Omnivore RNA pyrophosphatase

Abstract

Provided are proteins capable of modifying 5' ends of RNA polynucleotides. The RNA polynucleotides are modified such that all or a portion of a 5' protecting group is removed by the protein. Methods of using the proteins to modify RNA polynucleotides, and kits containing the modified proteins are also provided.

Description

[0001]Attorney Docket No.: 058636.00838 OMNIVORE RNA PYROPHOSPHATASE REFERENCE TO RELATED APPLICATION This application claims priority to U.S provisional application no.63 / 705,202, filed October 9, 2024, the entire disclosure of which is incorporated herein by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under R01 GM035769 and R35 GM145359 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy was created on October 1, 2025, is named “058636.00838.xml” and is 2,751 bytes in size. BACKGROUND Cellular and synthetic mRNAs typically bear a protecting group at the 5' end, such as a cap or oligophosphate. A variety of important methods for analyzing individual RNAs or entire transcriptomes, for synthesizing modified RNAs, or for degrading RNAs require the removal of this protecting group to generate a reactive 5'-terminal monophosphate. However, no enzyme that is currently available is able to efficiently remove diverse 5' protecting groups of many kinds, and some of these enzymes are known to be inhibited by 5'-terminal base pairing. There is accordingly an unmet need for new enzymes and methods of using them that can remove 5’ protecting groups. The present disclosure is pertinent to this need. BRIEF SUMMARY This disclosure provides proteins and methods of using the proteins to cleave a phosphoanhydride bond within a 5’ protecting group that is an oligophosphate or a cap that is covalently attached to the 5’ end of an RNA polynucleotide. A representative protein of the disclosure is referred to herein as “Omnivore.” In examples, exposure of the protein to an RNA that does not have a monophosphate at its 5 end, but has another structure as described herein at its 5’ end, generates an RNA polynucleotide with a monophosphate at its 5’ end. In examples, an isolated or recombinant protein of this disclosure may comprise at least one modification relative to a naturally occurring protein. In an example, the modification comprises a protein purification tag. In examples, a protein of this disclosure is capable of removing all or a portion of a 5' protecting group that comprises an oligophosphate. In examples, a protein of this disclosure is capable of removing all or a portion of a 5' protecting group that is methylated or non- methylated. In examples, a protein of this disclosure can modify a 5' protecting group that is present on a eukaryotic RNA, a bacterial RNA, an archaeal RNA, a viral RNA, or a synthetic RNA. In examples, a 5’ cap of a polynucleotide that is modified using a described protein comprises an oligophosphate or any chemical moiety covalently joined to the 5′ end of the polynucleotide via an oligophosphate bridge. In examples, a described protein is used to modify a polynucleotide having a 5’ protecting group that comprises a 7-methylguanosine triphosphate (m7Gp3), or a nicotinamide adenine dinucleotide (NAD or NADH), or an N- acetylglucosamine-, glucose-, or galactose-containing cap structure, or a flavin adenine dinucleotide (FAD or FADH2), or a nucleoside tetraphosphate (Np4), or a 3′-dephospho- coenzyme A (dpCoA). Representative structures that can be modified by the proteins of this disclosure are provided below. In an example, the disclosure provides a method of using the described proteins. In an example, the method comprises performing a reaction that comprises exposing RNA polynucleotides comprising 5' protecting groups to a described protein to thereby facilitate removal of all or a portion of the 5' protecting group from the RNA polynucleotides. This results in the RNA polynucleotides comprising 5’ monophosphates at their 5’ ends. In examples, the method is performed at a temperature that is higher than 37°C. In examples, the method is performed at a temperature that is approximately 55°C - 75°C. The method may further include separating polynucleotides from which all or a portion of 5' protecting groups have been removed from the reaction. In an example, the disclosure provides kits. The kits include a described protein and at least one sealed or sealable container in which the protein is held. The