RNA capping efficiency assay

Abstract

Provided is a method for quantifying RNA capping efficiency, comprising: (a) providing a sample of capped RNA; (b) contacting the capped RNA with a protein nuclease, thereby hydrolyzing the RNA to produce hydrolysis products including capped products containing dinucleotides and uncapped products containing nucleotides, wherein step (b) is performed in the absence of a nucleic acid having a base sequence complementary to the sequence of the capped RNA; (c) separating the hydrolysis products by chromatography; and (d) determining the concentrations of the hydrolysis products by triple quadrupole mass spectrometry, thereby quantifying RNA capping efficiency.

Description

[Technical Field]

[0001] The present invention relates to a method for quantifying RNA capping efficiency. [Background technology]

[0002] Messenger RNA ("mRNA") therapy is becoming an increasingly important approach for the treatment of various diseases. Effective mRNA therapy requires the efficient delivery of the mRNA to a patient and the efficient production of the protein encoded by the mRNA within the patient's body.

[0003] To optimize mRNA delivery and protein production in vivo, a suitable cap is typically required at the 5' end of the construct, which protects the mRNA from degradation and facilitates successful protein translation. The cap structure at the 5' end of the mRNA can be introduced co-transcriptionally with so-called cap analogs or with the aid of enzyme complexes, such as the vaccinia capping system (post-transcriptional).

[0004] Accurate characterization of the percentage of capped RNA (capping efficiency) is particularly important for determining the quality of mRNA for therapeutic applications, to assess expected translation efficiency, and to optimize the RNA production process.

[0005] WO 2014 / 152659 and EP 2971102 describe capping assays in which the 5' end of an mRNA is first hybridized with a DNA oligonucleotide probe complementary to a sequence in the 5' untranslated region of the mRNA adjacent to the cap. The resulting DNA anneals to the mRNA, and treatment with one or more nucleases generates 5' fragments, which are then analyzed by chromatography to determine the relative amounts of capped and uncapped fragments. However, these publications do not disclose a method in which the step of hydrolyzing the capped RNA is performed in the absence of a nucleic acid having a base sequence complementary to the capped RNA sequence. Furthermore, this assay has the disadvantage that a complementary DNA probe must first be generated and its functionality tested for each different sequence at the 5' end. The need to synthesize complementary DNA for each capped RNA sequence to be analyzed makes this method slow, inefficient, and difficult to adapt to a variety of capped mRNAs. Furthermore, because transcribed RNA is not uniform at the 5' end, peaks found in the chromatogram must be assigned to capped or uncapped species.

[0006] Beverly et al., Anal. Bioanal. Chem., 2016, 408(18), 5021-30, describes a similar method involving hybridization of an RNA oligonucleotide containing several DNA nucleotides at the 3' end to the 5' end of RNA. This creates a short (approximately 6 nucleotide) DNA-RNA hybrid duplex that can be specifically hydrolyzed at this site using RNase H enzyme. This hydrolysis results in 5' fragments of the same length, which can be isolated using a biotin tag on the probe and tested using chromatography coupled to mass spectrometry. However, this method suffers from the same drawbacks as WO 2014 / 152659 in that a complementary RNA probe must first be generated for each different sequence at the 5' end of the mRNA and tested for functionality, and the hydrolysis fragments are not homogeneous.

[0007] Furthermore, in both of these methods, evaluation is hindered by transcripts that are one nucleotide shorter or longer. Depending on the cap analog used, these transcripts can occur, particularly in the case of sequences starting with two or three guanosines, which are recommended for efficient transcription. In addition, short truncated transcripts resulting from the natural mechanism of T7 RNA polymerase can affect the assay. Furthermore, in both methods, new cleavage oligonucleotides must be designed and validated for each new 5' sequence. This can be problematic for highly structured sequences.

[0008] Trotman et al. Bio Protoc. 2018, 8(6), e2767) describe enzymatic capping with radiolabeled guanosine triphosphate (GTP). To determine the efficiency of subsequent enzymatic methylation, RNA is first hydrolyzed with nuclease P1, and the degradation products are separated using thin-layer chromatography. Quantitation of methylated and unmethylated cap species is based on radioactivity. However, only the efficiency of the methyltransferase is determined, and this method requires the use of radiolabeled products, which means that additional safety protocols must be followed when performing this method.

