Cap formation assay

Abstract

The present disclosure relates to a method for quantifying mRNA capping efficiency. The method uses a detectable label to quantify the amount of unlabeled mRNA in a sample. The method is applicable to RNA molecules of any sequence, and can be used to obtain information on the physical properties of an RNA sample. The present disclosure also relates to a kit that can be used to quantify mRNA capping efficiency.

Description

[Technical Field]

[0001] Related application data This application claims priority from U.S. Provisional Patent Application No. 63 / 482,343, filed January 31, 2023, entitled "Capping Assay," the entire contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application is filed with an electronic Sequence Listing, the entire contents of which are incorporated herein by reference.

[0003] The present disclosure relates to methods for quantifying mRNA capping efficiency. The present disclosure also relates to kits that can be used to quantify mRNA capping efficiency. [Background technology]

[0004] Vaccines are an important health intervention to prevent infectious diseases. The recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has seen the unprecedented development of multiple vaccines in a very short period of time, with mRNA vaccines being used for the first time as part of a global vaccination strategy.

[0005] mRNA vaccines contain synthetic mRNA molecules that encode antigens that generate immune responses. Synthetic mRNAs have a structure similar to endogenous mRNAs, typically containing, from 5' to 3', a 5' cap, a 5' untranslated region (5' UTR), an open reading frame encoding the antigen, a 3' UTR, and a poly(A) tail. Therapeutic use of mRNA, including in vaccines, requires the synthesis of large quantities of mRNA, and thorough characterization of properties such as cap formation. At this scale, characterization of mRNA requires quantitative, robust, accurate, and rapid processing of large numbers of samples. Previous methods for studying the 5' cap have traditionally incorporated the 5' cap as a phosphate. 32Previous methods have relied on radiolabeled detection of P. More recently, capping efficiency has been measured using biotin-tagged RNase H cleavage probes complementary to the 5' end of the mRNA. A drawback of this method is that it requires a specific probe for each mRNA being analyzed. Thus, there remains a need for assays that can be used to characterize mRNA for therapeutic purposes, particularly assays that can be used to quantitatively measure the percentage of mRNA that has a 5' cap. Summary of the Invention

[0006] The present application provides methods for quantitatively determining the capping efficiency of mRNA, particularly mRNA synthesized using in vitro transcription. Capping efficiency can be expressed, for example, as the percentage or percent of capped mRNA present in a sample relative to the total mRNA present in the sample.

[0007] Accordingly, the present disclosure provides a method for quantifying mRNA capping efficiency, the method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy labeling reagent to form a treated mRNA sample; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; The treated mRNA sample optionally comprises a heavily labeled 5' cap.

[0008] In one example, the heavy labeling reagent comprises a heavily labeled substrate and / or cofactor. In one example, the heavily labeled substrate and / or cofactor comprises heavy GTP or heavy SAM, or a combination thereof. In one example, the heavily labeled substrate and / or cofactor comprises heavy GTP and / or heavy SAM. In one example, the heavily labeled substrate and / or cofactor comprises 13 C. 15In one example, the heavily labeled substrate and / or cofactor comprises: N-GTP, and / or CD3-SAM. 13 C 10 , 15 The present disclosure also provides a method for quantifying mRNA capping efficiency, the method comprising: treating the mRNA sample with a capping enzyme in the presence of heavy GTP to form a treated mRNA sample; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; The treated mRNA sample optionally comprises a heavily labeled 5' cap.

[0009] The present disclosure also provides a method for quantifying mRNA capping efficiency, the method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy SAM to form a treated mRNA sample; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; The treated mRNA sample optionally comprises a heavily labeled 5' cap.

[0010] The present disclosure also provides a method for quantifying mRNA capping efficiency, the method comprising: treating the mRNA sample with a capping enzyme in the presence of heavy GTP and heavy SAM to form a treated mRNA sample; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; The treated mRNA sample optionally comprises a heavily labeled 5' cap.

[0011] In one example, the mRNA sample includes mRNA with an unlabeled 5' cap. In one example, the processed mRNA sample includes mRNA with a heavy-labeled 5' cap. In one example, the processed mRNA sample includes mRNA with an unlabeled 5' cap and a heavy-labeled 5' cap.

[0012] In one example, heavy GTP is 13 C. 15 In one example, heavy GTP is 13 C 10 , 15 In one example, the heavy GTP is represented by Formula I: [ka] The compound has the formula:

[0013] In one example, the heavy SAM comprises a D-SAM. In one example, the heavy SAM comprises a CD3-SAM. In one example, the heavy SAM has Formula II: [ka] The compound has the formula:

[0014] In one example, the nuclease comprises RNAse T1, nuclease NP1, or RNAse A, or a combination thereof. In one example, the nuclease comprises RNAse T1. In one example, the nuclease comprises nuclease NP1.

[0015] In one example, the step of quantitatively determining the amount of heavy-labeled and unlabeled 5' caps in the sample includes analyzing the released 5' caps by liquid chromatography / mass spectrometry (LC-MS) and measuring the relative amounts of heavy-labeled and unlabeled 5' fragments.

[0016] In one example, the capping enzyme comprises a vaccinia capping enzyme or a poxvirus capping enzyme. In one example, the capping enzyme comprises a vaccinia capping enzyme. In one example, the capping enzyme comprises a triphosphatase, a guanyltransferase, or a guanine methyltransferase, or a combination thereof. In one example, the capping enzyme comprises an mRNA cap 2'-O-methyltransferase.

[0017] In one example, the mRNA sample is synthesized by in vitro transcription. In one example, the mRNA sample includes capped mRNA produced by a post-transcriptional or co-transcriptional capping reaction. In one example, the mRNA sample includes capped mRNA, uncapped mRNA, or unmethylated capped mRNA, or a combination thereof.

[0018] In one example, an mRNA sample (i.e., an RNA sample used in the methods described herein) may contain any of the following cap analogs: cap 0, cap 1, cap 2, cap 4, anti-reverse cap analog (ARCA), inosine, N7,2'-O-dimethyl-guanosine (mCAP), N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (mCAP), ...'-fluoro-guanosine, 7 G), and CAP-003-CAP-225. In one example, the 5' cap is cap 0 or cap 1. In one example, the 5' cap is cap 0. In one example, the 5' cap is cap 1.

[0019] In one example, the mRNA sample (i.e., the RNA sample used in the methods described herein) comprises capped mRNA having five caps selected from the group consisting of m7GTPG, m7GTPGp, m7GTPA, m7GTPAp, m7GTPGm, and m7GTPAm.

[0020] In one example, the mRNA sample comprises a molecule of formula (III): [ka] wherein B is a nucleobase and R 1 is selected from H, halogen, OH, and OCH3; R 2 is selected from H, OH, and OCH3; R 3 is CH3, CH2CH3, CH2CH2CH3, or absent, and R 4 is NH2 and R 5 is OH, n is 1, 2, or 3, M is a nucleotide of mRNA, and The nuclease is RNAse T1.

[0021] In one example, B is guanine or adenine. In one example, B is guanine.

[0022] In one example, R 1 is selected from halogen, OH, and OCH. In one example, R 1 is selected from H, OH, and OCH. In one example, R 1 is selected from OH and OCH. In one example, R 1 is OH.

[0023] In one example, R 1 , R 2 , and R 5 are independently OH.

[0024] In one example, R 3 is CH3. In one example, R 3 does not exist.

[0025] In one example, n=1.

[0026] In one example, the mRNA sample includes capped mRNA with a 5' cap that is cap 0, and the nuclease is RNAse T1.

[0027] In one example, the mRNA sample comprises a molecule of formula (III): [ka] wherein B is a nucleobase and R 1 is selected from H, halogen, OH, and OCH3; R 2 is selected from H, OH, and OCH3; R 3 is CH3, CH2CH3, CH2CH2CH3, or absent, and R 4 is NH2 and R 5 is OH or OCH3, n is 1, 2, or 3, M is a nucleotide in mRNA, and The nuclease is nuclease NP1.

[0028] In one example, R 1 is selected from halogen, OH, and OCH. In one example, R 1 is selected from H, OH, and OCH. In one example, R 1 is selected from OH and OCH. In one example, R 1 is OH.

[0029] In one example, B is guanine or adenine. In one example, B is guanine.

[0030] In one example, R 1 and R 2 are independently OH.

[0031] In one example, R 5 is OH. In one example, R 5 is OCH3.

[0032] In one example, R 3 is CH3. In one example, R 3 does not exist.

[0033] In one example, n=1.

[0034] In one example, the mRNA sample includes capped mRNA having a 5' cap that is cap0 or cap1, and the nuclease is nuclease NP1.

[0035] In one example, the mRNA sample includes uncapped mRNA that has a 5' triphosphate group or a 5' diphosphate group, or a combination thereof.

[0036] In one example, the mRNA sample includes unmethylated capped mRNA with a 5' GpppN group, where N is any nucleotide. In one example, N is G. In one example, N is A.

[0037] In one example, quantifying mRNA capping efficiency includes quantifying the absolute amount of capped mRNA in an mRNA sample. In one example, quantifying mRNA capping efficiency includes quantifying the total amount of labeled and unlabeled capped mRNA in an mRNA sample. In one example, quantifying mRNA capping efficiency includes quantifying the percentage of labeled 5' caps in a digested mRNA sample. In one example, quantifying mRNA capping efficiency includes quantifying the percentage of labeled 5' caps relative to total mRNA in a digested mRNA sample. In one example, quantifying mRNA capping efficiency includes quantifying the percentage of unlabeled 5' caps relative to total mRNA in a digested mRNA sample.

[0038] 1. A kit for quantifying mRNA capping efficiency, the kit comprising one or more of the following: heavy-labeled substrates and / or cofactors, capping enzymes, and nuclease Also provided is the above kit, comprising:

[0039] In one example, the kit comprises a heavy labeling reagent and a capping enzyme. In one example, the kit comprises a heavy labeling reagent and a capping enzyme. In one example, the heavy labeling reagent is a heavy labeling reagent described herein. In one example, the capping enzyme is a capping enzyme described herein. In one example, the nuclease is a nuclease described herein.

[0040] The drawings are for illustrative purposes only and are not intended to be limiting in any way. [Brief explanation of the drawings]

[0041] [Figure 1] The following describes an exemplary method for quantifying mRNA capping efficiency described herein. In this exemplary method, an mRNA sample containing capped and uncapped mRNA is treated with a capping enzyme in the presence of heavy-labeled GTP and unlabeled SAM. The treated mRNA is digested using a nuclease (e.g., the dinucleotide m7GpppGp) to release the heavy-labeled and unlabeled 5' caps. The amount of heavy-labeled and unlabeled 5' caps is quantified using LC-MS, and capping efficiency can be quantified by calculating the relative abundance of unlabeled 5' caps to total 5' caps in the sample, expressed as a percentage (e.g., of total mRNA). [Figure 2] The methods described herein for quantifying mRNA capping efficiency are exemplified. The exemplary method involves treating an mRNA sample containing capped, unmethylated capped, and uncapped mRNA with a capping enzyme in the presence of heavy-labeled GTP and heavy-labeled SAM. The exemplary method can be used to quantify the abundance of cap-0 capped mRNA in a sample. The exemplary method can also be used to quantify the amount of uncapped mRNA, unmethylated capped mRNA, and capped mRNA present in a sample, which can be expressed as a percentage (e.g., of total mRNA). [Figure 3]The methods described herein for quantifying mRNA capping efficiency are exemplified. The exemplary method can be used to quantify the abundance of mRNA capped with Cap 1 in a sample. The exemplary method can be used to quantify the amount of uncapped mRNA, unmethylated capped (Cap 0 or Cap 1) mRNA, and capped (Cap 0 or Cap 1) mRNA present in a sample, which can be expressed as a percentage (e.g., of total mRNA). [Figure 4] 1 shows a proposed reaction mechanism for nucleases such as RNase A or T1, which involves the formation of a cyclic phosphate intermediate. [Figure 5] Figure 1 shows the use of the method described herein to measure mRNA capping efficiency by converting uncapped mRNA to labeled Cap 0. Each trace shown is the total ion chromatogram (TIC) from the corresponding MRM transition for each capped species. A shows results from T1 digestion of uncapped mRNA. B shows results from NP1 digestion of uncapped mRNA. For the uncapped mRNA sample, only the labeled Cap 0 peak is observed, while the unlabeled Cap 0 peak is below the detection limit. C and D show results from T1 (C) and NP1 (D), respectively, for an mRNA sample with unknown capping efficiency. For this mRNA sample, there is a trace labeled peak and a prominent unlabeled peak in the TIC. The areas of the labeled and unlabeled Cap 0 peaks correspond to the abundance of uncapped and capped mRNA in the sample, respectively. [Figure 6]This example demonstrates the use of the methods described herein to quantify mRNA capped species. This example uses dual labeling (heavy methylation with heavy SAM and heavy capping with heavy GTP). (A) shows the results of an exemplary assay on an unmethylated G-capped sample (some uncapped species are present in the background). The results demonstrate the incorporation of heavy methyl groups via heavy SAM into G-capped and uncapped species. (B) shows the results of an exemplary assay on a test article with unknown capping efficiency. The percentage of each capped species can be quantified using relative peak area. (C) shows a blended sample of uncapped, unmethylated capped, and capped mRNA. The measured percentage capping of the blended sample is compared with the expected percentage. Results from RNAse T1 (i) and nuclease P1 (ii) digestion are consistent in all three samples. (iii) summarizes the results. [Figure 7] This example demonstrates the use of the methods described herein to quantify mRNA capped species in serial blend samples. Dual labeling (heavy methylation with heavy SAM and heavy capping with heavy GTP) is used in this example. Figures A, B, and C show the results of an exemplary assay using RNAse T1 on a blend containing uncapped, unmethylated G-capped samples, and m7G-capped species. Figures D, E, and D show the results of an exemplary assay using Nuclease P1 on a blend containing uncapped, unmethylated G-capped samples, and m7G-capped species. The measured percentages of uncapped, G-capped, and m7G-capped species in the blend samples are consistent with expected values ​​(R2 ≥ 0.98).

[0042] Explanation of the sequence listing [Table 1] DETAILED DESCRIPTION OF THE INVENTION

[0043] General Throughout this specification, unless specifically stated otherwise or the context requires otherwise, references to a single step, configuration of events, group of steps or group of events shall be interpreted as encompassing one and more (i.e., one or more) of that step, configuration of events, group of steps or group of events.

[0044] Those skilled in the art will recognize that the present disclosure is susceptible to variations and modifications other than those specifically described, and it is to be understood that the present disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds referred to or indicated herein, individually or collectively, and any combination or any two or more of such steps or features.

[0045] The present disclosure is not to be limited in scope by the specific examples described herein, as such specific examples are intended for illustrative purposes only. Functionally equivalent objects, compositions, and methods are clearly within the scope of the present disclosure.

[0046] Any example of the present disclosure should be construed as applicable mutatis mutandis to any other example of the present disclosure, unless specifically stated otherwise, i.e., any specific example of the present disclosure can be combined with any other specific example of the present disclosure (except where mutually exclusive).

[0047] Any example of the present disclosure disclosing a specific feature or group of features, or a method or method step, is deemed to provide explicit support for disclaiming the specific feature or group of features, or method or method step.

[0048] Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, biochemistry, mass spectrometry, analytical chemistry, and separation science).

[0049] Unless otherwise indicated, the molecular biology, chemistry, biochemistry, cell culture, mass spectrometry, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H. Memes (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions), Ed. Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988), and J.E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all current editions), and other sources.

[0050] The term "and / or," e.g., "X and / or Y," should be understood to mean either "X and Y" or "X or Y," and should be interpreted as providing clear support for both meanings or either meaning.

[0051] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0052] Any discussion of documents, acts, materials, devices, articles or the like contained in this specification should not be construed as an admission that any or all of such matters form part of the prior art or were general general knowledge in the art relevant to this disclosure as existing prior to the priority date of each appended claim.

[0053] As used herein, the term "derived from" should be understood to indicate that the identified end product is obtained from a particular source, but is not necessarily obtained directly from that source. Similarly, the term "based on" should be understood to indicate that the identified end product is derived from or used in a particular source, but is not necessarily derived from or used directly from that source.

[0054] As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to the stated reference value. In one example, the term "approximately" or "about" refers to a range of values ​​that is within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such number may exceed 100% of the possible values).

