment, the invention includes a method for producing pseudouridine comprising catalyzing the formation of pseudouridine-5'-monophosphate (ΨMP) from uracil nucleobase and ribose-5-phosphate by a pseudouridine-5'-phosphate glycosidase (PsuG) enzyme.

[0110] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said PsuG is selected from SEQ ID NOs: 1-2, or a fragment or variant thereof.

[0111] In another preferred embodiment, the invention comprises a method for producing pseudouridine, wherein the concentrations of uracil nucleobase and ribose-5-phosphate substrate, and reaction temperature cause reverse catalysis of the substrate by PsuG to form ΨMP.

[0112] In another preferred embodiment, the present invention includes a method for producing pseudouridine, further comprising the step of contacting pseudouridine-5'-triphosphate (ΨTP) with PPK2 in the presence of inorganic polyphosphate to generate said ΨTP from ΨMP.

[0113] In another preferred embodiment, the invention comprises a method for producing pseudouridine, wherein the inorganic polyphosphate comprises sodium hexametaphosphate.

[0114] In another preferred embodiment, the present invention includes a method for producing pseudouridine further comprising the step of methylating said ΨTP to form N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP).

[0115] In another preferred embodiment, the invention includes a method of producing pseudouridine, wherein the methylating step comprises contacting the ΨTP with an N1-pseudouridine methyltransferase, or a fragment or variant thereof, to form N1-methyl-pseudouridine (m1Ψ).

[0116] In another preferred embodiment, the present invention includes a method for producing pseudouridine, wherein the N1-pseudouridine methyltransferase comprises the N1-pseudouridine methyltransferase set forth in SEQ ID NO:8.

[0117] In another preferred embodiment, the present invention includes a method of producing pseudouridine, wherein the generating step comprises contacting the ΨMP with a nucleoside diphosphate kinase (NdK) in the presence of an adenosine triphosphate (ATP) donor.

[0118] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said NdK is adenosyl kinase (AdK) as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0119] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, further comprising the step of isolating said ΨTP.

[0120] In another preferred embodiment, the present invention includes a method for producing pseudouridine further comprising the step of regenerating the ATP donor by contacting adenosine monophosphate (AMP) with a polyphosphate kinase (PPK) or enzyme in the presence of inorganic polyphosphate.

[0121] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said PPK2 is selected from the group consisting of SEQ ID NO: 4, 6, or a fragment or variant thereof.

[0122] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein one or more of the steps of the present invention are carried out in an in vitro transcription system.

[0123] In another preferred embodiment, the present invention comprises a method for producing pseudouridine, wherein said regenerating step is carried out in an in vitro transcription system.

[0124] In another preferred embodiment, the invention comprises a method for producing pseudouridine, wherein the in vitro transcription system comprises an in vitro RNA production system.

[0125] In a preferred embodiment, the present invention includes a system for producing pseudouridine, said system comprising: - a constant amount of the enzyme pseudouridine-5'-phosphate glycosidase (PsuG), a substrate, - a certain amount of uracil nucleobase; - a certain amount of ribose-5-phosphate, A substrate; - the PsuG catalyzes the formation of pseudouridine-5'-monophosphate (ΨMP) from the substrate.

[0126] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the PsuG is selected from SEQ ID NOs: 1-2, or a fragment or variant thereof.

[0127] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the concentrations of uracil nucleobase and ribose-5-phosphate substrate, and reaction temperature cause reverse catalysis of the substrate by PsuG to form ΨMP.

[0128] In another preferred embodiment, the present invention includes a system for producing pseudouridine, further comprising an amount of PPK2 and inorganic polyphosphate, wherein the PPK2 and inorganic polyphosphate catalyze the formation of pseudouridine-5'-triphosphate (ΨTP) from ΨMP.

[0129] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the inorganic polyphosphate comprises sodium hexametaphosphate.

[0130] In another preferred embodiment, the present invention includes a system for producing pseudouridine, further comprising an amount of N1-pseudouridine methyltransferase, or a fragment or variant thereof, which methylates the ΨTP to form N1-methyl-pseudouridine-5'-triphosphate (m1ΨTP).

[0131] In another preferred embodiment, the present invention includes a system for producing pseudouridine, wherein the N1-pseudouridine methyltransferase comprises the N1-pseudouridine methyltransferase set forth in SEQ ID NO:8.

[0132] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, further comprising an ATP regeneration system, said ATP regeneration system comprising: - Polyphosphate kinase (PPK2), - adenosyl kinase (AdK), - inorganic polyphosphates, - adenosine monophosphate (AMP).

[0133] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said NdK is an adenosyl kinase as set forth in SEQ ID NO: 10, or a fragment or variant thereof.

[0134] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said PPK2 is selected from the group consisting of SEQ ID NO: 4, 6, or a fragment or variant thereof.

[0135] In another preferred embodiment, the present invention includes a system for producing pseudouridine, further comprising an in vitro transcription system.

[0136] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein the regenerating step is carried out in an in vitro transcription system.

[0137] In another preferred embodiment, the present invention comprises a system for producing pseudouridine, wherein said in vitro transcription system comprises an in vitro RNA production system.