[0009] WO 2017 / 149139 describes a method for analyzing samples containing mRNA molecules, which involves completely hydrolyzing RNA molecules, thereby releasing nucleosides, and then separating and quantifying the released nucleosides by HPLC. To completely hydrolyze the mRNA, phosphodiesterase I from the Eastern diamondback rattlesnake (Crotalus adamanteus) and shrimp alkaline phosphatase are added to nuclease P1. However, this method requires the use of three separate enzymes, thereby requiring many additional enzymatic steps and increasing the risk of cross-reactivity between enzymes. Furthermore, this method requires the determination of the RNA copy number in the sample to calculate the percent capping, and this additional calculation step introduces an additional level of uncertainty.

[0010] Muthmann et al., Methods, 2022, 203, 196-206, describes a method for quantifying mRNA cap modifications. The described method involves first completely hydrolyzing RNA molecules to nucleosides using nuclease P1 in conjunction with snake venom phosphodiesterase, followed by dephosphorylation with alkaline phosphatase. The cap modifications are then quantified by liquid chromatography coupled to triple quadrupole mass spectrometry. However, this method also requires the use of three separate enzymes, thereby requiring many additional enzymatic steps and increasing the risk of cross-reactivity between enzymes. Furthermore, this method aims to determine the level of mRNA modification rather than capping efficiency, thereby requiring the quantification of different analytes.

[0011] U.S. Patent Application Publication No. 2020 / 0032274 discloses synthetic thermostable polynucleotides and describes in general terms that capped polynucleotides can be treated with nucleases to obtain a mixture of free nucleotides, and that the capped 5',5-triphosphate cap structures can be detected by LC-MS, with the amount of capped product in the LC-MS spectrum corresponding to the efficiency of the capping reaction. This document does not disclose a method for determining the concentration of hydrolysis products by triple quadrupole mass spectrometry.

[0012] European Patent Publication No. 3090060 describes a method for measuring RNA capping efficiency by hydrolyzing RNA with the hammerhead ribozyme HHNUH2d, an RNA motif that catalyzes reversible cleavage and ligation reactions at specific sites within the RNA molecule. The products can be separated by HPLC, and the presence or absence of the cap structure can be determined using several methods, including quantitative mass spectrometry. This publication does not disclose a method in which the agent used to hydrolyze the RNA is a protein. Furthermore, this method has the disadvantage that a specific ribozyme or probe must be designed for each RNA sequence to be analyzed.

[0013] It would be desirable to provide a simplified method for determining RNA capping efficiency that is applicable to RNAs with any 5'-end sequence, allows for unambiguous and standardized evaluation, and does not require radioactive labeling, without the need to provide specific complementary nucleic acid probes for all RNAs to be analyzed. It would also be desirable to use a single enzyme, rather than multiple enzymes, to hydrolyze RNA.

[0014] These objectives are achieved by the present invention as defined and described herein. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] International Publication No. 2014 / 152659 Brochure [Patent Document 2] European Patent No. 2971102 [Patent Document 3] International Publication No. 2017 / 149139 Brochure [Patent Document 4] US Patent Application Publication No. 2020 / 0032274 [Patent Document 5] European Patent No. 3090060 [Non-patent literature]

[0016] [Non-Patent Document 1] Beverly et al. Anal.Bioanal.Chem., 2016, 408(18), 5021-30 [Non-patent document 2] Trotman et al. Bio Protoc.2018, 8(6), e2767) [Non-patent document 3] Muthmann et al. Methods, 2022, 203, 196-206 Summary of the Invention [Means for solving the problem]

[0017] According to one aspect of the present invention, there is provided a method for quantifying RNA capping efficiency, comprising the steps of: (a) providing a sample of capped RNA; (b) contacting the capped RNA with a nuclease, thereby hydrolyzing the RNA to produce hydrolysis products that include a capped product that includes a dinucleotide and a non-capped product that includes a nucleotide; (c) separating the hydrolysis products by chromatography; and (d) determining the concentration of the hydrolysis products by mass spectrometry, thereby quantifying the RNA capping efficiency.