[0055] Selected Definitions As used herein, the term "nucleoside" or "nucleobase" refers to adenine ("A"), guanine ("G"), cytosine ("C"), uracil ("U"), thymine ("T"), and their analogs, such as D-ribose (in RNA) or 2'-deoxy-D-ribose (in DNA), which are linked to a carbohydrate by an N-glycosidic bond between the anomeric carbon of the carbohydrate (the 1'-carbon atom of the carbohydrate) and the nucleobase. The carbon atom of the ribose present in a nucleotide is indicated with a prime (') to distinguish it from the backbone numbering of the base. When the nucleobase is a purine, e.g., A or G, the ribose sugar is generally attached to the N9 position of the purine heterocycle. When the nucleobase is a pyrimidine, e.g., C, T, or U, the sugar is generally attached to the N1 position of the heterocycle. The carbohydrate can be substituted or unsubstituted. Substituted ribose sugars include, but are not limited to, those in which one or more of the carbon atoms, e.g., the 2'-carbon atom, is replaced with one or more of the same or different Cl, F, -R, -OR, -NR2, or halogen groups, where each R is independently H, C1-C6 alkyl, or C5-C 14Examples of riboses include ribose, 2'-deoxyribose, 2',3'-dideoxyribose, 2'-haloribose, 2'-fluororibose, 2'-chlororibose, and 2'-alkylribose, such as 2'-O-methyl, 4'-α-anomeric nucleotides, 1'-α-anomeric nucleotides (Asseline et al., Nucl. ACIDS RES., 19:4067-74

[1991] ), 2'-4'- and 3'-4'-linked and other "locked" or "LNA" bicyclic sugar modifications (WO98 / 22489, WO98 / 39352, WO99 / 14226).

[0056] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds). An alkyl group can have 1 to 20 carbon atoms (wherever it appears herein, a numerical range such as "1 to 20" refers to each integer within the given range. For example, "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to 20 carbon atoms, but this definition also covers occurrences of the term "alkyl" when no numerical range is specified). An alkyl group can also be a medium-sized alkyl, having 1 to 9 carbon atoms. An alkyl group can also be a lower alkyl, having 1 to 4 carbon atoms. An alkyl group can also be a "C 1-4 By way of example only, "C 1-4 "Alkyl" indicates that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

[0057] As used herein, the term "nucleotide" refers to a nucleoside in phosphorylated form (phosphate ester of the nucleoside), either as a monomer unit or within a polynucleotide polymer. "Nucleotide 5'-triphosphate" refers to a nucleotide having a triphosphate group at the 5' position, sometimes designated as "NTP," "dNTP," and "ddNTP" to specifically point out the structural features of the ribose sugar. The triphosphate group may contain sulfur substitutions for various oxygen moieties, e.g., α-thio-nucleotide 5'-triphosphate. Nucleotides may exist in mono-, di-, or tri-phosphorylated forms.

[0058] As used interchangeably herein, the terms "nucleic acid," "nucleic acid molecule," "polynucleotide," or "oligonucleotide" refer to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single- or double-stranded form, including polymers containing DNA, RNA, and hybrids thereof. The nucleotides may be genomic, synthetic, or semi-synthetic in origin. Unless otherwise indicated, the term encompasses nucleic acid-like structures with synthetic backbones as well as amplification products. As will be understood by those skilled in the art, the length of these polymers (i.e., the number of nucleotides they contain) can vary widely, often depending on their intended function or use. Polynucleotides can be linear, branched linear, or circular molecules. Polynucleotides also contain associated counterions, such as H + , NH4 + , trialkylammonium, Mg 2+ , Na + etc. A polynucleotide can be composed entirely of deoxyribonucleotides, entirely of ribonucleotides, or chimeric mixtures thereof. A polynucleotide can be composed of internucleotide nucleobase and sugar analogs.

[0059] In some examples, the term "oligonucleotide" is used to refer to a polynucleotide comprising from about 2 to about 150 nucleotides, e.g., from about 10 to about 100 nucleotides, from about 15 to about 75 nucleotides, or from about 15 to about 50 nucleotides.

[0060] Throughout this specification, whenever an oligonucleotide is represented by a sequence of letters, the nucleotides are represented in the order of 5'→3' from left to right. "Polynucleotide sequence" refers to the sequence of nucleotide monomers along a polymer. Unless otherwise indicated, whenever a polynucleotide sequence is represented, it will be understood that the nucleotides are in the direction of 5'→3' from left to right.

[0061] The term "3'" refers to a region or position in a polynucleotide or oligonucleotide 3' (i.e., downstream) from another region or position in the same polynucleotide or oligonucleotide. The term "5'" refers to a region or position in a polynucleotide or oligonucleotide 5' (i.e., upstream) from another region or position in the same polynucleotide or oligonucleotide. The terms "3' end" and "3' terminus," when used herein in reference to a nucleic acid molecule, refer to the end of a nucleic acid containing a free hydroxyl group attached to the 3' carbon of the terminal pentose sugar. The terms "5' end" and "5' terminus," when used herein in reference to a nucleic acid molecule, refer to the end of a nucleic acid molecule containing a free hydroxyl or phosphate group attached to the 5' carbon of the terminal pentose sugar. In some embodiments of the invention, the oligonucleotide primer comprises a polyadenosine tract at its 5' end.

[0062] As used herein, the term "stability" of an RNA refers to the "half-life" of the RNA. "Half-life" refers to the period of time required to remove half of the activity, amount, or number of a molecule. In one example, the half-life of an RNA indicates the stability of the RNA.

[0063] As used herein, "poxvirus" (or Poxviridae) refers to a member of a family of brick-shaped or oval-shaped viruses containing a double-stranded DNA genome. For example, but not limited to, poxviruses include vaccinia virus, smallpox virus, rabbitpox virus, monkeypox virus, ectromelia virus, camelpox virus, cowpox virus, mule deerpox virus, myxoma virus, rabbit fibroma virus, swinepox virus, lumpy skin disease virus, sheeppox virus, canarypox virus, infectious epithelioma virus, ovine thrush virus, and bovine papular stomatitis virus, as well as related species, offspring, variants, and derivatives of such poxviruses.

[0064] As used herein, "MS" refers to mass spectrometry, an analytical chemistry technique that helps identify the amount and type of chemicals present in a sample by measuring the mass-to-charge ratio and abundance of gas-phase ions. A mass spectrum (plural: spectra) is a plot of ion signals as a function of mass-to-charge ratio. Many MS techniques are known in the art. For more information, see MS Primer (2015), available from Waters Corporation, Milford MA USA. See also Basiri et al., Bioanalysis 1525-1542 (2014).

[0065] As used herein, "LC" refers to liquid chromatography, a technique used to separate a sample into its individual parts. This separation occurs based on the interaction of the sample with a mobile phase and a stationary phase. Many LC techniques are known in the art. For more information, see Beginners Guide to UPLC (2015) and HPLC Primer (2015), both available from Waters Corporation, Milford MA USA.

[0066] Methods for quantifying cap formation efficiency Typically, mature mRNAs have a "cap" structure at their 5' end, which plays an important role in translation and stability. For example, the 5' cap plays a key role in mRNA metabolism and is required for varying degrees of processing and maturation of RNA transcripts in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA into protein. The 5' cap also protects mRNA from exonucleolytic degradation, such that mRNA lacking a 5' cap is rapidly degraded and is involved in recognition by the translation initiation factor eIF4E, promoting the formation of the translation initiation machinery (Svitkin YV, Cheng YM, Chakraborty T., Presnyak V., John M., Sonenberg N. N1-methyl-pseudouridine in mRNA enhances translation through eIF2alpha-dependent and independent mechanisms by increasing ribosome density. Nucleic Acids Res. 2017;45:6023-6036; Sonenberg N., Gingras A.C. The mRNA 5' cap-binding protein eIF4E and control of cell growth. Curr. Opin. Cell Biol. 1998;10:268-275).

[0067] Several variations exist in naturally occurring 5' cap structures (Wang J., Alvin Chew BL, Lai Y., Dong H., Xu L., Balamkundu S., Cai WM, Cui L., Liu CF, Fu XY, et al. Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA. Nucleic Acids Res. 2019;47:e130;Henderson JM, Ujita A., Hill E., Yousif-Rosales S., Smith C., Ko N., McReynolds T., Cabral CR, Escamilla-Powers JR, Houston ME Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap((R)) Analog by In Vitro Transcription. Curr. Protoc. 2021;1:e39). One example is 7-methylguanosine (m), which is attached via a 5'-5' triphosphate bridge to the 5' end of the first transcribed nucleotide. 7 G), which is m 7 This results in a dinucleotide cap of GpppN, where N is any nucleoside (e.g., G, C, A, or U) and is the first transcribed nucleotide. This is often referred to as Cap 0. Another example is Cap 1 (m), which is methylated at the 2'O position of the first nucleotide. 7 GpppNmpN), and cap 2 (m 7 GpppNmpNm).

[0068] mRNA produced by in vitro transcription for use in vaccines and other therapeutics must also be capped in order for the mRNA to be translated. Generally, there are two methods for capping mRNA produced from in vitro transcription. Post-transcriptional capping involves treating the RNA formed by in vitro transcription with a capping enzyme (usually from vaccinia virus) in the presence of GTP or other capping nucleotides. Co-transcriptional capping involves adding a cap analog to the in vitro transcription reaction, which RNA polymerase incorporates into the 5' end of the mRNA in place of GTP. As will be appreciated by those skilled in the art, both types of capping reactions may not be 100% efficient, and the resulting mixture may contain capped and uncapped mRNA. The resulting reaction mixture may also contain unmethylated capped mRNA. The methods described herein can be used to measure the amount of capped, uncapped, and / or unmethylated capped mRNA in a sample.

[0069] While methods for estimating capping efficiency have been described in the art, the present application provides, inter alia, improved methods for quantifying mRNA capping efficiency. The methods described herein are particularly useful for quantifying the capping efficiency of mRNA synthesized using in vitro translation, e.g., without the need for sequence-specific oligonucleotides or radioisotopes. The described methods can be used as part of a quality control process, e.g., as part of a process to investigate the safety, efficacy, and / or homogeneity of mRNA for therapeutic use.

[0070] In some examples, a method for quantifying mRNA capping efficiency, the method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy labeling reagent to form a treated mRNA sample, optionally comprising mRNA with a heavy-labeled 5' cap; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; This can be expressed, for example, as a proportion or percentage (%) of the total mRNA present in the sample.

[0071] In one example, the mRNA sample includes mRNA with an unlabeled 5' cap. In one example, the processed mRNA sample includes mRNA with a heavy-labeled 5' cap. In one example, the processed mRNA sample includes mRNA with an unlabeled 5' cap and a heavy-labeled 5' cap.

[0072] The methods described herein involve treating an mRNA sample with a capping enzyme and a heavy labeling reagent (e.g., heavy GTP and / or heavy SAM). Depending on the efficiency of the original capping reaction, the mRNA sample will contain one or more of capped mRNA, uncapped mRNA, and unmethylated capped mRNA.

[0073] As used herein, a "capped mRNA" comprises at least one 5' cap structure. 5' cap structures are known to those skilled in the art. In one example, the 5' cap structure is as described herein. In one example, a capped mRNA is an mRNA comprising an mRNA fragment of ... 7 In one example, the capped mRNA contains GpppN (cap 0). 7 Contains GpppmN (cap 1).

[0074] As will be understood by those skilled in the art, "uncapped mRNA" includes any mRNA that does not contain a 5' cap structure, including, for example, mRNAs with a 5' triphosphate, 5' diphosphate, or 5' monophosphate group. In one example, an "uncapped mRNA" is an mRNA with a 5' triphosphate and / or a 5' diphosphate group. In one example, an "uncapped mRNA" is an mRNA with a 5' triphosphate group.

[0075] GMP from GTP is added to the 5' end of the RNA by guanyltransferase, generating a guanosine (GpppN) cap. This is followed by methylation of the 7-nitrogen of the guanine in the cap nucleotide by guanine-7-methyltransferase. If the subsequent reaction is not 100% complete, the reaction mixture will contain uncapped mRNA, unmethylated capped RNA (e.g., RNA with a GpppN cap, also referred to herein as G-capped RNA), and m 7 It may contain RNA with a GpppN cap (also referred to herein as m7G capping). Thus, in one example, unmethylated capped mRNA is an intermediate formed in the synthesis of mRNA with cap 0. In one example, unmethylated capped mRNA includes mRNA with a GpppN cap, where N is the first transcribed nucleotide. In one example, N is G. In one example, N is A. In other words, the 5' guanine is N 7 Using the methods described herein, methylation efficiency, e.g., N 7 The efficiency with which the ribose 2'OH is methylated to form cap 0 or the 2'OH of ribose is methylated to form cap 1, for example, can also be measured.

[0076] Using the methods described herein, the amount of capped mRNA, uncapped mRNA, and / or unmethylated capped mRNA in a sample can be measured, which can then be used to quantitatively measure capping efficiency. A processing step forms a processed mRNA sample. The processing step is carried out under conditions that allow for the formation of mRNA containing a heavy-labeled 5' cap. As will be understood by those skilled in the art, capped mRNA in an mRNA sample will not react with labeled GTP and will remain unlabeled. On the other hand, uncapped mRNA will react with labeled GTP in the presence of a capping system to form labeled mRNA. Therefore, the processed mRNA sample contains a mixture of labeled capped mRNA and unlabeled capped mRNA. The amount of labeled capped mRNA depends on the amount of uncapped mRNA in the mRNA sample being tested. If the mRNA sample being tested is entirely capped, the processed mRNA sample should not contain any labeled capped mRNA. This is illustrated in Figure 1.

[0077] The method further comprises contacting the processed mRNA with a nuclease. As will be appreciated by those skilled in the art, the contacting step comprises digesting the processed mRNA with a nuclease. Any nuclease suitable for digesting single-stranded RNA can be used. Digestion of the mRNA with a nuclease liberates the 5' cap. Depending on the nuclease used and the sequence of the mRNA, the liberated 5' cap can form part of a 5' cap-containing oligonucleotide (e.g., m 7 GpppN(N) a-p, where each occurrence of N is independently any nucleotide, a is an integer, and p is phosphate. As will be appreciated, nuclease treatment of a labeled mRNA sample results in a mixture of free nucleotides and heavy-labeled and unlabeled 5' caps that can be detected, for example, by LC-MS. As an example, RNAse T1, a nuclease that cleaves the phosphodiester bond between the 3'-guanyl and 5'-OH residues of adjacent nucleotides, can be used to produce m 7 The released 5' cap can be used to form an oligonucleotide with a 3' Cp or Up, e.g., m 7 GpppG(N) m (C / U)p, where each occurrence of N is independently any nucleotide and m is a positive integer, e.g., 0 to 10. In one example, N is G or A. As will be understood by one of skill in the art, the amount of labeled cap or labeled cap-containing oligonucleotide produced will be related to the amount of uncapped and / or unmethylated mRNA in the original sample.

[0078] The ratio of labeled and unlabeled capped mRNA in a sample can be measured by quantifying the amount of unlabeled caps and / or labeled caps present in the digested sample. This ratio can be used to quantify mRNA capping efficiency. The percentage of labeled and / or unlabeled capped mRNA relative to total mRNA in a sample can be measured by quantifying the amount of unlabeled caps and / or labeled caps present in the digested sample. The amount of labeled and unlabeled capped mRNA in a sample can be measured using any method known to those skilled in the art. In one example, the amount of capped and uncapped mRNA is measured by mass spectrometry, e.g., high-resolution mass spectrometry. In one example, mass spectrometry analysis quantifies labeled and unlabeled 5' caps by targeted multiple reaction monitoring (MRM). In one example, a triple quadrupole mass spectrometer is used.

[0079] Various mass spectrometry systems can be used in the methods described herein to quantify biomolecules in samples, such as mRNA 5' caps. Mass analyzers with high mass accuracy, high sensitivity, and high resolution include, but are not limited to, matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometers, electrospray ionization time-of-flight (ESI-TOF) mass spectrometers, Fourier transform ion cyclotron mass spectrometers (FT-ICR-MS), and ORBITRAP™ analytical instruments. Other modes of MS include ion traps and triple quadrupole mass spectrometers. In ion trap MS, analytes are ionized by electrospray ionization or MALDI and then transferred to the ion trap. The trapped ions can then be individually analyzed by MS upon selective release from the ion trap. Ion traps can also be combined with the other types of mass spectrometers mentioned above.

[0080] For high-resolution separation, liquid chromatography ESI-MS / MS or automated LC-MS / MS can be used, which utilizes capillary reverse-phase chromatography as the separation method. Other separation systems known to those skilled in the art can be used to separate the products of interest prior to MS analysis. Systems that can be coupled to MS include, but are not limited to, capillary electrophoresis and gas chromatography. Exemplary techniques applicable to characterizing RNA include ion mobility separation, EXD (EAD / ECD / ETD) fragmentation, and MS / MS analysis.

[0081] Mass spectrometry analysis of biomolecules in a sample can be targeted, increasing sensitivity by, for example, 10-100 times or more using SIM (selected ion monitoring) scans. SIM scans selectively accumulate only specific mass windows, increasing sensitivity. For example, SIM scans can scan a mass range that includes the predicted masses of labeled and / or unlabeled 5' caps. Using SIM scans, accurate quantification can then be achieved even for biomolecules present in very low amounts in a sample. Other fragmentation methods, such as ETD, ECT, and EAD, can be used to analyze RNA (e.g., RNA with cap 1 or cap 2) with or without an ion mobility gas separation module.