kits may further include printed material providing instructions for using the protein for removal of all or a portion of the 5' protecting groups from polynucleotides bearing the 5' protecting groups. Isolated polynucleotides encoding a described protein are included. The isolated polynucleotides may be in the form of an expression vector, such as a plasmid, or other polynucleotide that can be introduced into cells such that the cells produce the protein. In an example, a cDNA encoding a described protein is included provided. In an example, the disclosure provides a method of making a described protein by expressing the protein from an expression vector within cells such that the recombinant protein is produced by the cells. The protein may be separated from the cells and purified to any desired degree of purity. The purified protein may be frozen in a buffer that comprises any suitable components, non-limiting examples of which include glycerol, and / or other preservative agents. The protein may be provided in a dry form for reconstitution and use in the described methods. The kits may also include buffers for performing a described method, and / or reagents for preparing the buffer. BRIEF DESCRIPTION OF FIGURES FIG.1. Image of a blot of an electrophoresis gel showing conversion of triphosphorylated and diphosphorylated RNA 5’ ends to a 5′ monophosphate. In vitro transcribed yeiP RNA bearing a 5′ triphosphate (TriP) or diphosphate (DiP) was treated with Omnivore RNA pyrophosphatase (O), E. coli RppH (R), or mRNA Decapping Enzyme (D) or left untreated (–) and then tested for the ability of the resulting 5’ terminus to undergo splinted ligation to a DNA oligonucleotide by T4 DNA ligase. For comparison, yeiP RNA bearing a 5′ monophosphate (MonoP) was subjected to ligation without prior treatment with a pyrophosphatase (PPase). Only Omnivore and RppH efficiently converted the 5′ triphosphate and 5′ diphosphate to a ligatable 5′ monophosphate. FIG.2. Images of blots of electrophoresis gels showing decapping of RNA 5’ ends. (Panel A) Removal of m7Gp3, Ap4, and NAD caps. In vitro transcribed yeiP RNA bearing an m7Gp3, Ap4, or NAD cap was treated with Omnivore RNA pyrophosphatase (O), E. coli RppH (R), or mRNA Decapping Enzyme (D) or left untreated (–) and then subjected to boronate gel electrophoresis to separate capped from uncapped transcripts. PPase, pyrophosphatase. (Panel B) Removal of N-acetylglucosamine and glucose caps. In vitro transcribed ompX RNA bearing an N-acetylglucosamine (GlcNAc) or glucose (Glc) cap was treated with Omnivore RNA pyrophosphatase, E. coli RppH, or mRNA Decapping Enzyme and then subjected to electrophoresis on a 10% polyacrylamide-urea gel to separate capped from uncapped transcripts. (Panel C) Removal of galactose caps. In vitro transcribed ompX RNA bearing a galactose (Gal) cap was treated with Omnivore RNA pyrophosphatase and then subjected to electrophoresis on a 10% polyacrylamide-urea gel to separate capped from uncapped transcripts. (Panel D) Removal of FAD caps. In vitro transcribed yeiP RNA bearing an FAD cap was treated with Omnivore RNA pyrophosphatase, E. coli RppH, or mRNA Decapping Enzyme and then tested for the ability of the resulting 5 terminus to undergo splinted ligation to a DNA oligonucleotide by T4 DNA ligase. The capped RNA substrate also contained a significant amount of uncapped yeiP RNA which evidently was monophosphorylated and therefore underwent ligation even without prior treatment with a pyrophosphatase. Only Omnivore efficiently removed all seven types of caps. FIG.3. Images of blots of electrophoresis gels showing sequence-independent decapping of RNA 5’ ends by Omnivore. (Panel A) In vitro transcribed yeiP RNA sequence variants bearing an m7Gp3cap followed immediately by G at the first position and A, G, C, or U at the second position were treated with Omnivore RNA pyrophosphatase (O) to convert the 5′ end to a monophosphate and then with the 5′-monophosphate-dependent exonuclease XRN1 to selectively degrade monophosphorylated RNA products. The cap of these RNA substrates was 3′-O-methylated as a result of synthesis in vitro with an anti- reverse cap analog (ARCA). (Panel B) In vitro transcribed yeiP RNA bearing an m7Gp3cap followed immediately by A at the first position and G at the second position was treated with Omnivore (O) and then subjected to boronate gel electrophoresis to separate capped from uncapped RNA. The cap of