[0138] In another preferred embodiment, the present invention comprises an isolated nucleotide sequence encoding a peptide selected from the group consisting of SEQ ID NOs: 1-21, or a combination, fragment or variant thereof.

[0139] In another preferred embodiment, the present invention comprises an isolated expression vector having a nucleotide sequence operably linked to a promoter and encoding a peptide selected from the group consisting of SEQ ID NOs: 1-21, or a combination, fragment or variant thereof.

Claims

1. A method for producing pseudouridine, The steps include protecting pseudouridine-5'-monophosphate (ΨMP) with a protective agent, and the protective agent reacting with the hydroxyl group of the ΨMP to form protected ΨMP, The steps include: methylating the protected ΨMP using a methylating agent to form protected N1-methyl-pseudolidine-5'-monophosphate (protected m1ΨMP); The steps include: deprotecting the protected m1ΨMP using a deprotecting agent to form N1-methyl-pseuduridine-5'-monophosphate (m1ΨMP); A method comprising the step of sequentially phosphorylating the m1ΨMP using a phosphorylating agent to form N1-methyl-pseuduridine-5'-diphosphate (m1ΨDP) and N1-methyl-pseuduridine-5'-triphosphate (m1ΨTP).

2. The method according to claim 1, further comprising the step of isolating the m1ΨTP.

3. The method according to claim 1, further comprising the step of combining a uracil nucleic acid base with ribose-5-phosphate to form a ΨMP, wherein the formation of the ΨMP is catalyzed by pseudouridine-5'-phosphate glycosidase, or a functional fragment or variant thereof.

4. The method according to claim 3, wherein, depending on the concentrations of the uracil nucleic acid base and the ribose-5-phosphate, and the reaction temperature, the uracil nucleic acid base undergoes a reverse catalytic reaction with the ribose-5-phosphate to form ΨMP.

5. The method according to claim 3, wherein the pseudouridine-5'-phosphate glycosidase is derived from thermophilic bacteria or enterobacteria.

6. The method according to claim 3, wherein the pseudouridine-5'-phosphate glycosidase comprises a sequence selected from the polypeptide sequences of SEQ ID NOs: 1 and 3, or a sequence having at least 90% homology to SEQ ID NOs: 1 or 3 that catalyzes the formation of pseudouridine-5'-monophosphate (ΨMP) from a uracil nucleic acid base and ribose-5-phosphate.

7. The method according to claim 3, further comprising the step of catalyzing the formation of ribose-5-phosphate from ribose and an adenosine triphosphate (ATP) donor, wherein a ribokinase (RbsK) enzyme, or a functional fragment or variant thereof, catalyzes the transfer of a phosphate group from the ATP donor to the ribose, thereby forming ribose-5'-phosphate and adenosine diphosphate (ADP).

8. The method according to claim 7, wherein ATP is enzymatically regenerated from ADP in the presence of inorganic polyphosphate.

9. The method according to claim 7, wherein the RbsK comprises RbsK from thermophilic bacteria.

10. The method according to claim 7, wherein RbsK comprises the sequence described in SEQ ID NO: 20, or a sequence having at least 90% homology to SEQ ID NO: 20 which catalyzes the formation of ribose-5-phosphate from ribose in the presence of an adenosine triphosphate (ATP) donor.

11. The method according to claim 1, wherein the protective agent comprises an N-silyl compound.

12. The method according to claim 11, wherein the N-silyl compound comprises hexamethyldisilazane (HMDS), and the HMDS reacts with the ΨMP to form a protected ΨMP.

13. The method according to claim 1, wherein the methylating agent comprises iodomethane.

14. The method according to claim 1, wherein the deprotecting agent is selected from ammonia, methanol, or a combination thereof.

15. The method according to claim 1, wherein the phosphorylating agent comprises polyphosphate kinase (PPK2) or a functional fragment thereof, and inorganic polyphosphate, and the PPK2 or the functional fragment catalyzes the sequential phosphorylation of m1ΨMP to form m1ΨDP and m1ΨTP.

16. The method according to claim 15, wherein the concentration of inorganic polyphosphate is excessive, thereby promoting the sequential phosphorylation reaction of m1ΨMP to form m1ΨDP and m1ΨTP.

17. The method according to claim 15, wherein the sequential phosphorylation of m1ΨMP by PPK2 is carried out at a temperature and reaction conditions that cause the sequential phosphorylation of m1ΨMP to cause a sequential reaction in which m1ΨDP and m1ΨTP are formed.

18. The method according to claim 15, wherein the PPK2 comprises PPK2 from thermophilic bacteria.

19. The method according to claim 18, wherein the thermophilic bacterium is selected from Deinococcus diothermalis, Deinococcus radiodurans, or Meiothermus ruba.

20. The method according to claim 15, wherein the PPK2 includes a sequence selected from the polypeptide sequences of SEQ ID NOs: 4, 6, and 21, or a sequence having at least 90% homology to SEQ ID NOs: 4, 6, or 21, which catalyzes the sequential phosphorylation of m1ΨMP in the presence of inorganic polyphosphate to form m1ΨDP and m1ΨTP.

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