[0018] According to another aspect of the present invention, there is provided a method for quantifying RNA capping efficiency, comprising the following steps (a) to (d): (a) providing a sample of capped RNA; (b) contacting the capped RNA with a protein nuclease, thereby hydrolyzing the RNA to produce hydrolysis products that include capped products that include dinucleotides and uncapped products that include nucleotides, wherein step (b) is performed in the absence of a nucleic acid having a base sequence complementary to that of the capped RNA; (c) separating the hydrolysis products by chromatography; and (d) determining the concentration of the hydrolysis product by triple quadrupole mass spectrometry, thereby quantifying the RNA capping efficiency.

[0019] Benefits and Surprising Findings Surprisingly, the present inventors have discovered that a capped RNA assay involving hydrolysis of capped RNA using a nuclease as the sole hydrolytic enzyme, thereby generating hydrolysis products containing nucleotides, followed by determination of the analytes by chromatography (particularly liquid chromatography) coupled to mass spectrometry (particularly tandem mass spectrometry), allows for the determination of capped and uncapped products, thus enabling the determination of RNA capping efficiency in a manner that is simple and applicable to any RNA sequence and can be achieved in a highly selective, flexible, and sensitive manner. This method can be used with a wide variety of different RNA cap structures to determine RNA capping efficiency. Because the hybridization step is eliminated, there is no need to first prepare a nucleic acid probe complementary to the RNA. This assay method only requires the generation of a corresponding external standard.

[0020] The present inventors have found that analyte determination using hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry, particularly using a triple quadrupole mass spectrometer, is particularly advantageous for nucleotide quantification. HILIC allows for the retention and separation of very non-polar analytes such as nucleotides. Triple quadrupole mass spectrometers, particularly when operated in multiple reaction monitoring (MRM) mode, allow for highly sensitive, selective, accurate, and universal detection of nucleotides compared to non-selective detection using, for example, but not exclusively, ultraviolet spectroscopy. The combination of HILIC with tandem mass spectrometry, particularly when operated in MRM mode, allows for highly selective, sensitive, and accurate measurement of nucleotides in complex matrices.

[0021] Furthermore, in contrast to the methods described in the above-mentioned WO 2017 / 149139 and Muthmann et al., the present method can be performed using a single enzyme rather than multiple enzymes, thereby simplifying the method by reducing the number of enzymatic steps and reducing or eliminating the risk of cross-reactivity between enzymes. In particular, the method of the present invention has the advantage over the method described in Muthmann et al. that it directly measures the nucleotides resulting from the hydrolysis of RNA by a nuclease, thus avoiding the need for the use of alkaline phosphatase to hydrolyze nucleotides to nucleosides.

[0022] Furthermore, in contrast to the method described in EP 3090060, which uses hammerhead ribozymes to hydrolyze RNA, the use of protein nucleases according to the present invention means that the method is equally applicable to any RNA sequence, thereby avoiding the need to design specific ribozymes or probes for each RNA sequence. [Brief explanation of the drawings]

[0023] [Figure 1] 1 is an LC chromatogram showing the products of β-S-ARCA-capped RNA hydrolyzed using the method of Example 1 and analyzed using the method of Example 2. [Figure 2] 1 is an LC chromatogram showing the products of CleanCap® 413-capped RNA hydrolyzed using the method of Example 1 and analyzed using the method of Example 2. [Figure 3] FIG. 3 is an LC chromatogram of standard nucleotides and dinucleotides used for comparison with FIGS. 1 and 2. [Figure 4] Figure 1 shows the capping efficiency of various cap amounts in in vitro transcription. [Figure 5]The capping efficiency of RNA with two different cap structures is shown, where "CC413 Cap" refers to CleanCap® 413 as defined herein, and "D1 Cap" refers to β-S-ARCA. DETAILED DESCRIPTION OF THE INVENTION

[0024] definition Although the present disclosure is described in detail below, it is to be understood that the disclosure is not limited to the particular methodology, protocols, and reagents described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is limited only by the appended claims.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, Basel, Switzerland, (1995). The practice of the present disclosure employs, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0026] Each element of the present invention is described below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered disclosed by this description unless the context indicates otherwise.