this RNA substrate was not 3′-O-methylated. (Panel C) Human Rps12 mRNA, which begins m7Gp3CC..., was extracted from cultured cells, treated with Omnivore (O), and then subjected to boronate gel electrophoresis to separate capped from uncapped RNA. Omnivore efficiently removed the cap in every case, irrespective of the 5′- terminal sequence of the RNA and methylation. FIG.4. Image of a blot of an electrophoresis gel showing structure-independent decapping of RNA 5 ends by Omnivore. In vitro transcribed yeiP RNA bearing an m7Gp3cap followed immediately by a 5’-terminal stem-loop (AGCGAAAAUAAUUUCGCU (SEQ ID NO:2)) was treated with Omnivore RNA pyrophosphatase (O), E. coli RppH (R), or mRNA Decapping Enzyme (D) and then subjected to boronate gel electrophoresis to separate capped from uncapped transcripts. Only Omnivore and mRNA decapping enzyme efficiently removed the cap despite the base-paired 5’ end. FIG 5. Images of blots of electrophoresis gels showing effects of temperature on decapping by Omnivore. In vitro transcribed yeiP RNA bearing (Panel A) an m7Gp3cap or (Panel B) an NAD cap was treated with Omnivore RNA pyrophosphatase at various temperatures and then subjected to boronate gel electrophoresis to separate capped from uncapped transcripts. Omnivore is active at every temperature tested and most effective at 55-75°C. DETAILED DESCRIPTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For instance, in examples, the disclosure provides an enzyme that is used to remove a 5’ cap from an RNA polynucleotide. The enzyme may be referred to herein as a protein, and as “Omnivore.” Caps other than oligophosphates are referred to in the art using various terminologies, some of which refer only to the chemical moiety joined to the first RNA nucleotide, while the names of certain other caps refer to both the additional chemical moiety and the first RNA nucleotide, such as NAD, FAD, UDP-GlcNAc, and dpCoA. But regardless of variations in nomenclature, the disclosure demonstrates removal of a wide variety of 5’ end cap structures from RNA polynucleotides such that a monophosphate remains at the 5′ end of the polynucleotide. Thus, any discrepancy in the chemical name of a cap that exists in the art is governed by the presence of a 5’ monophosphate at the 5’ end of the RNA polynucleotide once the RNA polynucleotide is acted on by a described enzyme. Likewise, the manner of cleavage is such that a 5’ monophosphate is present at the 5’ end of an RNA polynucleotide after the RNA polynucleotide is acted on by a described enzyme. In an example, a described enzyme cleaves a phosphoanhydride bond to leave a 5’ monophosphate at the 5’ end of an RNA polynucleotide. In another example, a described enzyme cleaves a phosphoanhydride bond to leave a 5’ oligophosphate at the 5’ end of an RNA polynucleotide, which then reacts again with the enzyme to convert the 5’ oligophosphate to a 5’ monophosphate. In examples, a described enzyme cleaves a phosphoanhydride bond within an oligophosphate or cap that is covalently attached to the 5’ end of an RNA polynucleotide. In examples, an oligophosphate and other structures that are comprised by a 5’ cap are referred to herein as a 5’ protecting group. As used in the specification and the appended claims, the singular forms “a” "and” and “the" include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example + / -10%. This disclosure includes every amino acid sequence described herein and all nucleotide sequences encoding the amino acid sequences. Every sequence having 50-99% identity, inclusive, and including and all numbers and ranges of numbers there between, with the sequences provided here are included in the invention. All of the amino acid sequences described herein can include amino acid substitutions, such as conservative substitutions, that do not adversely affect the function of the protein that comprises the amino acid sequences. All sequences that are described by reference to a database are incorporated herein by reference as the sequences exist in the database as of the effective filing date of this application or patent. All sequences referred to in publications are incorporated herein by reference. To demonstrate examples of the invention, an enzyme isolated from Aquifex aeolicus is used to modify RNA polynucleotides that comprise a variety of different 5’ caps, illustrating a broadly applicable utility. This enzyme, previously referred to as aq_158 