[0082] Similarly, those skilled in the art will appreciate that targeted analysis using selected / multiple reaction monitoring (SRM / MRM) techniques, well known in the art (see, e.g., Picotti, P., Bodenmiller, B., Mueller, LN, Domon, B., and Aebersold, R. (2009): Full dynamic range proteome analysis of S. cerevisiae by targeted proteomics. Cell 138, 795-806), can be used to increase the sensitivity of measurements. Instead of monitoring a narrow mass range, specific transitions from precursors to specific fragments can be monitored. The predicted MRM transitions for nuclease digestion of mRNAs with m7GpppG (cap 0), m7GpppGm (cap 1), m7GpppA, and m7GpppAm structures are shown in Table 1(a), (b), (c), (d), and (e). [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0083] In one example, measuring the ratio of labeled and unlabeled capped mRNA in a sample involves measuring the area ratio of one or more peaks of labeled caps to one or more corresponding peaks of unlabeled caps. For example, the ratio can be obtained by first integrating the signal of the labeled caps, then integrating the signal of the unlabeled caps, and then measuring the ratio by dividing one signal by the other, or vice versa. The amount of unlabeled product in an LC-MS spectrum can be expressed as a percentage of total mRNA (labeled and unlabeled 5' caps) and corresponds to the efficiency of the capping reaction.

[0084] The present inventors have also discovered that the amount of unmethylated capped mRNA present in a sample can be quantified by using a heavy-labeled methyl donor (e.g., a heavy-labeled SAM, e.g., CD3-SAM). Accordingly, a method for quantifying mRNA capping efficiency is provided, the method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy-labeled methyl donor to form a treated mRNA sample, optionally comprising mRNA with a heavy-labeled 5' cap; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; In one example, quantifying mRNA capping efficiency involves measuring the amount of unmethylated capped mRNA in a sample. This can be expressed, for example, as a percentage or percentage of capped mRNA present in the sample relative to the total mRNA present in the sample. This method (using heavy-labeled GTP and heavy-labeled SAM) is shown in Figure 2.

[0085] In one example, the mRNA sample includes mRNA with an unlabeled 5' cap. In one example, the processed mRNA sample includes mRNA with a heavy-labeled 5' cap. In one example, the processed mRNA sample includes mRNA with an unlabeled 5' cap and a heavy-labeled 5' cap.

[0086] Any suitable methyl donor can be used as long as the transferred methyl group contains a heavy atom, e.g., CD3. In one example, the heavy-labeled methyl donor is a heavy-labeled SAM, e.g., CD3-SAM. In one example, the capping enzyme comprises a guanine-7-methyltransferase or an enzyme having guanine-7-methyltransferase activity. In one example, the capping enzyme comprises an mRNA cap 2'-O-methyltransferase or an enzyme having mRNA cap 2'-O-methyltransferase activity.

[0087] In one example, the method further comprises treating the mRNA sample with a capping enzyme and heavy-labeled nucleotides. As will be appreciated by those skilled in the art, this reaction can be performed prior to or in the same reaction mixture as treatment with the capping enzyme and heavy methyl donor. The inventors have also discovered that the amount of uncapped mRNA and unmethylated capped mRNA present in a sample can be quantified in a one-spot assay using a heavy-labeled methyl donor (e.g., heavy-labeled SAM, e.g., CD3-SAM) and a heavy-labeled substrate (e.g., heavy nucleotide, e.g., heavy-labeled GTP). In one example, the method described herein comprises treating an mRNA sample containing capped RNA with a capping enzyme, a heavy-labeled methyl donor, and heavy-labeled nucleotides. Thus, a method for quantifying mRNA capping efficiency is provided, the method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy-labeled methyl donor and a heavy-labeled nucleotide to form a treated mRNA sample, optionally comprising mRNA with a heavy-labeled 5' cap; digesting the treated mRNA sample with a nuclease to release the 5' cap; Quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify mRNA capping efficiency; Also provided are methods as described above, comprising: (a) determining whether a nucleotide sequence is present in a sample, (b) determining whether a nucleotide sequence is present in a sample, (c) determining whether a nucleotide sequence is present in a sample, or (d) determining whether a nucleotide sequence is present in a sample. This can be expressed, for example, as a percentage or percentage (%) of total mRNA present in the sample, or as a percentage or percentage (%) of capped mRNA present in the sample. In one example, the heavy-labeled methyl donor is as described herein (e.g., CD3-SAM). In one example, the heavy-labeled nucleotide is as described herein (e.g., 13 C. 15N-GTP). This method can be used when the 5' cap is Cap 0. This method is shown in Figure 2. In one example, the capping enzyme includes VCE and an mRNA cap 2'-O-methyltransferase or an enzyme with mRNA cap 2'-O-methyltransferase activity. This assay can be used when the 5' cap is Cap 1. This method is shown in Figure 3.

[0088] Capping enzyme mRNA cap formation is a three-step process catalyzed by capping enzymes (e.g., RNA triphosphatase, guanyltransferase, and methyltransferase). Through a series of three steps, a cap is added to the 5' hydroxyl group of the first nucleotide of an mRNA strand. This can occur co-transcriptionally (i.e., while the mRNA is still being synthesized) or after completion of mRNA transcription. The latter is more frequently the case for RNA prepared using in vitro transcription, as RNA can be first synthesized from a DNA template using in vitro transcription, and then the 5' cap can be added using capping enzymes. First (for addition of a 7-methylguanosine cap, or cap 0), RNA 5' triphosphatase hydrolyzes the 5' triphosphate group and removes the terminal phosphate group, producing 5' diphosphate-RNA. Next, guanyltransferase adds GMP from GTP to the diphosphate end of the RNA, creating a guanosine (GpppN) cap. Third, an RNA methyltransferase transfers a methyl group from a methyl donor to the N7 position of the cap guanosine, resulting in a 7-methylguanosine cap attached to the 5' end of the transcript. These reactions usually require GTP and a methyl donor, e.g., S-adenosylmethionine (SAM). GTP is required for the addition of GMP by guanyltransferase. The methyl donor (e.g., SAM) is required for the RNA methyltransferase to add a methyl group to the guanosine cap.

[0089] The enzymatic reactions involved in capping eukaryotic mRNA are described in more detail below: (1) RNA triphosphatase cleaves the 5'-triphosphate of mRNA to form a diphosphate: pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi; (2) RNA guanyltransferase catalyzes the addition of GMP (from GTP) to the 5'-diphosphate of the 5'-most nucleotide (N1) of mRNA: ppN1(pN)x-OH(3')+GTP→GpppN1(pN)x-OH(3')+PPi; and finally, (3) Using S-adenosylmethionine (AdoMet) as a cofactor, guanine-7-methyltransferase catalyzes the methylation of the 7-nitrogen of guanine in the cap nucleotide to form the cap 0 structure: GpppN1(pN)x-OH(3')+AdoMet→m7GpppN1(pN)x-OH(3')+AdoHyc.

[0090] To form cap 1, mRNA cap 2'-O-methyltransferase catalyzes the methylation of the 2'-hydroxy group of the first ribose sugar using S-adenosylmethionine (AdoMet) as a cofactor. m 7 GpppN1(pN)x-OH(3') +AdoMet→m 7 GpppN1 * (pN)x-OH(3')+AdoHyc(where N1 * is methylated at the 2' hydroxy group)

[0091] The methods described herein include treating an mRNA sample with a capping enzyme to form a treated mRNA sample. As used herein, a "capping enzyme" or "CE" is an enzyme that catalyzes one or more of the reactions involved in attaching a 5' cap to a messenger RNA. Capping enzymes include RNA triphosphatase, guanyltransferase (or CE), and methyltransferase. A capping enzyme or capping enzyme system refers to a combination of one or more polypeptides having enzymatic activity that, in the presence of a cap nucleotide, including a modified cap nucleotide, and suitable reaction conditions, results in the synthesis of a capped RNA, including a modified nucleotide-capped RNA, e.g., a capped RNA having a cap 0 structure. Generally, a capping enzyme system or capping enzyme suitable for use in the methods described herein comprises RNA triphosphatase and RNA guanyltransferase enzymatic activity; optionally, the capping enzyme system or capping enzyme can also comprise RNA guanine-7-methyltransferase enzymatic activity. In one example, a capping enzyme system, or capping enzyme, suitable for use in the methods described herein comprises RNA triphosphatase, RNA guanyltransferase, and RNA guanine-7-methyltransferase enzyme activities.

[0092] Capping enzyme systems that can be used in the methods of the present disclosure are well known in the art (see, e.g., Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001; Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 50:101-129, 1995; Bisaillon, M and Lemay, G, Virology 236:1-7, 1997; Banerjee, AK, Microbiol. Rev. 44: 175-205, 1980). These include, but are not limited to, vaccinia virus capping enzyme, Faustovirus capping enzyme, and poxvirus capping enzyme, both full-length and enzymatically active portions thereof, for which capping enzymes have been identified, purified, characterized, and recombinantly expressed (see, e.g., Martin SA et al., J. Biol. Chem. 250: 9322-9329, 1975; Shuman, J. Biol. Chem. 265: 11960-11966, 1990; Shuman and Morham, J. Biol. Chem. 265: 11967-11972, 1990; MA Higman et al., J. Biol. Chem. 267: 16430, 1992; Myette, JR and Niles, EG, J. Biol. Chem. 271: 11936, 1996). The present disclosure is not limited by the type of capping enzyme utilized.

[0093] The active sites for RNA triphosphatase, RNA guanyltransferase, and guanine-7-methyltransferase enzymatic activity can be present in a single component polypeptide, a two component polypeptide (typically having RNA triphosphatase and RNA guanyltransferase activity), or a three component polypeptide derived from a cloned or wild-type source. The genes encoding the RNA triphosphatase, RNA guanyltransferase, and guanine-7-methyltransferase from one source can be component deletions in one or all of these genes from another source; the capping enzyme system can originate from a single wild-type source; and one or more of the RNA triphosphatase, RNA guanyltransferase, and / or guanine-7-methyltransferase activities can comprise polypeptides from different sources, each of which can be encoded by a DNA sequence derived from the same biological source or from a different biological source. In one example, the RNA triphosphatase component of the capping enzyme comprises a divalent cation-dependent RNA triphosphatase encoded by a DNA virus or fungus having the conserved motifs A, B, and C. In one example, the RNA triphosphatase is encoded by a poxvirus gene. In one example, the RNA triphosphatase is encoded by a vaccinia virus gene. However, any suitable RNA triphosphatase can be used. In one example, the RNA triphosphatase comprises a divalent cation-independent RNA triphosphatase encoded by DNA from a nematode, mammal, or other metazoan source, so long as the RNA triphosphatase removes the gamma phosphate of a triphosphate-terminated RNA to form RNA with a 5'-diphosphate terminus.Given the fact that vaccinia virus RNA triphosphatase-deficient mutants transfer GMP to the 5' triphosphate RNA end to generate a cap with a tetraphosphate bond (Yu, L and Shuman, S, J. Virology 70: 6162-6168, 1996), the capping enzyme system may, in some instances, lack RNA triphosphatase activity.

[0094] The RNA guanyltransferase of the capping enzyme system is structurally and mechanistically conserved among fungi, metazoans, protozoans, and DNA viruses (Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001). In one example, a capping enzyme has a conserved motif I consisting of the amino acid sequence KxDGxx (SEQ ID NO: 1), where X is any amino acid. In one example, the sixth position of motif I is not arginine. Motif I contains the active site for covalent binding of GMP to the capping enzyme within the RNA guanyltransferase portion of the capping enzyme. In one example, motif I of a capping enzyme has the amino acid sequence KTDG(I / V)(P / G) (SEQ ID NO: 2). In one example, motif I of a capping enzyme has the amino acid sequence KTDG(I / V)X (SEQ ID NO: 3), where the sixth amino acid (i.e., X) of motif I is an amino acid selected from the group consisting of phenylalanine, serine, and leucine. Another conserved motif in capping enzymes is motif III, which is also present in the RNA guanyltransferase portion of capping enzymes. In one example, the first position of motif III is valine, isoleucine, or tyrosine, the sixth position of motif III is glutamic acid, and the fourth position of motif III is phenylalanine, tyrosine, or tryptophan. In one example of a capping enzyme, motif III has the amino acid sequence VVVFGEAV (SEQ ID NO: 4). In one example, motif III has the amino acid sequence YRLWCEAV (SEQ ID NO: 5). In one example, motif III has the amino acid sequence VT(L / I)YGEA(I / V) (SEQ ID NO: 6). In one example, motif III has the amino acid sequence (V / I)YLYAEMR (SEQ ID NO: 7). In one example, motif III has the amino acid sequence (V / I)XL(Y / F)GEA(I / V) (SEQ ID NO: 8), where X is any amino acid. In one example, the RNA guanyltransferase is encoded by a poxvirus gene. In one example, the RNA guanyltransferase is encoded by a vaccinia virus gene.

[0095] A "capping enzyme" or "capping enzyme system" useful in the methods described herein optionally includes a guanine-7-methyltransferase. While the RNA guanyltransferase reaction step is reversible, the methylation step catalyzed by the guanine-7-methyltransferase activity of the capping enzyme is essentially irreversible. Therefore, guanine 7-methylation is useful because it completes the reaction toward cap formation. Cap methylation is also useful for improving RNA translation. The amino acid sequence and structure of the capping enzyme guanine-7-methyltransferase enzyme are highly conserved, from DNA viruses to yeast to humans and other metazoans. This is also demonstrated by the fact that full-length and several truncated guanine-7-methyltransferase genes encoding S. pombe, C. albicans, and human capping enzymes can complement deletions in the S. cerevisiae capping enzyme, guanine-7-methyltransferase, by the fact that the guanine-7-methyltransferase domain of the vaccinia virus capping enzyme can function in vivo in place of the yeast methyltransferase enzyme (Saha, N et al., J. Virology 77: 7300-7307, 2003), and by the concordance of mutation effects between the yeast, human, and vaccinia enzymes (Shuman, S, Prog. Nucleic Acid Res. Mol. Biol. 66: 1-40, 2001). Thus, the guanine-7-methyltransferase portion of the capping enzyme of the present invention can comprise wild-type or recombinant enzymes derived from any of a variety of sources. In one example, the guanine-7-methyltransferase is encoded by a poxvirus gene. In one example, the guanine-7-methyltransferase is encoded by a vaccinia virus gene. In one example, the guanine-7-methyltransferase has a conserved IHF amino acid motif.In one example, a guanine-7-methyltransferase has a motif consisting of the amino acid sequence VL(D / E)XGXGXG (SEQ ID NO: 9), where X is any amino acid.

[0096] In one example, a "capping enzyme" or "capping enzyme system" useful in the methods described herein includes an mRNA cap 2'-O-methyltransferase (2'OMTase), or an enzyme with mRNA cap 2'-O-methyltransferase activity. mRNA cap 2'-O-methyltransferase adds a methyl group at the 2'-O position of the first nucleotide adjacent to the cap structure at the 5' end of the RNA. In one example, the enzyme utilizes S-adenosylmethionine (SAM) as the methyl donor to methylate the capped RNA at the 2'OH group of the ribose (cap-O), forming the cap-I structure. Substrates for mRNA cap 2'-O-methyltransferase include mRNA cap 2'-O-methyltransferase, which utilizes m as a substrate. 7 The substrate is RNA with a GpppN cap. This substrate cannot utilize RNA with pN, ppN, pppN, or GpppN at the 5' end. 7 RNAs bearing a GpppN cap can be prepared using methods known to those skilled in the art, for example, by in vitro transcription using a cap analog or by enzymatic capping using vaccinia capping enzyme or other capping enzymes. mRNA cap 2'-O-methyltransferases can be produced recombinantly or are available from commercial sources (New England Biolabs). In one example, the capping enzymes include cap methyltransferases 1 and 2 for forming cap 1 and cap 2, respectively, at the 5' end of the RNA.

[0097] In one example, the capping enzyme comprises vaccinia virus capping enzyme (VCE). This is a complete system for enzymatic capping based on vaccinia virus capping enzyme and is available from suppliers such as New England Biolabs and KAC.TUS. VCE is composed of two subunits (D1 and D12). The D1 subunit contains RNA triphosphatase, guanyltransferase, and guanine methyltransferase. The D12 subunit binds to and stimulates methyltransferase. VCE can be used to add a 7-methylguanylate cap structure (Cap 0) to the 5' end of RNA generated by in vitro transcription. In one example, the capping enzyme is vaccinia virus capping enzyme (VCE), an enzyme with VCE-like biochemical activity, or a VCE-based enzyme system. In one example, the capping enzyme is vaccinia virus capping enzyme (VCE). In some instances where VCE is used, the capping enzyme is purified from vaccinia virus, while in other instances, the vaccinia capping enzyme is purified recombinant vaccinia virus capping enzyme. In some instances, the vaccinia virus capping enzyme is a mutant or variant of the wild-type enzyme (e.g., one that exhibits greater enzymatic activity compared to the wild-type capping enzyme). Variants (including allelic variants, muteins, analogs, and fragments) that can function as the provided capping enzymes are well known in the art and are also contemplated by the present invention.