[0027] The term "about" means approximately or approximately, and in the context of numerical values ​​or ranges described herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0028] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the," and similar expressions, should be construed to encompass both the singular and the plural, unless otherwise stated herein or otherwise clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range. Unless otherwise stated herein, each individual value is incorporated herein as if it were individually recited herein.

[0029] The term "absent," when used in reference to an enzyme composition, preferably means that the described enzyme is not present in the enzyme composition. However, it should be understood that trace amounts of an enzyme described as not being present in the composition (e.g., less than 1 wt.%, less than 0.5 wt.%, less than 0.1 wt.%, less than 0.05 wt.%, less than 0.01 wt.%, less than 0.05 wt.%, less than 0.01 wt.%, less than 0.005 wt.% of the total weight of the enzyme composition) may be present in the enzyme composition as long as they do not affect the course of the enzymatic reaction or result in undesirable side reactions. In one embodiment, the term "absent" means that a substance is not added to the composition.

[0030] "Alkyl" generally refers to straight- and branched-chain saturated hydrocarbon groups having a specified number of carbon atoms (e.g., C 1-4 Alkyl refers to an alkyl group having 1 to 4 (i.e., 1, 2, 3, or 4) carbon atoms, C 1-6 (Alkyl refers to an alkyl group having 1 to 6 carbon atoms, etc.) Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-1-yl, n-hexyl, and the like. In some embodiments, alkyl is C 1-6 In some embodiments, alkyl is C 1-4 In some embodiments, alkyl is C 1-3 In some embodiments, alkyl is C 1-2 It means alkyl.

[0031] "Halo," "halogen," and "halogeno" may be used interchangeably and refer to fluoro, chloro, bromo, and iodo.

[0032] Annealing or Hybridization: As used herein, the terms "annealing," "hybridization," and grammatically equivalent terms refer to the formation of a complex (also called a duplex or hybrid) between nucleotide sequences that are sufficiently complementary to form a complex through Watson-Crick base pairing or non-canonical base pairing. It is understood that annealing or hybridizing sequences need not be perfectly complementary to provide a stable hybrid. In many situations, stable hybrids are formed when less than about 10% of the bases are mismatched. Thus, as used herein, the term "complementary" refers to a nucleic acid molecule that forms a stable duplex with its complement under specific conditions, generally with about 90% or more homology (e.g., about 95% or more, about 98% or more, or about 99% or more homology). Those skilled in the art understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least the desired level of complementarity stably hybridize, while sequences with lower complementarity do not hybridize. For examples of hybridization conditions and parameters, see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," 1989, Second Edition, Cold Spring Harbor Press: Plainview, NY, and Ausubel, "Current Protocols in Molecular Biology," 1994, John Wiley & Sons: Secaucus, NJ. Complementarity between two nucleic acid molecules is said to be "complete," "total," or "perfect" if all nucleic acid bases match; otherwise, it is said to be "partial."

[0033] Chromatography: As used herein, the term "chromatography" generally refers to a technique for the separation of mixtures. Typically, the mixture is dissolved in a fluid called the "mobile phase" or eluent, which carries the mixture through a structure that holds another material called the "stationary phase." More specific chromatographic techniques are defined in more detail herein.

[0034] Nucleoside: As used herein, the term "nucleoside" refers to a nucleobase, which may be adenine ("A"), guanine ("G"), cytosine ("C"), uracil ("U"), or thymine ("T"), linked to a carbohydrate, e.g., D-ribose (in RNA—this unit is called a "ribonucleoside") or 2'-deoxy-D-ribose (in DNA—this unit is called a "deoxyribonucleoside"), via a glycosidic bond between the anomeric carbon of the carbohydrate (the 1'-carbon atom of the carbohydrate) and the nucleobase. 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 may be substituted or unsubstituted. Substituted ribose sugars include, but are not limited to, those in which one or more carbon atoms, such as the 2'-carbon atom, are substituted with one or more of the same or different Cl, F, R, OR, NR, or halogen groups, where each R is independently H, C-C alkyl, or C-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'-alpha-anomeric nucleotide, 1'-alpha-anomeric nucleotide (Asseline et al., Nucl. Acids Res., 1991, 19, 4067-74), and 2'-O-[2-(N-methylcarbamoyl)ethyl]ribose (Yamada et al., J. Org. Chem. 2011, 76, 3042-53).