protein, comprises the sequence: MKKEFSAGGVLFKDGEVLLIKTPSNVWSFPKGNIEPGEKPEETAVREVWEETGVKGEILDYI GEIHYWYTLKGERIFKTVKYYLMKYKEGEPRPSWEVKDAKFFPIKEAKKLLKYKGDKEIFEK ALKLKEKFKL (SEQ ID NO:1). While examples of using the protein comprising SEQ ID NO:1 are presented in this disclosure, it is expected that enzymes with similar capacity can be identified and used in a described method. In examples, the protein is at least 50% identical to the sequence MKKEFSAGGVLFKDGEVLLIKTPSNVWSFPKGNIEPGEKPEETAVREVWEETGVKGEILDYI GEIHYWYTLKGERIFKTVKYYLMKYKEGEPRPSWEVKDAKFFPIKEAKKLLKYKGDKEIFEK ALKLKEKFKL (SEQ ID NO:1) but retains the same or similar activity for the protein of SEQ ID NO:1. The disclosure includes all amino acid sequences that are 50%-99% identical to SEQ ID NO:1 across its entire length. Proteins comprising or consisting of the sequence of SEQ ID NO:1 are included in the disclosure, with the proviso that if a purification tag is included, it can be excluded from the protein. In examples, the disclosure includes the proviso that the described enzymes and methods of using them may exclude the E. coli- derived RNA 5’ pyrophosphohydrolase RppH and any plant-derived acid pyrophosphatases. The disclosure includes any protein with a described activity that is obtained or derived from a member of the phylum Aquificota or Nitrospirota. In examples, the protein is isolated from a microorganism. In an example, a recombinant polynucleotide that encodes a described protein is introduced into host cells that do not encode the same described protein. In example, the host cells may of any microorganism type. Cells modified to encode a described protein that is not encoded by the cells absent the modification are included in the disclosure. In examples, a polynucleotide that is modified by a described protein is capable of being further modified by an RNA ligase or DNA ligase that requires a 5’ monophosphate to exhibit its ligase activity, by a 5’-to-3’ exoribonuclease that requires a 5’ monophosphate to exhibit its 5’-to-3’ exoribonuclease activity, or by a phosphatase that requires one or more unprotected phosphates to exhibit its phosphatase activity. In examples, a described enzyme is capable of removing a plurality of distinct 5’ caps. In examples, a described enzyme is capable of removing a plurality of distinct 5’ caps from a mixture of RNA polynucleotides wherein individual polynucleotides in the mixture each have distinct cap structures. In examples, a described enzyme is capable of removing all or a portion of a 5' cap from a polynucleotide that contains one or more methylated nucleotides. In examples, a described enzyme is used to modify an RNA polynucleotide wherein the 5’ cap comprises an oligophosphate or any chemical moiety covalently joined to the 5′ end of the polynucleotide via an oligophosphate bridge. In examples, the 5' protecting group is present on a eukaryotic RNA, a bacterial RNA, an archaeal RNA, a viral RNA, or a synthetic RNA. In examples, the RNA is an RNA that is capable of being translated into protein, i.e., an mRNA. In examples, a described protein is capable of removing all or a portion of a 5' protecting group that comprises an oligophosphate, including but not limited to di- and tri- phosphates. In examples, the 5’ protecting group comprises 7-methylguanosine triphosphate (m7Gp3), or nicotinamide adenine dinucleotide (NAD or NADH), or an N-acetylglucosamine-, glucose-, or galactose-containing cap structure, or flavin adenine dinucleotide (FAD or FADH2), or a nucleoside tetraphosphate (Np4), or 3′-dephospho-coenzyme A (dpCoA). In an example, the described enzyme is capable of removing all or a portion of a 5’ protecting group, wherein the 5' segment of the polynucleotide covalently joined to the protecting group is base paired. In examples, a described enzyme is used to modify an RNA polynucleotide which, prior to modification, comprises any of the following 5’ protecting groups. In these structural diagrams, the potential for variation in ribonucleoside identity (“Any nucleoside”) or the lack thereof (“Adenosine” or “Uridine”) is indicated. 