[0098] In one example, the capping enzyme includes Faustwill capping enzyme (FCE). FCE catalyzes the addition of an N7-methylguanosine cap to the 5' end of triphosphorylated and diphosphorylated transcripts, generating uncapped RNA (Ramanathan, A. et al. (2016). Nucleic Acids Res. 44 (16), 7511-7526). FCE is a single-subunit enzyme containing triphosphatase, guanyltransferase, and (guanine-N7)-methyltransferase activities. FCE retains significant capping activity at low temperatures and tolerates reaction temperatures below 55°C. In one example, the capping reaction is performed between 15 and 55°C. In another example, the capping reaction is performed between 25 and 55°C. In some examples where FCE is used, the capping enzyme is recombinantly expressed, for example, in E. coli. In some examples, the FCE is a mutant or variant of a wild-type enzyme (e.g., one that exhibits greater enzymatic activity compared to the wild-type capping enzyme). Variants (including allelic variants, muteins, analogs, and fragments) that can function as the provided capping enzymes are well known in the art and are also contemplated by the present disclosure. Suitable FCEs and their variants are described in WO2022164428 (New England Biolabs, Inc.) and WO2021041260 (New England Biolabs, Inc.). FCEs are compatible with mRNA cap 2'-O-methyltransferase, enabling one-pot synthesis of mRNAs with Cap 1 structures. Thus, in some examples, the capping enzyme comprises an FCE and an mRNA cap 2'-O-methyltransferase. The use of an FCE and an mRNA cap 2'-O-methyltransferase allows for quantification of Cap 1 capping efficiency in a single-pot reaction.

[0099] Although a full system for enzymatic capping can be used in the methods and kits described herein, in some examples, the capping enzyme includes an RNA triphosphatase, an RNA guanyltransferase, and / or a guanine-7-methyltransferase. In one example, the capping enzyme includes an RNA guanyltransferase. In some examples, the capping enzyme includes an RNA triphosphatase and an RNA guanyltransferase. In some examples, the capping enzyme includes an RNA triphosphatase, an RNA guanyltransferase, and a guanine-7-methyltransferase. In one example, the capping enzyme includes a guanine-7-methyltransferase.

[0100] Labeling Reagents The methods described herein include treating an mRNA sample with a capping enzyme and one or more labeling reagents (e.g., heavy GTP and / or heavy SAM). As used herein, the term "label" refers to the attachment of a detectable signal, agent, or moiety to a compound. As used herein, the term "detectable signal" refers to a signal that can be detected or measured by a human or machine. In one example, a detectable signal can be quantified such that the intensity of the signal is related (e.g., proportional) to the amount of compound associated with the signal. Depending on the nature of the signal, a detectable signal can be detected, measured, or quantified by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. A "detectable signal" can also be referred to as a "detectable agent" or a "detectable moiety" in this application. In one example, a "detectable signal" is detectable by mass spectrometry.

[0101] In one example, the labeling reagent is labeled with a label that can be detected by mass spectrometry. In one example, the labeling reagent is labeled with a stable isotope. In one example, the labeling reagent is labeled with a heavy isotope. As used herein, a "heavy" isotope is one or more stable isotopes of an element that are heavier than the most abundant isotopes. In other words, a "heavy isotope" is a stable atom that has more neutrons than the normal isotope of the element, resulting in a larger mass. For example, 18 O is the most abundant 16 It is considered heavy oxygen compared to O. The heavy isotopes of nitrogen are: 15 N( 14 The heavy isotopes of carbon are 13 C, a heavy isotope of hydrogen, is also called deuterium 2 In one example, the labeling reagent includes one or more 15 N, 13 C, and / or D ( 2 H) atoms. These reagents are often referred to as "heavy" reagents, e.g., heavy GTP and heavy SAM. Heavy reagents can also be referred to as "isotopically labeled" reagents, e.g., isotopically labeled GTP, isotopically labeled SAM, etc.

[0102] In one example, the labeling reagent is a heavy labeling reagent (also referred to as a heavy reagent or isotopic labeling reagent). In one example, the heavy labeling reagent is a heavy-labeled substrate and / or cofactor of a capping enzyme. Any heavy-labeled substrate and / or cofactor suitable for use with a capping enzyme can be used. In one example, the heavy reagent comprises a heavy nucleotide, such as heavy ATP, heavy GTP, heavy CTP, or heavy UTP. In one example, the reagent is heavy GTP. As will be appreciated by those skilled in the art, the molecular weight of heavy GTP is greater than the molecular weight of GTP, and the difference in molecular weight can be detected by mass spectrometry. In one example, heavy GTP is 15 N-GTP, 13 C-GTP, D-GTP, 15 N, 13 C-GTP, 15 N,D-GTP, 13 C, D-GTP, or15 N, 13 C, D-GTP. In one example, heavy GTP is 15 N, 13 C-GTP. As will be understood by those skilled in the art, it is not necessary for all atoms in the molecule to be substituted with a heavy isotope. In one example, each carbon and / or nitrogen atom in the molecule is substituted with a heavy isotope. In one example, a subset of each carbon and / or nitrogen atom in the molecule is substituted with a heavy isotope. In one example, heavy GTP is 13 C 10 , 15 N5-GTP. 13 C 10 , 15 The mass of N5-GTP is 15 Daltons greater than normal / light GTP. In one example, heavy GTP is the compound of Formula I shown below, available from commercial sources such as CortecNet (Paris-Saclay, France). [ka]

[0103] In one example, the labeling reagent comprises a heavy-labeled methyl donor. Any suitable methyl donor can be used as long as the transferred methyl group comprises one or more heavy atoms, such as CD3. In one example, the reagent is a heavy SAM. SAM is also called S-adenosylmethionine or AdoMet. In one example, the heavy SAM is D-SAM, 13 C-SAM, or D, 13 In one example, the heavy SAM is a D-SAM. In one example, the heavy SAM is a D3-SAM. In one example, the heavy SAM is a compound of formula (II) shown below: [ka]

[0104] In one example, the labeling reagent comprises a heavy-labeled nucleotide and a heavy-labeled methyl donor. In one example, the labeling reagent comprises a nucleotide and a heavy-labeled methyl donor. In one example, the labeling reagent comprises a heavy-labeled nucleotide and a methyl donor. In one example, the labeling reagent comprises13 C. 15 Contains N-GTP and / or D3-SAM.

[0105] In one example, the labeling reagent is a radioisotope (e.g., 32 In one example, the labeling reagent is not labeled with a fluorescent label. In one example, the labeling reagent is not labeled with biotin.

[0106] The methods described herein provide those skilled in the art with a method for quantifying the amount of uncapped mRNA in a sample without the need for radiolabeling. Using heavy-labeled reagents and liquid chromatography coupled to electrospray mass spectrometry (LC-MS), the amount of uncapped mRNA in a sample can be accurately and with high resolution determined by detecting mass differences. The use of CD3-SAM in addition to heavy GTP allows for the quantification of various unmethylated capped mRNAs, including G-capped species.

[0107] nuclease The methods described herein include digesting treated mRNA samples with a nuclease (e.g., RNase) to release the 5' cap. As will be appreciated, depending on the nuclease and / or the sequence of the mRNA, the released 5' cap may be a dinucleotide (e.g., m 7The 5' cap can be a nucleotide (GpppG) or can form part of an oligonucleotide. An oligonucleotide containing a 5' cap can contain 2, 3, 4, 5, or more nucleotides, including the first nucleotide. While the exemplary method is not limited by the identity of the nuclease, it can be understood that a particular nuclease can offer one or more advantages. Any nuclease capable of cleaving or digesting mRNA, and particularly single-stranded mRNA, and releasing the 5' cap (including oligonucleotides containing a 5' cap) can be used. It will also be understood by those skilled in the art that the identity of the cap and / or sequence at the 5' end of the mRNA can affect the selection of nuclease. In addition, the specificity of the nuclease can also affect the selection of nuclease. For example, nuclease NP1 may be preferred when the mRNA is capped with cap 0, cap 1, or cap 2. In analyzing mRNA with cap 0, RNase A can be used when the first nucleotide following the triphosphate bond is cytosine (or uracil). In analyzing mRNAs with a cap of 0, RNAse T1 can be used when the first nucleotide following each triphosphate bond is a guanosine. In some instances, the cleavage site can be further shifted to the third, fourth, etc. nucleotide depending on the nature of the mRNA, the nuclease, and / or the test requirement(s). In one example, the nuclease is an RNase. Suitable nucleases include, but are not limited to, RNase A, RNAse T1, NP1, barnase, colicin E5, and mazF.

[0108] In one example, a suitable nuclease is RNAse T1 or an enzyme with RNAse T1-like biochemical activity. RNAse T1 is an endonuclease that cleaves single-stranded RNA after a guanine residue, i.e., the 3' side of the G. Without wishing to be bound by theory, one advantage of RNAse T1 is that it does not require metal ions for activity. Digestion by RNAse T1 results in the formation of m7 GpppGp was produced, which was detected using LC-MS as background m 7 The inventors have also found that cap-0 is more easily distinguishable from GpppG. The reaction products include oligonucleotides with a terminal 3' Gp. In the case of cap-0 capped mRNA, the reaction products are m 7 *G* includes pppGp. In examples where heavy GTP is used, G* can be heavily labeled, and the ratio of heavily labeled caps to unlabeled caps in the digested sample can be measured as described herein to determine the percentage of capped mRNA in the original sample. In examples where heavy SAM is used, m* can also be heavily labeled, and the ratio of heavily labeled methylated caps to unlabeled caps in the digested sample can be measured as described herein to determine the percentage of unmethylated capped mRNA in the original sample. In one example, RNAse T1 is derived from the mold Aspergillus oryzae. RNAse T1 and its variants can be produced using recombinant techniques or are commercially available from sources such as Thermo Fisher Scientific and Sigma-Aldrich. In some embodiments, it may be desirable to heat the sample (e.g., to about 37°C) to facilitate RNA digestion. In some examples, the pH of the nuclease reaction mixture is between about 5 and 8, e.g., pH 7.5. In some instances, the reaction is carried out according to the manufacturer's instructions.

[0109] In some instances, a suitable nuclease is nuclease P1 (NP1) or an enzyme with NP1-like biochemical activity. NP1 is a zinc-dependent endonuclease that cleaves single-stranded RNA or DNA without base specificity and can convert single-stranded DNA or RNA to 5' mononucleotides. Reaction products include oligonucleotides with terminal 3' Gp and terminal 3'-G. In the case of capped mRNA, the reaction products are m 7*G*pppG. Depending on whether heavy GTP or heavy SAM is used, G* and / or m* can be heavily labeled. The ratio of heavily labeled caps to unlabeled caps in the digested sample can be measured using techniques known to those of skill in the art (including mass spectrometry) to determine the percentage of capped mRNA in the original sample. In one example, NP1 is derived from the fungus Penicillium citrinum. NP1 and its variants can be produced recombinantly or are commercially available from sources such as Thermo Fisher Scientific and New England Biolabs. In some embodiments, it may be desirable to heat the sample (e.g., to about 37°C or to about 60°C) to facilitate RNA digestion. In some examples, the pH of the nuclease reaction mixture is between about 5 and 8, e.g., pH 5.5. In some examples, the reaction is performed according to the manufacturer's instructions.

[0110] In some instances, a suitable nuclease is RNase A or an enzyme with RNase A-like biochemical activity. RNase A is an endoribonuclease that specifically degrades single-stranded RNA at C and U (pyrimidine) residues. RNase A cleaves the phosphodiester bond between the 5'-ribose of a nucleotide and the phosphate group attached to the 3'-ribose of an adjacent C or U (pyrimidine) nucleotide. The resulting 2',3'-cyclic phosphate is hydrolyzed to the corresponding 3'-nucleoside phosphate. Reaction products include oligonucleotides with a terminal 3'-(C / U)p. In the case of capped mRNA, the reaction products include oligonucleotides containing a 5'-cap, e.g., m 7 *G*pppG(N) x(C / U)p, where N is A or U, and x is an integer starting with 0. G* and / or m* can be heavily labeled, and the ratio of heavily labeled caps to unlabeled caps in the digested sample can be measured to determine the percentage of capped mRNA in the original sample. In one example, RNase A is derived from bovine pancreas. RNase A and its variants can be isolated from bovine pancreas, produced using recombinant technology, or are commercially available from sources such as Thermo Fisher Scientific and Sigma-Aldrich. In some embodiments, it may be desirable to heat the sample (e.g., to about 37°C, to about 50°C, or to about 60°C) to facilitate RNA digestion. In some examples, the nuclease reaction mixture contains NaCl at a concentration of 0.3 M or greater. In some examples, the reaction is performed according to the manufacturer's instructions.

[0111] In some instances, two or more nucleases can be used in combination, for example, RNAse T1 and NP1, RNAse T1 and RNase A, NP1 and RNase A, or RNAse T1, NP1 and RNase A.

[0112] As will be appreciated by those skilled in the art, nucleases (e.g., ribonucleases) can be categorized into several groups based on the intermediates formed during RNA hydrolysis. In one example, RNA hydrolysis by nucleases proceeds via a 2',3'-cyclic phosphate intermediate (see Figure 4). RNA cleavage by these nucleases involves the formation of a cyclic phosphate intermediate followed by a transesterification (phosphorylation) step in which the cyclic phosphate intermediate is hydrolyzed to produce the 3'-phosphate. Nucleases that form a 2',3'-cyclic phosphate intermediate include, but are not limited to, RNase A, RNAse T1, and RNAse T2. In another example, RNA hydrolysis by nucleases does not proceed via a 2',3'-cyclic phosphate intermediate. Nucleases that do not form a 2',3'-cyclic phosphate intermediate include, but are not limited to, NP1.

[0113] In one example, the nuclease used in the methods described herein is a nuclease that forms a 2',3'-cyclic phosphate intermediate, such as RNase A, RNAse T1, or RNAse T2. Due to the hydrolysis mechanism, these nucleases are believed to be only suitable for quantifying the capping efficiency of RNA with a hydroxyl (-OH) group at the 2' position of the ribose, e.g., RNA with Cap 0 but not Cap 1. In one example, the nuclease is RNase A, RNAse T1, and / or RNAse T2, and the 5' cap is Cap 0. In one example, the nuclease is RNase A and / or RNAse T1, and the 5' cap is Cap 0. In one example, the nuclease is RNase A, and the 5' cap is Cap 0. In one example, the nuclease is RNAse T1, and the 5' cap is Cap 0. As will be appreciated by those skilled in the art, the cleavage product from such nucleases is m7GTPGp or m7GTPAp.

[0114] In one example, the nuclease used in the methods described herein is a nuclease that does not form a 2',3'-cyclic phosphate intermediate, such as NP1. Due to their hydrolysis mechanism, these nucleases are believed to be suitable for quantifying the capping efficiency of RNA with a hydroxyl (-OH) or -OR (where R is C1-C4 alkyl) (e.g., -OCH3) at the 2' position of the ribose, e.g., RNA with cap 0 or cap 1. In one example, the nuclease is NP1, and the 5' cap is cap 0 or cap 1. As will be understood by those skilled in the art, the cleavage products from such nucleases are m7GTPG (or m7GTPA) and m7GTPGm (or m7GTPAm) for cap 0 and cap 1 RNA, respectively.

[0115] 5' cap structure The methods described herein involve treating an mRNA sample with a capping enzyme and a heavy labeling reagent (e.g., heavy GTP and / or heavy SAM). Depending on the efficiency of the original capping reaction, the mRNA sample may contain one or more of capped mRNA, uncapped mRNA, and unmethylated capped mRNA. Capped mRNA is said to have a 5' cap structure, or 5' cap. As used herein, the term "5' cap structure" or "5' cap" refers to a structure at the 5' end of an mRNA. The 5' cap structure is known to stabilize mRNA by allowing CBP to bind to poly(A)-binding protein to form mature mRNA. Therefore, the presence of a 5' cap structure in the mRNA of the present disclosure may further increase the stability of the mRNA compared to mRNA without a 5' cap. The 5' cap structure provides resistance to 5'-exonuclease activity, and its absence results in rapid degradation of the mRNA.