[0035] Nucleoside Analog: As used herein, the term "nucleoside analog" is intended to encompass compounds in which the carbohydrate portion of a nucleoside is replaced with a non-natural group. In one embodiment, the 2'-O and 4'-C or 3'-O and 4'-C positions of the ribose group are linked by a covalent bond or linker (typically a methylene or ethylene group); such groups are referred to as "locked nucleic acids" or "LNAs." The structure of LNAs can be found, for example, in Wengel, et al., Chemical Communications (1998) 455; Koshkin et al., Tetrahedron (1998) 54:3607; Jesper Wengel, Accounts of Chem. Research (1999) 32:301; Obika, et al., Tetrahedron Letters (1997) 38:8735; Obika, et al., Tetrahedron Letters (1998) 39:5401; and Obika, et al., Bioorganic Medicinal Chemistry (2008) 16:9230, as well as in WO 98 / 22489; WO 98 / 39352 and WO 99 / 14226.

[0036] In other embodiments, the carbohydrate moiety of the nucleotide is replaced with an N-(2-aminoethyl)glycine unit; such groups are referred to as "peptide nucleic acids" or "PNAs." PNAs can be synthetically produced using any technique known in the art. See, e.g., U.S. Pat. Nos. 6,969,766; 7,211,668; 7,022,851; 7,125,994; 7,145,006; and 7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 for the preparation of PNAs. Further teachings of PNA compounds can be found in Nielsen et al., Science, 254:1497-1500, 1991.

[0037] In other embodiments, the C2'-C3' bond of the carbohydrate moiety is broken, and such groups are referred to as "unlocked nucleic acid" or "UNA" moieties. UNAs are disclosed, for example, in WO 2016 / 070166.

[0038] In other embodiments, the carbohydrate moiety of the nucleotide is replaced with a morpholino group, the nucleobase is at position 3 of the morpholino group, and position 6 of the adjacent morpholino group is bonded to the phosphorus of the intersubunit linkage (via a -CH-O- bond), which in turn is bonded to the nitrogen of the adjacent morpholino group. Typically, in such compounds, the negatively charged oxygen of the phosphate intersubunit linkage is replaced by an amide or substituted amide group; such compounds having both a morpholino backbone and a phosphorodiamidate intersubunit linkage are referred to as "phosphorodiamidate morpholinos" (or simply "morpholino" groups). Their general structure is as set forth in Figure 2 of Summerton, J., et al., Antisense & Nucleic Acid Drug Development, 7:187-195 (1997), and their synthesis, structure, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063; 5,506,337; 8,076,476; and 8,299,206.

[0039] Nucleotide: As used herein, the term "nucleotide" refers to a nucleoside (or nucleoside analog) in phosphorylated form (phosphate ester of the nucleoside or nucleoside analog) as a monomer unit or within a polynucleotide polymer. The phosphate group can be located on any oxygen atom on the sugar moiety of the nucleotide. Typically, the phosphate group is located at the 3' or 5' position, preferably the 5' position. The phosphate group can contain any number of phosphate units, typically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units. Preferably, the phosphate group is monophosphate (one phosphate unit), diphosphate (two phosphate units), or triphosphate (three phosphate units). Sulfur can replace the oxygen atom in any or all of the phosphate groups to form a thiophosphate group. "Nucleotide 5'-triphosphate" refers to a nucleotide having a triphosphate group at the 5' position, and is sometimes referred to as "NTP," or "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., alpha-thio-nucleotide 5'-triphosphate). Nucleotides may exist in mono-, di-, or tri-phosphorylated forms. The carbon atom of ribose present in a nucleotide is indicated with a prime letter (') to distinguish it from the backbone numbering in the base. For a review of the chemistry of polynucleotides and nucleic acids, see Shabarova, Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH, New York, 1994.