5 G G′ H 5 In examples, a described enzyme of this disclosure may be modified relative to its wild type form. In examples, the enzyme comprises any suitable purification tag, including but not limited to a polyhistidine tag such as 5-10 histidine residues, a Glutathione-S- Transferase tag, Maltose Binding Protein, Calmodulin Binding Peptide, an Intein-Chitin Binding Domain, a streptavidin component, e.g., a strep-tag, biotinylation, a thioredoxin molecule that has a cluster of histidines, an epitope tag, a SUMO tag, a mutant haloalkane dehalogenase, or the protein may be biotinylated, or include other modifications for use as protein purification tags that will be known to those skilled in the art. In an aspect, the disclosure provides a method comprising performing a reaction, the reaction comprising exposing RNA polynucleotides comprising 5' protecting groups to a described enzyme, to thereby facilitate removal of all or a portion of the 5' protecting group from the polynucleotides such that the resulting RNA polynucleotides comprise a 5’ monophosphate at their 5’ ends. The reaction may be performed at a range of temperatures, and using any suitable buffer. In a non-limiting example, a suitable buffer comprises one or a combination of a salt, a reducing agent, magnesium, a pH buffering agent such as HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), or other components. The buffer may be used at a pH of approximately 7.0 – 8.0. In examples, the method is performed at temperature that is more than 37°C. In examples, the method is performed at temperature that is between approximately 55°C and 75°C. The method may further comprise separating RNA polynucleotides that have been processed according to the method, and purifying them to any desired degree of purity. The method may further comprise determining the sequence of the separated RNA polynucleotides. In an aspect, the disclosure provides articles of manufacture, such as kits, that contain a described enzyme. A kit may comprise one or more sealed or sealable containers that contain the described enzyme, and may also comprise such containers that contain a suitable buffer for performing a described reaction. The kit may also include printed information that provides instructions for using a described enzyme in a described method. A described enzyme may be provided in a form that can be used for reconstitution of the protein, such as a lyophilized or powder form. A described enzyme may also be provided in a frozen form. Non-limiting representations of the disclosure are provided by the accompanying figures. The enzyme designated “Omnivore” in the figure legends comprises the sequence of SEQ ID NO:1. FIG.1 provides representative results demonstrating conversion of triphosphorylated and diphosphorylated RNA 5’ ends to a monophosphate. As shown in the photographic representation of the gel blot, only Omnivore and RppH efficiently converted the 5′ triphosphate and 5′ diphosphate to a ligatable 5′ monophosphate. The yeiP transcript is an E. coli mRNA which encodes a paralog of elongation factor EF-P and is rapidly degraded by a 5′-end-dependent mechanism in cells that express the E. coli RNA pyrophosphohydrolase RppH. FIG.2 provides representative results demonstrating decapping of 7 differently capped RNA 5’ ends, and a comparison of decapping by Omnivore, E. coli RppH, or mRNA Decapping Enzyme. As shown by the photographic representations of the gel blots, only Omnivore efficiently removed all seven types of caps. mRNA Decapping Enzyme is a tradename of a product sold by New England Biolabs. It is an engineered version of a protein complex present in Schizosaccharomyces pombe. FIG.3 demonstrates sequence-independent decapping of RNA 5′ ends by Omnivore. The photographic representations of the gel blots show that RNA variants bearing an m7Gp3cap are efficiently decapped, regardless of whether the nucleotide at the first position is A, G, or C, regardless of whether the nucleotide at the second position is A, G, C, or U, regardless of whether the cap is 3′-O-methylated, regardless of whether the RNA is synthetic or extracted from mammalian cells, and regardless of whether the nucleotide at the first position is 2′-O-methylated, as would be expected for mRNA extracted from mammalian cells. FIG.4 demonstrates structure-independent decapping of base-paired RNA 5′ ends by Omnivore despite the presence of a stem-loop at the RNA 5’ end, as shown in the photographic representation of the gel blot. FIG.5 demonstrates that Omnivore is active at every temperature tested and most effective at 55-75°C, as shown in the photographic representation of the gel blots. The disclosure is illustrated by the examples discussed above and shown in the figures, which are not intended to be limiting.