[0116] Typically, endogenous mRNAs are 5'-capped with guanosine via a (5)'-ppp-(5)'-triphosphate linkage attached to the 5'-terminal nucleotide of the mRNA. The guanosine cap is then replaced by 7-methylguanosine (m7 G), which can be methylated to m 7 GpppN1p-, where N1 represents the first 5'-terminal nucleotide of the mRNA (e.g., Cap 0), is further 2'-O-methylated to form m 7 GpppN l mp- (e.g., Cap1), and / or m 7 G-pppN l mpN2mp (e.g., cap2) can be generated.

[0117] Although the examples provided herein below refer to the quantification of cap 0 caps, one of skill in the art will understand that the methods described herein can be used to quantify any of the cap structures and cap analogs described herein, as well as various modifications within the cap.

[0118] The methods described herein are generally suitable for quantification of any type of mRNA cap. Exemplary 5' cap structures include cap0, cap1, cap2, cap4, anti-reverse cap analog (ARCA), inosine, N7,2'-O-dimethyl-guanosine (mCAP), N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (mCAP), and 5' cap structures. 7 G), and CAP-003-CAP-225.

[0119] In one example, a capped mRNA of the present disclosure includes an endogenous cap. As used herein, the term "endogenous cap" refers to a 5' cap that can be synthesized within a cell. For example, an endogenous cap is a natural 5' cap or a wild-type 5' cap. For example, an endogenous cap is a Cap0, Cap1, or Cap2 structure. In one example, the 5' cap is Cap0. In one example, the 5' cap is Cap1. In one example, the 5' cap is Cap2. In one example, the 5' cap is Cap0 or Cap1.

[0120] In one example, the 5' cap is m 7 GpppN1p-, where N1 represents the first 5'-terminal nucleotide of the mRNA. N1 can be any nucleotide. In one example, N1 is A, m 6 It includes A, G, C, or U. In one example, N1 is A or m 6 In one example, N1 is A. In one example, N1 is G. In one example, the N1 nucleotide can be methylated at the 2'OH. For example, the 5' cap can be further 2'-O-methylated, resulting in m 7 GpppN l mp- and / or m 7 G-pppN l mpN2mp can be generated.

[0121] In one example, a capped mRNA of the present disclosure comprises an analog of an endogenous cap (also referred to as a cap analog). As used herein, the term "analog" or "cap analog" in the context of an endogenous cap refers to a synthetic 5' cap. Cap analogs can be used to generate 5' capped mRNA in an in vitro transcription reaction. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or attached to a nucleotide (e.g., the 5' terminal nucleotide of an mRNA). Suitable cap analogs include m 7 GpppG, m 7 GpppA, m 7 GpppC, 3′-O-Me-m 7GpppG, unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m 2,7 GpppG), trimethylated cap analogs (e.g., m 2,2,7 GpppG), dimethylated symmetric cap analogs (e.g., m 7 Gpppm 7 G), or anti-reverse cap analogs (e.g., ARCA; m 7,2’Ome GpppG, m 7,2’d GpppG, m 7,3’Ome GpppG, m 7,3’d Examples of suitable cap analogs include, but are not limited to, GpppG, GpppG, and their tetraphosphate derivatives (see, for example, Jemielity, J. et al., "Novel 'anti-reverse' cap analogs with superior translational properties," RNA, 9: 1108-1122 (2003)). In one example, the cap analog is N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (i.e., anti-reverse cap analog (ARCA)). ARCA can only be inserted in the proper orientation, thereby allowing, for example, m 7 This results in capped mRNA that is translated twice as efficiently as that displayed by GpppG.

[0122] In one example, the 5' cap has the structure of formula (III): [ka] where B is a nucleobase and R 1 is selected from H, halogen, OH, and OCH3; R 2 is selected from H, OH, and OCH3; R 3 is CH3, CH2CH3, CH2CH2CH3, or absent, and R 4 is NH2 and R 5 is selected from OH, OCH3, or halogen, n is 1, 2, or 3, and M is a nucleotide of mRNA. 1is selected from halogen, OH, and OCH. In one example, R 1 is selected from H, OH, and OCH. In one example, R 1 is selected from OH and OCH. In one example, R 1 is OH. In one example, R 5 is OCH3 or OH. In one example, R5 is OH. In one example, R 5 is OCH3. In one example, R 1 is OH and R 2 is OH and R 3 is CH3 and R 4 is NH2 and R 5 is OH or OCH3 and n is 1. In one example, R 1 is OH and R 2 is OH and R 3 is CH3 and R 4 is NH2 and R 5 is OH and n is 1. In one example, nucleobase B is guanine, cytosine, uracil, or adenine. In one example, nucleobase B is adenine. In one example, nucleobase B is guanine. In one example, the cap is m 7 GpppmG, where the ribose ring 2'OH group of base 1 is methylated (i.e., R 5 is OCH3). In one example, the cap is m 7 GpppG, where R of the ribose ring of base 1 5 The group is -OH.

[0123] In one example, the cap has the structure of formula (IV): [ka] In the formula, R 2 is H, OH, or OCH3; R 4 is NH2; R 5 is OH or OCH3, and R 6 is H or CH3; M is a nucleotide of mRNA. 2 is OH and R 4 is NH2 and R 5is OH or OCH3, and R 6 is H. In one example, R 2 is OH and R 4 is NH2 and R 5 is OH and R 6 is H. In one example, R 5 is OH. In one example, R 5 is OCH3. In one example, R 5 is OCH3 or OH.

[0124] In one example, the 5' cap is an unmethylated cap having the structure of formula (IVa): [ka] where M is a nucleotide of the mRNA.

[0125] In one example, the 5' cap is an unmethylated cap having the structure of formula (IVb): [ka] where M is a nucleotide of the mRNA.

[0126] In one example, the cap has the structure of formula (V): [ka] In the formula, R 2 is H, OH, or OCH3; R 4 is NH2; R 5 is OH or OCH3, and M is a nucleotide in mRNA. 2 is OH and R 4 is NH2 and R 5 is OH or OCH3. In one example, R 2 is OH and R 4 is NH2 and R 5 is OH. In one example, R 5 is OH. In one example, R 5 is OCH3. In one example, R 5is OCH3 or OH.

[0127] In one example, the 5' cap has the structure of formula (VI): [ka] In the formula, R 7 is OH or OP(O)2O-M, where M is a nucleotide of mRNA. The 5' cap is also called CleanCap AG (N-7113) (TriLink Biotechnologies, Inc.) and is a 5' N 7 -methylguanosine structure.

[0128] In one example, the 5' cap has the structure of formula (VII): [ka] In the formula, R 7 is OH or OP(O)2O-M, where M is a nucleotide of the mRNA.

[0129] In one example, the 5' cap has the structure of formula (VIIIa): [ka] In the formula, R 7 is OH or OP(O)2O-M, where M is a nucleotide in the mRNA. The 5' cap is also called CleanCap AG (3'OMe) - (N-7413) (TriLink Biotechnologies, Inc.) and contains a 5' N7-methyl-3'-O-methylguanosine, which is commonly found in mRNA capped using ARCA.

[0130] In one example, the 5' cap has the structure of formula (VIIIb): [ka] In the formula, R 7is OH or OP(O)2O-M, where M is a nucleotide of the mRNA.

[0131] In one example, the 5' cap has the structure of formula (IXa): [ka] In the formula, R 7 is OH or OP(O)2O-M, where M is a nucleotide of mRNA. The 5' cap can be CleanCap AG - (N-7114) (TriLink Biotechnologies, Inc.) or (m 7 Also called Gppp(2'OMeA)pU.

[0132] In one example, the 5' cap has the structure of formula (IXb): [ka] In the formula, R 7 is OH or OP(O)2O-M, where M is a nucleotide of the mRNA.

[0133] 5' caps are commercially available, for example, from TriLink Biotechnologies, Inc., San Diego CA USA. In some instances, the 5' cap can be synthesized using techniques known to those of skill in the art.

[0134] In one example, the 5' cap structure is a non-hydrolyzable cap structure. The non-hydrolyzable cap structure can prevent mRNA decapping and increase the half-life of the mRNA. Because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond, modified nucleotides can be used during the cap formation reaction. In one example, one or more of the oxygen atoms in the phosphorodiester bond can be replaced with, for example, a sulfur atom. In one example, the cap structure can include a phosphorothioate bond. In one example, a 5'-phosphorothioate dinucleotide cap analog is used to form the cap (see, e.g., Blazej A. Wojtczak, Pawel J. Sikorski, Kaja Fac-Dabrowska, Anna Nowicka, Marcin Warminski, Dorota Kubacka, Elzbieta Nowak, Marcin Nowotny, Joanna Kowalska, and Jacek Jemielity, J. Am. Chem. Soc. 2018, 140, 18, 5987-5999). In one example, vaccinia capping enzyme (New England Biolabs) can be used with α-thioguanosine nucleotides according to the manufacturer's instructions to create phosphorothioate linkages within the 5'-ppp-5' cap. In one example, the non-hydrolyzable cap structure comprises a modified nucleotide selected from the group consisting of α-thioguanosine nucleotides, α-methyl-phosphonates, seleno-phosphates, and combinations thereof. In one example, modified nucleotides are attached to the 5' end of an mRNA via an α-phosphorothioate linkage. Methods for attaching modified nucleotides to the 5' end of an mRNA would be apparent to those skilled in the art, for example, using vaccinia capping enzyme (New England Biolabs) or poxvirus capping enzyme. Additional modifications include, but are not limited to, 2'-O-methylation of the 5'-terminus of the mRNA on the 2'-hydroxyl group of the sugar ring and / or the ribose sugar of the 5'-pre-terminal nucleotide.Multiple unique 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.

[0135] mRNA The present disclosure provides a method for quantifying mRNA capping efficiency. As used herein, the term "messenger RNA" (also referred to as mRNA) refers to any polynucleotide that encodes a polypeptide of interest and can be translated (e.g., translated directly or indirectly) to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo. mRNA may or may not be chemically modified. mRNA of the present disclosure encompasses non-self-replicating mRNA (also referred to as conventional mRNA (cRNA)), self-replicating RNA (sa-mRNA), and any RNA that requires a 5' cap. In one example, the mRNA is sa-mRNA. In one example, the mRNA is cRNA.

[0136] Typically, a cRNA comprises, in 5' to 3' order, a 5' cap structure, a 5'-UTR, a nucleotide sequence encoding a polypeptide of interest, a 3'-UTR, and a tailing sequence (e.g., a polyadenylation signal or polyA tail). The cRNA of the present disclosure may further comprise a translational internal ribosome entry site (e.g., a Kozak consensus sequence or IRES). In some embodiments, the cRNA may also comprise strand terminating nucleotides and / or a stem-loop.

[0137] As used herein, the term "self-replicating RNA" refers to a construct based on an RNA virus that has been engineered to enable the expression of heterologous RNA and proteins. Self-replicating RNAs can also be referred to as replicons. Self-replicating RNAs can be amplified within a host cell to express a desired gene product within the host cell. In one example, the present disclosure provides a monocistronic self-replicating RNA. In one example, the present disclosure provides a bicistronic self-replicating RNA. In one example, the present disclosure provides a multicistronic self-replicating RNA. The sa-mRNA of the present disclosure includes one or more features of a cRNA, but further includes a nucleotide sequence encoding a nonstructural protein (NSP), enabling the sa-mRNA to direct its self-amplification. The nonstructural protein includes at least one gene selected from the group consisting of a viral replicase (or viral polymerase), a viral protease, a viral helicase, and other nonstructural viral proteins. Those skilled in the art will understand that in one example, the self-replicating RNA can be based on the genomic RNA of an RNA virus. Because the RNA must be a plus (+) strand, it can be directly translated after delivery to a cell without the need for an intervening replication step (e.g., reverse transcription). Translation of the RNA results in the production of nonstructural proteins (NSPs) that combine to form a replicase complex (i.e., an RNA-dependent RNA polymerase). The replicase complex is a component of the sa-mRNA and amplifies the original RNA to produce both antisense and sense transcripts, resulting in the generation of multiple daughter RNAs and, subsequently, the encoded polypeptide of interest. For example, in one example, the self-replicating RNA comprises a viral replicase (or viral polymerase).

[0138] For example, the sa-mRNA comprises an NSP derived from (or based on) an alphavirus. Exemplary alphaviruses include, but are not limited to, Venezuelan equine encephalitis virus (VEEV; e.g., Trinidad donkey, TC83CR), Semliki Forest virus (SFV), Sindbis virus (SIN), Ross River virus, Western equine encephalitis virus, Eastern equine encephalitis virus, Chikungunya virus, SAAR86 virus, Everglades virus, Mucambo virus, Barmah Forest virus, Middelburg virus, Pixuna virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Banbanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus, and Buggy Creek virus. The term alphavirus can also include chimeric alphaviruses, which contain genomic sequences from more than one alphavirus (as described in Perri et al., (2003) J. Virol. 77(19):10394-403). In another example, the self-replicating RNA is derived from or based on a virus other than an alphavirus (e.g., a positive-strand RNA virus). Suitable positive-strand RNA viruses suitable for use in the present disclosure will be apparent to those of skill in the art and include, for example, a picornavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.

[0139] Typically, the sa-mRNA also includes a subgenomic (SG) promoter, which, upon binding to a nucleotide sequence encoding the NSP and / or polypeptide of interest, drives expression of the NSP and / or polypeptide of interest. The present disclosure provides a self-replicating RNA comprising a nucleotide sequence encoding an antigen operably linked to an SG promoter. SG promoters (also known as "junction region" promoters) suitable for use in the present disclosure will be apparent to those skilled in the art and / or are described herein. In one example, the SG promoter is derived from or based on an alphavirus SG promoter. For example, the SG promoter is a native alphavirus SG promoter. In one example, the native SG promoter is a minimal SG promoter. For example, a minimal SG promoter is the minimal sequence required for initiation of transcription. In one example, the self-replicating RNA includes nonstructural proteins, 5' and 3' untranslated regions (UTRs), and a native subgenomic promoter of an RNA virus. In another example, the self-replicating RNA includes 5'- and 3'-terminal UTRs of an RNA virus.

[0140] The mRNA of the present disclosure typically comprises a nucleotide sequence encoding a polypeptide of interest. The nucleotide sequence can encode any polypeptide known to those of skill in the art, including any naturally occurring, non-naturally occurring, or otherwise modified polypeptide. The polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA may have a therapeutic effect when expressed in a cell. In one example, the nucleotide sequence encodes an antigen (e.g., a pathogenic antigen). For example, the antigen can induce an immune response in a subject. In one example, the mRNA of the present disclosure comprises a nucleotide sequence encoding an antigen derived from a virus. Examples of viruses include, but are not limited to, influenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, human parainfluenza virus, Epstein-Barr virus, human papillomavirus, measles virus, varicella-zoster virus, and the like. In one example, the mRNA of the present disclosure comprises a nucleotide sequence encoding an antigen derived from a respiratory virus, such as influenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, or human parainfluenza virus. In one example, the antigen is derived from SARS-CoV-2. In one example, the antigen is derived from influenza. In one example, the antigen is derived from respiratory syncytial virus. In one example, the antigen is derived from human metapneumovirus. In one example, the antigen is derived from human parainfluenza virus.

[0141] In examples where the virus is influenza, the mRNA may have a coding region encoding at least one antigenic peptide or protein derived from influenza virus hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein 1 (M1), matrix protein 2 (M2), nonstructural protein 1 (NS1), nonstructural protein 2 (NS2), nuclear export protein (NEP), polymerase acidic protein (PA), polymerase basic protein PB1, PB1-F2, or polymerase basic protein 2 (PB2), or a fragment or variant thereof. In one example, the coding region encodes at least one antigenic peptide or protein derived from influenza virus hemagglutinin (HA) and / or neuraminidase (NA), or a fragment or variant thereof. The HA and / or NA may independently be derived from influenza A virus or influenza B virus, or a fragment of either.

[0142] In instances where the virus is a coronavirus, the mRNA molecule can have a coding region encoding at least one antigenic peptide or protein derived from the spike (S) protein and / or the nucleocapsid (N) protein, where S and / or N can independently be derived from a variant of SARS-CoV-2 (e.g., the original strain, alpha, beta, omicron), or a fragment of either.

[0143] In some instances, the mRNA may contain one or more intron sequences that can be excised from the mRNA.

[0144] modified mRNA In one example, an mRNA of the present disclosure includes one or more modifications. Typically, modifications are introduced into a polynucleotide (e.g., mRNA) to increase its translation efficiency and / or stability. Suitable modifications to polynucleotides will be apparent to those of skill in the art and / or are described herein. RNA can be modified in many ways, including chemically, structurally, and functionally, by methods known to those skilled in the art of biotechnology. Such RNA modifications can include, for example, modifications typically introduced into mammalian cell mRNA after transcription. Furthermore, mRNA molecules can be modified by the introduction of alternative nucleotides, nucleosides, or nucleotides during transcription, as described in U.S. Pat. No. 108,278,036 (Kariko et al.); U.S. Patent Application No. 2013 / 0102034 (Schrum); U.S. Patent Application No. 2013 / 0115272 (deFougerolles et al.); and U.S. Patent Application No. 2013 / 0123481 (deFougerolles et al.).