Claims

What is claimed is:

1. A recombinant or isolated protein for use in cleaving a phosphoanhydride bond within a 5’ protecting group that is an oligophosphate or a cap that is covalently attached to the 5’ end of an RNA polynucleotide, such that exposure to the recombinant protein generates an RNA polynucleotide with a monophosphate at its 5’ end, wherein optionally the recombinant protein comprises at least one modification relative to a naturally occurring protein, and wherein said modification optionally comprises a protein purification tag, and wherein the amino acid sequence of the recombinant protein is at least 50% identical to the sequence MKKEFSAGGVLFKDGEVLLIKTPSNVWSFPKGNIEPGEKPEETAVREVWEETGVKGEILDYI GEIHYWYTLKGERIFKTVKYYLMKYKEGEPRPSWEVKDAKFFPIKEAKKLLKYKGDKEIFEK ALKLKEKFKL (SEQ ID NO:1).

2. The recombinant protein of claim 1 wherein the protein is capable of removing all or a portion of a 5' protecting group that comprises the oligophosphate.

3. The recombinant protein of claim 1, wherein the protein is capable of removing all or a portion of a 5' protecting group that is methylated or non-methylated.

4. The recombinant protein of claim 1, wherein the 5' protecting group is present on a eukaryotic RNA, a bacterial RNA, an archaeal RNA, a viral RNA, or a synthetic RNA.

5. The recombinant protein of claim 1, wherein the 5’ cap comprises an oligophosphate or any chemical moiety covalently joined to the 5′ end of the polynucleotide via an oligophosphate bridge.

6. The recombinant protein of claim 1, wherein the 5’ protecting group comprises a 7- methylguanosine triphosphate (m7Gp3), or a nicotinamide adenine dinucleotide (NAD or NADH), or an N-acetylglucosamine-, glucose-, or galactose-containing cap structure, or a flavin adenine dinucleotide (FAD or FADH2), or a nucleoside tetraphosphate (Np4), or a 3′- dephospho-coenzyme A (dpCoA).

7. The recombinant protein of any one of claims 1-6, wherein the recombinant protein is capable of removing all or a portion of a 5’ protecting group comprised by the following structures:5H 8. The recombinant protein of claim 7, wherein the protein is capable of removing all or a portion of a 5’ protecting group, wherein the 5' segment of the polynucleotide covalently joined to the protecting group is base paired.

9. A method comprising performing a reaction, the reaction comprising exposing RNA polynucleotides comprising 5' protecting groups to a recombinant protein of claim 7, to thereby facilitate removal of all or a portion of the 5' protecting group from the polynucleotides such that the resulting RNA polynucleotides comprise a 5’ monophosphate at their 5’ ends.

10. The method of claim 9, wherein the method is performed at a temperature that is more than 37°C.

11. The method of claim 9, wherein the method is performed at a temperature that is between approximately 55°C and 75°C.

12. The method of claim 9, further comprising separating polynucleotides from which all or a portion of 5' protecting groups have been removed from the reaction.

13. A kit comprising a recombinant protein of claim 7, the kit further comprising at least one sealed or sealable container in which the recombinant protein is held.

14. The kit of claim 13, further comprising printed material providing instructions for using the recombinant protein for removal of all or a portion of the 5' protecting groups from polynucleotides bearing the 5' protecting groups.

15. A cDNA encoding a recombinant protein of claim 7.

16. A method of making a protein of claim 7, the method comprising expressing the protein from an expression vector within cells such that the recombinant protein is produced by the cells, and separating the recombinant protein from the cells.

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