[0145] In one example, a first nucleotide sequence comprising a 5'-UTR and / or a fragment thereof is modified. By modifying the first nucleotide sequence comprising a 5'-UTR and / or a fragment thereof, a variant of the 5'-UTR and / or a fragment thereof is obtained.

[0146] In one example, one or more nucleotide sequence(s) of the polynucleotide are codon-optimized. Methods of codon optimization would be apparent to one of skill in the art and / or are described herein. For example, tools for codon optimization of polynucleotides include, for example, GeneArt GeneOptimizer (Thermofisher®) or GenSmart® (GeneScript®).

[0147] In one example, a polynucleotide is modified to increase the amount of guanine (G) and / or cytosine (C) within the polynucleotide. The amount of G / C within a polynucleotide (i.e., G / C content) can affect the stability of the polynucleotide. Thus, a polynucleotide containing an increased amount of G / C nucleotides is functionally more stable than a polynucleotide containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. The G / C content is increased by substituting G or C nucleotides for A or T nucleotides.

[0148] In one example, the G / C content is increased in the nucleotide sequence encoding the polypeptide of interest. The modification(s) of the second nucleotide sequence utilizes the ability to replace codons containing unfavorable nucleotide combinations (in terms of mRNA stability) with alternative codons encoding the same amino acid or amino acid(s) with similar chemical properties (e.g., conservative amino acid substitutions). For example, the G / C content is increased by replacing codons containing A or T nucleotides with codons containing G or C nucleotides encoding the same amino acid. For example, the G / C content is increased by replacing codons containing A or T nucleotides with codons containing G or C nucleotides encoding amino acids with similar chemical properties.

[0149] In one example, the G / C content is increased in one or more nucleotide sequences of a polynucleotide that does not encode a polypeptide of interest. For example, the G / C content is increased in a portion of an mRNA that includes a 5'-UTR. For example, the G / C content is increased in a portion of an mRNA that includes a 3'-UTR.

[0150] In one example, mRNA contains one or more alternative nucleotides. Alternative nucleotides can include alternative nucleobases. The nucleobases of polynucleotides are organic bases such as purines, pyrimidines, or derivatives thereof. The nucleobases can be standard bases (e.g., adenine, guanine, uracil, thymine, and cytosine). These nucleobases can be modified or completely substituted to provide polynucleotide molecules with enhanced properties, for example, improved stability, such as nuclease resistance. Non-standard or modified bases can include one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkyloxy, and / or thio substitutions; one or more fused or open rings; oxidation; and / or reduction. Alternative nucleotides can be naturally occurring or non-naturally occurring.

[0151] Alternative nucleotide base pairs include not only canonical adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between nucleotides and / or alternative nucleotides containing non-standard or alternative bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases, or between two complementary non-standard base structures. One example of such a non-standard base pair is a base pair between the alternative nucleotide inosine and adenine, cytosine, or uracil.

[0152] In some embodiments, the nucleobase is a substituted uracil. Exemplary nucleobases and nucleosides having a substituted uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uracil, 6-aza-uracil, 2-thio-5-aza-uracil, 2-thio-uracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halo-uracil (e.g., 5-iodo-uracil or 5-bromo-uracil), 3-methyl-uracil (m3U), 5-Methoxy-uracil (mo5U), uracil 5-oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thio-uracil (mcm5s2U), 5-aminomethyl-2- Thio-uracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2-thio-uracil (mnm5s2U), 5-methylaminomethyl-2-seleno-uracil (mnm5se2U), 5-carbamoylmethyl-uracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinomethyl-uracil (τm 5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uracil (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (mψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ),2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrolauracil (D), dihydropseudouridine, 5,6-dihydrolauracil, 5-methyl-dihydrolauracil (m5D), 2-thio-dihydrolauracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-meth oxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uracil (inm5U), 5-(isopentenylaminomethyl)-2-thio-uracil (inm5s2U), 5,2 '-O-dimethyluridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm5Um), 3,2'-O-dimethyluridine (m3Um), and 5-(isopentenylaminomethyl) Examples of modified uracils include 2'-O-methyluridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-carbomethoxyvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyl-uracil, 5-methoxy-2-thiouracil, and 5-[3-(1-E-propenylamino)]uracil. In one example, the modified uracil is pseudouridine. In one example, the modified uracil is N1-methyl-pseudouridine.

[0153] In some embodiments, the nucleobase is a cytosine substitute. Exemplary nucleobases and nucleosides having a cytosine substitute include 5-aza-cytosine, 6-aza-cytosine, pseudoisocytidine, 3-methyl-cytosine (m3C), N4-acetyl-cytosine (ac4C), 5-formyl-cytosine (f5C), N4-methyl-cytosine (m4C), 5-methyl-cytosine (m5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytosine, pyrrolo-pseudoisocytidine, 2-thio-cytosine (s2C), 2-thio-5-methyl-cytosine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza- Pseudoisocytidine, Zebularine, 5-Aza-Zebularine, 5-Methyl-Zebularine, 5-Aza-2-Thio-Zebularine, 2-Thio-Zebularine, 2-Methoxy-Cytosine, 2-Methoxy-5-Methyl-Cytosine, 4-Methoxy-Pseudoisocytidine, 4-Methoxy-1-Methyl-Pseudoisocytidine, Lysidine (k2C), 5,2'-O-Dimethyl-Cytidine (m5Cm), N4- These include acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5-(3-azidopropyl)-cytosine, and 5-(2-azidoethyl)-cytosine. In one example, the modified cytosine is 5-methyl-cytosine.

[0154] In some embodiments, the nucleobase is an adenine substitute. Exemplary nucleobases and nucleosides having an adenine substitute include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azido-adenine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza- 2,6-Diaminopurine, 1-methyl-adenine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenine (m6A), 2-methylthio-N6-methyl-adenine (ms2m6A), N6-isopentenyl-adenine (i6A), 2-methylthio-N6-isopentenyl-adenine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenine (ms2io6A), N6-glycinylcarbamoyl N6-threonylcarbamoyl-adenine (g6A), N6-threonylcarbamoyl-adenine (t6A), N6-methyl-N6-threonylcarbamoyl-adenine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenine (ms2g6A), N6,N6-dimethyl-adenine (m62A), N6-hydroxynorvalylcarbamoyl-adenine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenine (ms2hn6A), N6-acetyl-adenine (ac6A), 7-methyl These include adenine, 2-methylthio-adenine, 2-methoxy-adenine, N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2-amino-N6-methyl-purine, 1-thio-adenine, 8-azido-adenine, N6-(19-amino-pentaoxanonadecyl)-adenine, 2,8-dimethyl-adenine, N6-formyl-adenine, and N6-hydroxymethyl-adenine.

[0155] In some embodiments, the nucleobase is a guanine substitute. Exemplary nucleobases and nucleosides having a guanine substitute include inosine (I), 1-methyl-inosine (mII), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o2yW), hydroxywyosine (OHyW), undermodified hydroxywyosine (OHyW*), 7-deaza-guanine, queuosine (Q ), epoxyqueuosin (oQ), galactosyl-queuosin (galQ), mannosyl-queuosin (manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine (preQ1), archeocysin (G+), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m7G), 6-thio-7 -methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m1G), N2-methyl-guanine (m2G), N2,N2-dimethyl-guanine (m22G), N2,7-dimethyl-guanine (m2,7G), N2,N2,7-dimethyl-guanine (m2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2- These include dimethyl-6-thioguanine, N2-methyl-2'-O-methylguanosine (m2Gm), N2,N2-dimethyl-2'-O-methylguanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (mlm), 1-thio-guanine, and O-6-methyl-guanine.

[0156] The alternative nucleobase of a nucleotide can independently be a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can be substituted for adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, nucleobases also include, for example, derivatives of naturally occurring and synthetic bases, such as pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioa alkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; or 1,3,5 triazine.

[0157] In one example, at least one naturally occurring nucleotide of the polynucleotide is replaced with a chemically modified nucleotide. In one example, at least 10%, or 20%, or 30%, or 40%, or 50%, or 60%, or 70%, or 80%, or 90%, or 100% of the nucleotides of the naturally occurring polynucleotide are replaced with a chemically modified nucleotide.

[0158] The mRNA may or may not be uniformly altered along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, C) may be uniformly modified or unmodified in a polynucleotide or in a given predetermined sequence region thereof. Those skilled in the art will understand that nucleotide analogs or other modification(s) may be located at any position(s) of the mRNA so as not to substantially reduce the function of the mRNA. The alteration may be at the 5' end or at the 3' end. In some examples, the mRNA includes a modification at the 3' end. In some examples, the mRNA includes a modification at the 5' end.

[0159] In one example, an mRNA can contain one or more naturally occurring components, including any of the classical nucleotides A (adenosine), G (guanosine), C (cytosine), or U (uridine). In one embodiment, all or substantially all of the nucleotides comprising (a) the 5'-UTR, (b) the open reading frame (ORF), (c) the 3'-UTR, (d) the polyA tail, and any combination of (a, b, c, or d above) contain the naturally occurring standard nucleotides A (adenosine), G (guanosine), C (cytosine), U (uridine), or T (thymidine). In one example, an mRNA can contain one or more alternative components that confer useful properties, including increased stability of a cell into which the polynucleotide is introduced and / or lack of substantial induction of an innate immune response, as described herein. For example, the alternative polynucleotide exhibits reduced degradation in a cell into which the polynucleotide is introduced, compared to a corresponding unmodified polynucleotide. These alternative species may increase the efficiency of protein production, the intracellular retention of the polynucleotide, and / or the viability of contacted cells, and may also be less immunogenic.

[0160] Production of capped mRNA mRNA suitable for use in the described methods can be prepared using any method known in the art, including, for example, synthetic methods such as solid-phase synthesis, and in vitro methods such as in vitro transcription reactions.

[0161] In one example, RNA is produced using in vitro transcription (IVT) from a corresponding DNA molecule. This method was originally developed by Krieg and Melton (Methods Enzymol., 1987, 155: 397-415) for the synthesis of RNA using RNA phage polymerase. RNA transcription usually begins with a nucleoside triphosphate (usually a purine, A, or G). In vitro transcription typically involves a phage RNA polymerase such as T7, T3, or SP6, a DNA template containing a phage polymerase promoter, nucleotides (ATP, GTP, CTP, and UTP), and a buffer containing magnesium salts under conditions that support polymerase activity. RNA synthesis yield can be optimized by increasing nucleotide concentration, adjusting magnesium concentration, and including inorganic pyrophosphatase (U.S. Patent No. 5,256,555; Gurevich, et al., Anal. Biochem. 195: 207-213 (1991); Sampson, JR and Uhlenbeck, OC, Proc. Natl. Acad. Sci. USA. 85, 1033-1037 (1988); Wyatt, JR, et al., Biotechniques, 11: 764-769 (1991)). Many in vitro transcription techniques are known in the field of biotechnology. See, for example, The Basics: In Vitro Transcription (2015), available from Thermo Fisher Scientific Inc., Waltham MA USA. In vitro transcription may be performed using various commercially available kits, including, but not limited to, RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription Kit (Life Technologies), as well as commercially available reagents such as RNA polymerase and rNTPs. Methodologies for in vitro transcription of mRNA are well known in the art.(See, e.g., Losick, R., 1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamaka, RT and Kraus, WL 2001. In Vitro Transcription. Current Protocols in Cell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B., (2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods in Molecular Biology v. 703 (Neilson, H. Ed), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter Five - In vitro transcription from plasmid or PCR-amplified DNA, Methods in Enzymology v. 530, 101-114; all of which are incorporated herein by reference).

[0162] Template DNA can be prepared for in vitro transcription from many sources using suitable techniques known in the art, including, but not limited to, plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template and Bowman, JC, Azizi, B., Lenz, TK, Ray, P., and Williams, LD, In RNA in vitro transcription and RNA purification by denaturing PAGE in Recombinant and in vitro RNA synthesis Methods v.941 Conn GL (ed), New York, NY Humana Press, 2012). For example, the DNA molecule can be plasmid DNA, a PCR product, dogbone DNA, etc. In one example, the DNA molecule comprises a suitable promoter for in vitro transcription, such as a T7, T3, or SP6 promoter, followed by the desired nucleotide sequence to be prepared, e.g., mRNA, and a termination signal for in vitro transcription. In one example, the DNA molecule comprises a T7 promoter. The desired nucleotide sequence comprises, in the 5' to 3' direction, a 5' UTR, an ORF, and a 3' UTR. The desired nucleotide sequence or a portion thereof (e.g., an ORF) can be codon-optimized.

[0163] In one example (e.g., plasmid DNA), the DNA molecule can be linearized before use in an in vitro transcription reaction. In this example, the DNA molecule includes a restriction cleavage and / or recognition sequence. Any suitable restriction enzyme (and corresponding restriction cleavage and / or recognition sequence) can be used for linearization. In one example, the restriction enzyme is a type II restriction enzyme (e.g., a type IIs restriction enzyme). In one example, the recognition sequence for a type IIS restriction endonuclease is located 5 to 26 base pairs, e.g., 24 to 26 base pairs, downstream of the 3' end of the nucleic acid sequence (e.g., downstream of the polyadenylation cassette). In one example, the DNA template is cleaved within the polyadenylation cassette, resulting in a transcript that terminates with an unmasked poly(A) sequence. It has been discovered that RNAs with open-ended poly(A) sequences are translated more efficiently than RNAs with masked ends (i.e., a nucleotide other than A at the 3' end) (see, e.g., WO2017 / 059902A1).

[0164] As used herein, "restriction endonucleases" or "restriction enzymes" refer to a class of enzymes that cleave phosphodiester bonds in both strands of a DNA molecule within a specific base sequence. They recognize specific binding sites on double-stranded DNA molecules, called recognition sequences. The location in DNA where the phosphodiester bond is cleaved by the enzyme is called the cleavage site. In the class of Type IIS enzymes, the cleavage site is located a fixed distance from the DNA binding site. According to the present invention, the term "restriction endonuclease" includes, for example, the enzymes Sapl, Ecil, Bpil, Aarl, Alol, Bael, BbvCI, Ppil and Psrl, BsrDl, Btsl, Earl, Bmrl, Bsal, BsmBI, Faul, Bbsl, BciVI, BfuAI, BspI, BseRI, Ecil, BtgZI, BpuEI, Bsgl, Mmel, CspCI, Bael, BsaMI, Mval269l, Pctl, Bse3DI, BseMI, Bst6I, Eamll04I, sp632I, Bfil, Bso31I, BspTNI, Eco31I, Esp3I, Bful, Acc36I, Aarl, Eco57I, Eco57MI, Gsul, Alol, Hin4I, Ppil, and Psrl.

[0165] In one example, the synthesis of capped RNA is initiated by the addition of a cap analog (e.g., m 7 This includes the incorporation of GpppG (GpppG). Cap analogs are incorporated only as the initial, or 5'-terminal G, of a transcript because their structure prevents incorporation at any other position in the RNA molecule. In some instances, RNA polymerases incorporate cap analogs as readily as any other nucleotide, i.e., there is no bias against cap analogs. In some instances, cap analogs will be incorporated at the 5' terminus by the enzyme guanylyltransferase. In some instances, cap analogs will be incorporated only at the 5' terminus because they do not have a 5' triphosphate. In some embodiments using T7, T3, and SP6 RNA polymerases, the +1 nucleotide of their respective promoters is typically a G residue, and GTP and a cap analog (e.g., m7 When both cap analogs (e.g., GpppG) are present at equivalent concentrations in a transcription reaction, they each have an equal chance of being incorporated at the +1 position. 7 GTP (GpppG) is present in these reactions at concentrations several times higher than GTP, increasing the chance that a transcript will have a 5' cap. In one example, an excess of cap analog (e.g., a 4:1 ratio of cap analog to GTP) is added. The excess cap analog is believed to increase the chance that each transcript contains a cap analog. In some instances, as the ratio of cap analog to GTP in a reaction increases, the ratio of capped to uncapped RNA increases proportionally. Capping efficiency considerations must be balanced with yield considerations. Increasing the ratio of cap analog to GTP in a transcription reaction results in a lower yield of total RNA because the concentration of GTP becomes limiting when maintaining constant total cap and GTP concentrations. Thus, the final RNA yield depends on the GTP concentration required for transcript elongation. Other nucleotides (ATP, CTP, UTP) are usually present in excess. Kits for capping in vitro transcribed mRNA are commercially available, including the mMESSAGE mMACHINE® kit (Ambion, Inc., Austin, Tex.). Typically, these kits result in 80% capped RNA to 20% uncapped RNA. In some instances, the mMESSAGE mMACHINE® kit (Catalog #1344, Ambion, Inc.) is used according to the manufacturer's instructions, which recommend a cap to GTP ratio of 4:1 (6 mM:1.5 mM). In one example, the DNA template can include the sequence 5'-TAATACGACTCACTATAAGG-3' (SEQ ID NO: 10) downstream of the promoter sequence to include a cap analog.

[0166] In one example, synthesis of capped RNA involves in vitro transcription from a DNA template, followed by the addition of a cap using a capping enzyme or capping system. In some examples, commercially available kits for large-scale synthesis of in vitro transcripts are utilized (e.g., MEGAscript®, Ambion). The RNA synthesized in these reactions is usually characterized by a 5'-terminal nucleotide bearing a triphosphate at the 5' position of the ribose. Typically, this nucleotide is guanosine, but it can also be adenosine, depending on the combination of RNA polymerase and promoter used (see, e.g., Coleman, TM, et al., Nucleic Acids Res., 32: el4 (2004)). In these reactions, all four nucleotides are typically included in equimolar concentrations, and none of them is limiting.

[0167] In one example of in vitro transcription, the reaction is a batch reaction, i.e., all components are combined and then incubated at approximately 37°C to promote RNA polymerization until the reaction is complete. Typically, batch reactions are used for convenience, obtaining the required amount of RNA from such reactions for their experiments. In some instances, a "fed-batch" system (see, e.g., Jeffrey A. Kern, "Batch and Fed-batch Strategies for Large-scale Production of RNA by in Vitro Transactions (University of Colorado) (1997)) is used to increase the efficiency of in vitro transcription reactions. All components are combined, while additional amounts of some reagents, such as nucleotides and magnesium, are added over time to attempt to maintain constant reaction conditions. Furthermore, in some embodiments, the pH of the reaction can be maintained at 7.4 by monitoring it over time and adding a base, such as KOH, as needed.

[0168] In vitro transcribed RNA can be further processed, for example, by adding a poly(A) tail. In one example, a poly(A) tail is added using methods known to those of skill in the art. The poly(A) tail can be contained in a plasmid, added by PCR, or added post-transcriptionally by enzymatic polyadenylation. In the latter case, the DNA template can include a polyadenylation signal. In one example, a poly(A) tail is introduced by including a poly(dT) stretch at the end of the transcription template. In one example, this is achieved by a PCR step utilizing primers containing poly(dT) stretches. In one example, a poly(A) tail is added after in vitro transcription. In some examples, a 3' poly(A) tail approximately 200 nucleotides in length (as determined, for example, by gel electrophoresis) is added after in vitro transcription by adding ATP along with poly(A) polymerase. As used herein, "poly(A) polymerase ("PAP")" refers to a template-dependent RNA polymerase found in most eukaryotes, prokaryotes, and eukaryotic viruses that, for example, selectively uses ATP to incorporate AMP residues into the 3'-hydroxylated end of RNA. Because PAP enzymes studied from plants, animals, bacteria, and viruses all catalyze the same overall reaction (see, e.g., Edmonds, M, Methods Enzymol., 181;161-180, 1990), are highly structurally conserved (see, e.g., Gershon, P, Nature Structural Biol. 7:819-821, 2000), and lack inherent specificity for RNA molecules of a particular sequence or size, purified wild-type and recombinant PAP enzymes from any of a variety of sources can be used in the kits and methods of the present disclosure. In some embodiments, the poly(A) tail is approximately 100-250 nucleotides in length. In some embodiments, the poly(A) tail is about 50-300 nucleotides in length.

[0169] In some embodiments, the in vitro transcription product comprises 5' and 3' untranslated regions. Any suitable 5' and 3' untranslated regions known to those skilled in the art can be used. Suitable 5' and 3' untranslated regions include those described in WO2017059902A1 (Biontech Rna Pharmaceuticals GmbH, Tron - Translationale Onkologie An Der Universitaetsmedizin Der Johannes Gutenberg-Universitaet Mainz Ggmbh), the entire contents of which are incorporated herein by reference.

[0170] A DNA molecule forming a template for at least one RNA of interest can be prepared by fermentation growth and subsequent isolation as part of a replicable plasmid in bacteria. In one example, a DNA plasmid encoding the RNA of interest is used to transform competent bacterial cells (e.g., Escherichia coli cells). Individual bacterial colonies are isolated, and the resulting plasmid DNA is amplified in E. coli culture. The plasmid DNA amplified in the E. coli culture can be isolated using techniques known to those skilled in the art. In one example, the plasmid DNA is isolated after fermentation. For example, the plasmid DNA is isolated using a commercially available kit (e.g., Maxiprep DNA kit) or other conventional methods known to those skilled in the art. After isolation, the plasmid DNA can be linearized by restriction digestion (i.e., using a restriction enzyme). The restriction enzyme is removed using methods known in the art (e.g., including phenol / chloroform extraction, ethanol precipitation, chromatography, etc.).

[0171] Plasmids that may be suitable for use as templates include, for example, the plasmid pT7T (GenBank accession number U26404; Lai et al., Development 1 995, 121:2349-2360), pGEM® species such as pGEM®-1 (GenBank accession number X65300; manufactured by Promega), and pSP64 (GenBank accession number X65327).

[0172] In one example, a DNA molecule is amplified from a template nucleic acid using PCR. In one example, at least one of the primers used for PCR can include a promoter sequence (e.g., a T7, T3, or SP6 promoter sequence). In one example, at least one of the primers used for PCR can include a polyadenylation cassette. In one example, at least one of the primers used for PCR can include a cleavage site for a restriction enzyme.

[0173] After restriction digestion or PCR, the DNA template can be purified to remove enzymes and reaction components. Commercially available column purification methods, such as the Plasmid Plus kit from QIAGEN, or phenol / chloroform extraction followed by ethanol precipitation can be used.

[0174] After in vitro transcription, the DNA template can be removed using any technique known to those of skill in the art. In one example, after in vitro transcription, the DNA template is removed by treatment with a DNase (e.g., DNase I).

[0175] In one example, mRNA is purified. This serves to separate mRNA from undesired components of transcription and related reactions. Various methods for purifying mRNA will be apparent to those skilled in the art. For example, mRNA is purified by LiCl precipitation, phenol:chloroform extraction followed by ethanol precipitation, precipitation with ether alcohol in the presence of monovalent cations, or using a spin-column-based method. In another example, mRNA is purified using tangential flow filtration (TFF), e.g., diafiltration. In one example, the purification step includes diafiltration into a suitable buffer. In one example, the purification step includes a spin-column-based method or column chromatography. A spin column can be used to remove unincorporated nucleotides, proteins, and salts. A spin-column-based method can be performed using a commercially available kit (e.g., Monarch RNA Cleanup Kit (New England Biolabs)) according to the manufacturer's instructions. In one example, the purification step includes phenol-chloroform extraction and ethanol precipitation. After purification, the mRNA is resuspended, for example, in nuclease-free water or a suitable buffer.

[0176] In one example, mRNA products are analyzed to assess reaction yield and quality. Various methods for analyzing mRNA will be apparent to those skilled in the art, including, but not limited to, mass spectrometry, gel electrophoresis, liquid chromatography, spectroscopic analysis (e.g., Nanodrop), or a combination thereof. In one example, mRNA products are analyzed by the methods disclosed herein.

[0177] use The methods and uses described herein are useful for quality control during mRNA manufacturing and for characterizing mRNA as an active pharmaceutical ingredient (API) in final therapeutic products, including vaccines. Without wishing to be bound by theory, the inventors have discovered that the use of heavy labeling reagents, such as heavy GTP and / or heavy SAM, allows for the measurement of capping efficiency without the use of oligonucleotides complementary to the 5' region of the mRNA being analyzed. As a result, the methods of the present disclosure are not limited by the sequence of the RNA molecule being analyzed. For example, the methods used herein do not require the RNA to contain a cleavage site for a catalytic nucleic acid molecule or the synthesis of a tagged probe complementary to the 5' end of the target RNA. Using the methods described herein, 32 It is also possible to quantify capping efficiency without the use of radioisotopes such as P. In one example, the methods described herein are performed prior to release of the mRNA lot.

[0178] In one example, the method described herein is carried out before incorporating mRNA into LNP. The mRNA can be formulated in lipid nanoparticles for administration. Methods for forming lipid nanoparticles are known to those skilled in the art. For example, suitable LNPs can be formed using microfluidics, including herringbone micromixing, and mixing processes such as T-junction mixing of two fluid streams, where one fluid stream typically contains polynucleotide in aqueous solution, and the other fluid stream contains various necessary lipid components (usually ethanol).

[0179] kit The present invention further provides kits containing various reagents and materials useful for carrying out the methods described herein. The quantitative procedures described herein can be performed by diagnostic laboratories, research laboratories, or commercial testing laboratories. The disclosed kits can be used in these various settings.

[0180] As used herein, the term "kit" refers to any supply system for providing materials. Such delivery systems can include systems that allow for the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, instructions for performing an assay, etc.) from one location to another. For example, a kit includes one or more enclosures (e.g., boxes) containing relevant reaction reagents and / or auxiliary materials. As used herein, the term "fragmentation kit" refers to a delivery system that includes two or more separate containers, each containing a subportion of the total kit components. The containers can be delivered to the intended recipient together or separately. For example, a first container can contain an enzyme (e.g., a capping enzyme) for use in the methods described herein, while a second container can contain one or more labeling reagents (such as heavy GTP and / or heavy SAM). Indeed, any delivery system comprising two or more separate containers, each containing a subportion of the total kit components, is encompassed by the term "fragmentation kit." In contrast, a "composite kit" refers to a supply system that contains all of the components in a single container (e.g., a single box containing each of the desired components). The term "kit" includes both fragmented kits and composite kits.

[0181] For example, materials and reagents for quantifying mRNA capping efficiency in an mRNA sample by enzymatic manipulation can be assembled together in a kit. In one example, the kit includes a labeling reagent (heavy reagent) and instructions for using the kit according to the methods described herein. In one example, the kit can further include a capping enzyme or system and instructions for using the same. The kit can also include a nuclease for degrading single-stranded RNA, such as RNAse T1, RNase A, and / or NP1, and instructions for using the same. In one example, the kit includes a capping enzyme or capping system and heavy GTP. In one example, the kit includes a capping enzyme or capping system, heavy GTP, and heavy SAM. In one example, the kit includes a capping enzyme or capping system, a nuclease, and heavy GTP. In one example, the kit includes a capping enzyme or capping system, a nuclease, heavy GTP, and heavy SAM.

[0182] The kit or other manufactured article described herein can include one or more containers for holding various reagents.Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampoules.Containers can be made of various materials, such as glass or plastic.

[0183] In some embodiments, the kits described herein can include a suitable control level or control sample for determining the control levels described herein. In some embodiments, the kits of the invention can include instructions for using the kit according to one or more methods of the invention, including instructions for 5' capping and / or nuclease treatment.

[0184] The invention is further disclosed in the following numbered paragraphs:

[0185] 1. A method for quantifying mRNA capping efficiency, said method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy labeling reagent to form a treated mRNA sample, optionally comprising mRNA with a heavy-labeled 5' cap; digesting the treated mRNA sample with a nuclease to release the 5' cap; quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify the mRNA capping efficiency; The method comprising:

[0186] 2. The method of paragraph 1, wherein the heavy-labeled reagent comprises a heavy-labeled substrate and / or a heavy-labeled cofactor.

[0187] 3. The method of paragraph 2, wherein the heavy-labeled substrate and / or cofactor comprises heavy GTP or heavy SAM, or a combination thereof.

[0188] 4. The heavy-labeled substrate and / or the heavy-labeled cofactor are 13 C. 15 4. The method of paragraph 2 or 3, comprising N-GTP.

[0189] 5. The method of paragraph 2 or 3, wherein the heavily labeled substrate and / or heavily labeled cofactor comprises CD3-SAM.

[0190] 6. The heavily labeled substrate and / or cofactor comprises heavy GTP and heavy SAM, e.g., the heavily labeled substrate and / or cofactor comprises: 13 C. 15 6. The method of any one of paragraphs 2 to 5, comprising N-GTP and CD3-SAM.

[0191] 7. The method of any one of paragraphs 1 to 6, wherein the step of quantitatively determining the amount of heavy-labeled and unlabeled 5' caps in the sample comprises analyzing the released 5' caps by liquid chromatography / mass spectrometry (LC-MS) and measuring the relative amounts of heavy-labeled and unlabeled 5' fragments.

[0192] 8. The method of any one of paragraphs 1 to 7, wherein the capping enzyme comprises a vaccinia capping system or a poxvirus capping enzyme.

[0193] 9. The method of paragraph 8, wherein the capping enzyme comprises the vaccinia capping system.

[0194] 10. The method of any one of paragraphs 1-9, wherein the capping enzyme comprises a triphosphatase, a guanyltransferase, or a guanine methyltransferase, or a combination thereof.

[0195] 11. The method of any one of paragraphs 1 to 10, wherein the mRNA sample is synthesized by in vitro transcription.

[0196] 12. The method of any one of paragraphs 1 to 11, wherein the mRNA sample comprises capped mRNA produced by a post-transcriptional or co-transcriptional capping reaction.

[0197] 13. The mRNA sample may contain any of the following: cap 0, cap 1, cap 2, cap 4, anti-reverse cap analog (ARCA), inosine, N7,2'-O-dimethyl-guanosine (mCAP), N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (mCAP), 7 13. The method of any one of paragraphs 1 to 12, comprising capped mRNA having a 5' cap selected from the group consisting of CAP-003-CAP-225, and CAP-003-CAP-225.

[0198] 14. The method of paragraph 13, wherein the 5' cap is Cap0 or Cap1.

[0199] 15. The method of paragraph 13, wherein the 5' cap is Cap1.

[0200] 16. The method of paragraph 13, wherein the 5' cap is Cap0.

[0201] 17. The method of any one of paragraphs 1 to 13, wherein the nuclease comprises RNAse T1, nuclease NP1, or RNAse A, or a combination thereof.

[0202] 18. The method of paragraph 17, wherein the nuclease is RNAse T1.

[0203] 19. The method of paragraph 17, wherein the nuclease is nuclease NP1.

[0204] 20. The mRNA sample comprises a sequence represented by formula (III): [ka] wherein B is a nucleobase and R 1 is selected from H, halogen, OH, and OCH3; R 2 is selected from H, OH, and OCH3; R 3 is CH3, CH2CH3, CH2CH2CH3, or absent, and R 4 is NH2 and R 5 is OH, n is 1, 2, or 3, M is a nucleotide of said mRNA, and 13. The method of any one of paragraphs 1 to 12, wherein the nuclease is RNAse T1.

[0205] 21. The method of paragraph 20, wherein the mRNA sample comprises capped mRNA having a 5' cap that is cap 0, and the nuclease is RNAse T1.

[0206] 22. The mRNA sample comprises a sequence represented by formula (III): [ka] wherein B is a nucleobase and R 1 is selected from H, halogen, OH, and OCH3; R 2 is selected from H, OH, and OCH3; R 3 is CH3, CH2CH3, CH2CH2CH3, or absent, and R 4 is NH2 and R 5 is OH or OCH3, n is 1, 2, or 3, M is a nucleotide of said mRNA, and 13. The method of any one of paragraphs 1 to 12, wherein the nuclease is nuclease NP1.

[0207] 23. The method of paragraph 22, wherein the mRNA sample comprises capped mRNA having a 5' cap that is Cap 0 or Cap 1, and the nuclease is nuclease NP1.

[0208] 24. The method of any one of paragraphs 1 to 23, wherein the mRNA sample comprises uncapped mRNA having a 5' triphosphate group or a 5' diphosphate group, or a combination thereof.

[0209] 25. The method of any one of paragraphs 1 to 24, wherein the mRNA sample comprises unmethylated capped mRNA having a 5' GpppN group, where N is any nucleotide.

[0210] 26. The method of any one of paragraphs 1 to 25, wherein quantitatively measuring the amount of labeled and unlabeled 5' caps in the sample comprises measuring the relative amounts of the labeled and unlabeled 5' caps.

[0211] 27. The method of any one of the preceding paragraphs, wherein quantifying mRNA capping efficiency comprises quantifying the absolute amount of capped mRNA in the mRNA sample.

[0212] 28. The method of any one of paragraphs 1 to 27, wherein quantifying the efficiency of mRNA cap formation comprises quantifying the percentage of unlabeled 5' caps relative to total 5' caps in the digested mRNA sample.

[0213] 29. The method of any one of paragraphs 1 to 27, wherein quantifying mRNA cap formation efficiency comprises quantifying the ratio of unlabeled 5' caps to total 5' caps in the digested mRNA sample.

[0214] 30. A kit for quantifying mRNA capping efficiency, the kit comprising: heavy-labeled substrates and / or cofactors, capping enzymes, and Optionally, a nuclease The kit comprises:

[0215] It will be apparent to those skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments without departing from the broad general scope of the present disclosure, and the present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0216] Example The present disclosure includes the following non-limiting examples.

[0217] Example 1 - RNA synthesis RNA synthesis was performed under ribonuclease-free conditions: all tubes, vials, pipette tips, pipettes, buffers, etc. had to be nuclease-free.

[0218] A DNA template encoding a self-replicating RNA was generated in competent Escherichia coli cells transformed with a DNA plasmid. Individual bacterial colonies were isolated, and the resulting plasmid DNA was amplified in E. coli cultures. After fermentation, the plasmid DNA was isolated and linearized by restriction digestion. The restriction enzyme was then removed using phenol / chloroform extraction and ethanol precipitation. mRNA was produced by in vitro transcription from the linearized DNA template using T7 RNA polymerase. The DNA template was subsequently removed by DNase digestion.

[0219] After in vitro transcription, a 7-methylguanylate cap structure (Cap 0) was added to the 5' end of the RNA using the vaccinia capping system according to the manufacturer's instructions (NEB). Briefly, purified mRNA products from the in vitro transcription reaction were denatured at 65°C for 5 minutes and then incubated on ice for 5 minutes. The following was added to the RNA in order while mixing: 10x capping buffer, 10 mM GTP, 2 mM S-adenosylmethionine, VCE). The reaction mixture was incubated at 37°C for 30 minutes. Upon completion, the final reaction mixture was purified. The purified mRNA was resuspended in nuclease-free water.

[0220] The following constructs were prepared: F602, which expresses the H5 and N1 antigens, and NSP1-4 (SEQ ID NO: 11). The sequence at the 5' end of the F602 construct comprises 5'-GAUAGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA (SEQ ID NO: 12).

[0221] Example 2 - Quantifying Capping Efficiency for Known Samples This example demonstrates the quantification of capping efficiency by converting uncapped mRNA to heavily labeled 0-capped mRNA. A schematic diagram illustrating the assay used in this example is shown in Figure 1.

[0222] mRNA samples containing uncapped mRNA were analyzed using the methods described herein. Briefly, a heavily labeled cap was added to the mRNA sample using the vaccinia capping system according to the manufacturer's instructions (New England Biolabs). More specifically, the in vitro synthesized RNA product was denatured at 65°C for 5 minutes and then incubated on ice for 5 minutes. While mixing, the following was added to the mRNA in order: 10x capping buffer, 10 mM GTP- 13 C 10 , 15 N5 (Sigma Aldrich, Cat. No. 645680), 2 mM S-adenosylmethionine, VCE). The reaction mixture was incubated for 30 minutes at 37°C.

[0223] After completion of the capping reaction, the labeled mRNA was treated with either RNAse T1 (ThermoFisher Scientific) or Nuclease P1 (New England Biolabs). For RNAse T1, the labeled mRNA product was denatured at 95°C for 5 minutes, then ramped down to 25°C for 5 minutes. RNAse T1 was added to the sample, and the mixture was incubated for 3 hours at 37°C. For Nuclease P1, the enzyme was added to the sample, and the mixture was incubated for 1 hour at 37°C. Upon completion, the final reaction mixture was optionally quenched by adding methanol.

[0224] LC-MS was used for quantitative analysis of capped and uncapped mRNA after RNAse digestion. Briefly, analysis of nuclease-treated mRNA samples was performed using a Vanquish UPLC (Thermo Scientific, Grand Island, NY, USA) connected to a TSQ Altis triple quadrupole mass spectrometer (Thermo Scientific, Grand Island, NY, USA). Mobile phase A consisted of 200 mM hexafluoroisopropanol, 5 mM N,N-dimethylhexylamine (pH 7.4), and mobile phase B was 100% methanol. A Waters Acquity™ Premier, Oligonucleotide, BEH C18, 130 Å, 1.7 μm, 2.1 × 50 mm column heated to 60°C at a flow rate of 0.400 mL / min was used for all analyses. General liquid chromatography parameters are listed in Table 2. The gradient profile for elution is shown in Table 3. [Table 7] [Table 8]

[0225] Mass spectra were obtained in negative ion mode using MS / MS multiple reaction monitoring. The mass spectrometer run parameters used are listed in Table 4. For this example, the multiple reaction monitoring (MRM) parameters for each molecule are listed in Table 5, including retention time (min), RT window (min), polarity, precursor (m / z), product (m / z), collision energy (V), minimum residence time (ms), and RF lens (V). Spectra were analyzed using Skyline software (available online at https: / / skyline.ms / project / home / software / Skyline / begin.view). [Table 9] [Table 10]

[0226] The LCMS results for T1-digested mRNA are shown in Figure 5A. The LCMS results for NP1-digested mRNA are shown in Figure 5B. Both show that only the labeled cap product is observed, which corresponds to 0% capping efficiency. This is consistent with the uncapped nature of the original mRNA sample. There is no background or interfering signal for Cap 0 in this uncapped sample.

[0227] Example 3 - Quantifying capping efficiency for unknown samples Generally, the absolute amount of capped species can be measured by the total ion chromatogram (TIC) from the corresponding transition. Analysis can be performed using a TSQ MS, or a Q-TOF or Orbitrap MS instrument, and the data can be processed using software provided by the supplier or third-party software such as Skyline. Data can be expressed in the form of peak area or the ratio of unlabeled peak area to labeled peak area. Here, peak area refers to the total peak area derived from all transitions for a particular precursor compound. Peak area can also refer to the specific transition selected for quantification. Capping efficiency (%) can be calculated using the following formula:

number

[0228] As an example, an mRNA sample with unknown capping efficiency was processed in the same manner as described in Example 2. The LCMS results for the T1-digested mRNA are shown in Figure 5C. The LCMS results for the NP1-digested mRNA are shown in Figure 5D. Both show mostly unlabeled capped product, which corresponds to the capped mRNA present in the original sample, while the small amount of labeled capped product observed corresponds to the uncapped mRNA present in the sample. Based on these peak areas, the capping efficiency is 94.4% for the T1 digest and 98.0% for the NP1 digest.

[0229] Example 4 - Quantification of capping efficiency relative to cap 0 using hGTP and hSAM This example demonstrates quantification of capped species for a Cap 0 reaction. In this example, the capped species of interest are Cap 0 (m7G-capped), unmethylated capped (G-capped), and uncapped mRNA. A schematic diagram illustrating the assay used in this example is shown in Figure 2.

[0230] Three types of samples were analyzed using the methods described herein: 1) a G-capped sample (1017 ng / mL), in which SMA remained in the capping reaction but the reaction efficiency was unknown; 2) a capped sample (2001 ng / mL), in which the capping reaction was performed with all reagents but the reaction efficiency was unknown; and 3) a blended sample consisting of an equal volume mixture of fully uncapped mRNA (1092 ng / mL), a G-capped sample (1017 ng / mL), and a capped sample (2001 ng / mL).

[0231] Briefly, mRNA samples were denatured at 65°C for 5 minutes and then incubated on ice for 5 minutes. While mixing, the following was added to the mRNA in order: 10x capping buffer, 10 mM heavy GTP- 13 C 10 , 15N5 (Sigma Aldrich, catalog number 645680), 2 mM heavy S-adenosylmethionine D3 (ChemCruz, catalog number sc-481746), and VCE). The reaction mixture was incubated at 37°C for 30 minutes.

[0232] After the capping reaction was complete, the mRNA product was denatured at 95°C for 5 minutes and then ramped down to 25°C for 5 minutes. The denatured mRNA was digested with RNAse T1 or nuclease P1. For RNAse T1 digestion, the enzyme was added to the sample and the mixture was incubated for 3 hours at 37°C. For nuclease P1 digestion, the enzyme was added to the sample and the mixture was incubated for 1 hour at 37°C.

[0233] The digested mRNA samples were analyzed by LC-MS as described in Example 2, with the MRM transition settings shown in Table 6. The results are shown in Figure 6. The percentage of each capped species present in the sample being analyzed is related to the relative peak areas of the unlabeled and labeled peaks. Of the two labeled peaks, the +3 Da heavy label results from methylation with heavy SAM and therefore represents the amount of unmethylated (G-capped) mRNA present in the original sample. The +18 Da heavy label results from incorporation of heavy SAM and heavy GTP and represents the uncapped mRNA present in the original sample.

[0234] As shown in Figure 6A, the G-capped sample (i.e., Sample 1) was determined to contain 84.2% G-capped mRNA and 15.8% uncapped mRNA (using RNAse T1) or 86.5% G-capped and 13.5% uncapped mRNA (using NP1), clearly demonstrating that the +3 Da heavy methylation was incorporated into both G-capped and uncapped mRNA.

[0235] As shown in Figure 6B (left panel), the capped sample (i.e., Sample 2) was determined to contain 87.4% capped, 1.7% G-capped, and 10.9% uncapped mRNA (using RNAse T1). As shown in Figure 6B (right panel), the capped sample (i.e., Sample 2) was determined to contain 90.9% capped, 1.4% G-capped, and 7.8% uncapped mRNA (using NP1). This demonstrates that the assay can be used to quantify the capping efficiency of samples with unknown capping efficiency. These two samples demonstrate that the efficiency of the capping reaction can be analyzed regardless of methylation status, and that the methylation status itself can also be quantified.

[0236] As shown in Figure 6C (left panel), the blend sample (i.e., Sample 3) was measured to contain 45.9% capped, 20% G-capped, and 34.1% uncapped mRNA (using RNAse T1). As shown in Figure 6C (right panel), the blend sample (i.e., Sample 3) was measured to contain 47.9% capped, 20.6% capped, and 31.5% uncapped mRNA (using NP1). The expected percentage of capped species in the blend sample can also be calculated based on the total mRNA concentration of each component sample and the measured percentage of capped species in each component sample (i.e., Figures 6A and 6B for the G-capped and uncapped samples, respectively). Assuming that the uncapped sample contained 100% uncapped mRNA, the blended sample was predicted to contain 42.5% cap 0 mRNA, 21.7% G-capped mRNA, and 35.8% uncapped mRNA (using RNAse T1) or 43.2% cap 0 mRNA, 23.9% G-capped mRNA, and 32.9% uncapped mRNA (using NP1). The measured values ​​of the blended sample (Figure 6C) were consistent with the predicted values ​​calculated from the constituent components.

[0237] Results from RNAse T1 and nuclease P1 digestion were consistent for all three samples. These examples demonstrate that the efficiency of capping and cap methylation reactions can be quantified by this method. [Table 11-1] [Table 11-2]

[0238] Example 5 - Quantification of capping efficiency using blend samples This example demonstrates the correlation between predicted and measured values ​​using blended samples containing varying amounts of capped and uncapped mRNA. Briefly, Samples A and B were blended at various ratios (V / V) (80%, 60%, 40%, 20%, including controls of 100% Sample A and 100% Sample B). The blended samples were analyzed according to the method described in Example 4. The predicted percentage of each species in each blend was calculated based on the following formula:

number

[0239] As shown in Figure 7, the measured capping percentages for the blend samples correspond well with the expected capping percentages (R 2 >0.98). This example further demonstrates that mRNA capping efficiency can be accurately quantified by this method.

[0240] array [Table 12-1] [Table 12-2] Table 12-3 Table 12-4 Table 12-5 Table 12-6

Claims

1. 1. A method for quantifying mRNA capping efficiency, the method comprising: treating the mRNA sample with a capping enzyme in the presence of a heavy labeling reagent to form a treated mRNA sample, optionally comprising mRNA with a heavy labeled 5' cap; digesting the treated mRNA sample with a nuclease to release the 5' cap; quantitatively determining the amount of heavy-labeled 5' cap and unlabeled 5' cap in the sample to quantify the mRNA capping efficiency; The method comprising:

2. The method of claim 1 , wherein the heavy-labeled reagent comprises a heavy-labeled substrate and / or a heavy-labeled cofactor.

3. 3. The method of claim 2, wherein the heavy-labeled substrate and / or cofactor comprises heavy GTP or heavy SAM, or a combination thereof.

4. The heavily labeled substrate and / or the heavily labeled cofactor are １３ C. １５ The method of claim 2, comprising N-GTP.

5. The heavily labeled substrate and / or the heavily labeled cofactor are ３ The method of claim 2, comprising:

6. 3. The method of claim 2, wherein the heavy-labeled substrate and / or cofactor comprises heavy GTP and heavy SAM.

7. 2. The method of claim 1, wherein the step of quantitatively determining the amount of heavy-labeled and unlabeled 5' caps in the sample comprises analyzing the released 5' caps by liquid chromatography / mass spectrometry (LC-MS) and measuring the relative amounts of heavy-labeled and unlabeled 5' fragments.

8. 2. The method of claim 1, wherein the capping enzyme comprises a vaccinia capping system or a poxvirus capping enzyme.

9. The method of claim 8 , wherein the capping enzyme comprises the vaccinia capping system.

10. 10. The method of claim 1, wherein the capping enzyme comprises a triphosphatase, a guanyltransferase, or a guanine methyltransferase, or a combination thereof.

11. 10. The method of claim 1, wherein the mRNA sample is synthesized by in vitro transcription.

12. 2. The method of claim 1, wherein the mRNA sample comprises capped mRNA produced by a post-transcriptional or co-transcriptional capping reaction.

13. The mRNA samples contained cap 0, cap 1, cap 2, cap 4, anti-reverse cap analog (ARCA), inosine, N7,2'-O-dimethyl-guanosine (mCAP), N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, 2-azido-guanosine, N6,2'-O-dimethyladenosine, 7-methylguanosine (mCAP), ... ７ 10. The method of claim 1, comprising capped mRNA having a 5' cap selected from the group consisting of CAP-003-CAP-225, CAP-003-CAP-G, and CAP-003-CAP-225.

14. The method of claim 13, wherein the 5' cap is Cap 0 or Cap 1.

15. The method of claim 13, wherein the 5' cap is Cap1.

16. The method of claim 13, wherein the 5' cap is Cap 0.

17. 2. The method of claim 1, wherein the nuclease comprises RNAse T1, nuclease NP1, or RNAse A, or a combination thereof.

18. 18. The method of claim 17, wherein the nuclease is RNAse T1.

19. 18. The method of claim 17, wherein the nuclease is nuclease NP1.

20. The mRNA sample has the formula (III): 【Chemistry 1】 wherein B is a nucleobase and R １ is H, halogen, OH, and OCH ３ and R ２ are H, OH, and OCH ３ and R ３ is CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ or absent, R ４ is NH ２ and R ５ is OH, n is 1, 2, or 3, M is a nucleotide of said mRNA, and The method of claim 1 , wherein the nuclease is RNAse T1.

21. 21. The method of claim 20, wherein the mRNA sample comprises capped mRNA having a 5' cap that is cap 0, and the nuclease is RNAse T1.

22. The mRNA sample has the formula (III): 【Chemistry 2】 wherein B is a nucleobase and R １ is H, halogen, OH, and OCH ３ and R ２ are H, OH, and OCH ３ and R ３ is CH ３ , C.H. ２ CH ３ , C.H. ２ CH ２ CH ３ or absent, R ４ is NH ２ and R ５ is OH or OCH ３ wherein n is 1, 2, or 3, and M is a nucleotide of said mRNA; and The method of claim 1, wherein the nuclease is nuclease NP1.

23. 23. The method of claim 22, wherein the mRNA sample comprises capped mRNA having a 5' cap that is cap 0 or cap 1, and the nuclease is nuclease NP1.

24. 2. The method of claim 1, wherein the mRNA sample comprises uncapped mRNA having a 5' triphosphate group or a 5' diphosphate group, or a combination thereof.

25. 2. The method of claim 1, wherein the mRNA sample comprises unmethylated capped mRNA having a 5' GpppN group, where N is any nucleotide.

26. 2. The method of claim 1, wherein quantitatively measuring the amount of labeled and unlabeled 5' caps in the sample comprises measuring the relative amounts of the labeled and unlabeled 5' caps.

27. 2. The method of claim 1, wherein quantifying mRNA capping efficiency comprises quantifying the absolute amount of capped mRNA in the mRNA sample.

28. 2. The method of claim 1, wherein quantifying mRNA capping efficiency comprises quantifying the percentage of unlabeled 5' caps relative to total 5' caps in the digested mRNA sample.

29. 2. The method of claim 1, wherein quantifying mRNA capping efficiency comprises quantifying the ratio of unlabeled 5' caps to total 5' caps in the digested mRNA sample.

30. 1. A kit for quantifying mRNA capping efficiency, the kit comprising: a heavily labeled substrate and / or cofactor; capping enzymes, and Optionally, a nuclease The kit comprises:

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