[0040] Dinucleotide: As used herein, the term "dinucleotide" refers to a nucleic acid containing two nucleosides or nucleoside analogs (as defined above) linked by a mono- or poly-phosphate ester group. The phosphate group may be present on any oxygen atom on the sugar moiety of the nucleotide. The phosphate group may contain any number of phosphate units, typically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units. Preferably, the phosphate group is monophosphate (one phosphate unit), diphosphate (two phosphate units), triphosphate (three phosphate units), or tetraphosphate (four phosphate units). Additionally, any one or all of the oxo (=O) groups in the phosphate units (preferably, only one oxo group in one phosphate unit) may be replaced with a thio (=S) group. The phosphate groups may be independently present at the 3' or 5' position of each nucleoside, and preferably at the 5' position of both nucleosides. In one embodiment, the dinucleotide comprises two nucleosides linked by a 5',5'-triphosphate bridge.

[0041] Nucleic Acid: The terms "nucleic acid," "nucleic acid molecule," "polynucleotide," or "oligonucleotide" may be used interchangeably herein. They refer to polymers of nucleotide monomers or analogs thereof, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), and combinations thereof. Nucleotides may be of genomic, synthetic, or semi-synthetic origin and may contain nucleosides (defined above) or nucleoside analogs (defined above), encompassing nucleic acid-like structures with synthetic backbones, as well as amplification products. As will be appreciated 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 may be linear, branched, or circular molecules. Polynucleotides also refer to polymers of nucleotides, such as nucleotides, nucleotides, and oligonucleotides. + , NH4 + , trialkylammonium, Mg 2+ , Na +A polynucleotide can be composed of only deoxyribonucleotides, only ribonucleotides, or chimeric mixtures thereof, or nucleotides containing nucleoside analogs. A polynucleotide can be composed of internucleotide nucleobase and sugar analogs.

[0042] In some embodiments, the term "oligonucleotide" is used herein to refer to a polynucleotide that includes from about 5 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.

[0043] Throughout this specification, whenever an oligonucleotide is represented by a sequence of letters (e.g., selected from the four base letters: A, C, G, and T, which represent adenosine, cytidine, guanosine, and thymidine, respectively), the nucleotides are presented in 5' to 3' order from left to right. A "polynucleotide sequence" refers to the sequence of nucleotide monomers along a polymer. Unless otherwise specified, whenever a polynucleotide sequence is represented, it is understood that the nucleotides are in 5' to 3' direction from left to right.

[0044] Nucleic acids, polynucleotides, and oligonucleotides may be composed of standard nucleotide bases or may be substituted with nucleotide isoform analogs, including, but not limited to, iso-C and iso-G bases, which may hybridize more or less acceptably than standard bases and hybridize preferentially with complementary isoform analog bases. Many such isoform bases are described, for example, by Benner et al., Cold Spring Harb. Symp. Quant. Biol. 1987, 52, 53-63. Analogs of naturally occurring nucleotide monomers include, for example, 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, 7-methyl-guanine, inosine, nebularine, nitropyrrole (Bergstrom, J. Amer. Chem. Soc., 1995, 117, 1201-1209), nitroindole, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine (Seela, U.S. Pat. No. 6,147,199), 7-deazaguanine (Seela, U.S. Pat. No. 5,990,303), 2-azapurine (Seela, WO 01 / 16149), ), 2-thiopyrimidine, 6-thioguanine, 4-thiothymine, 4-thiouracil, O-6-methylguanine, N-6-methyladenine, O-4-methylthymine, 5,6-dihydrothymine, 5,6-dihydrouracil, 4-methylindole, pyrazolo[3,4-d]pyrimidine, "PPG" (Meyer, U.S. Pat. Nos. 6,143,877 and 6,127,121; Gall, WO 01 / 38584), and ethenoadenine (Fasman (1989) in Practical Handbook of Biochemistry and Molecular Biology, pp. 385-394, CRC Press, Boca Raton, Fla.).

[0045] The term "3'" refers to a region or position in a polynucleotide or oligonucleotide that is 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 that is 5' (i.e., upstream) from another region or position in the same polynucleotide or oligonucleotide. The terms "3' end" and "3' terminus," as used herein with respect to a nucleic acid molecule, refer to the end of a nucleic acid that contains a free hydroxyl group attached to the 3' carbon of the terminal pentose sugar. The terms "5' end" and "5' terminus," as used herein with respect to a nucleic acid molecule, refer to the end of a nucleic acid molecule that contains 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 region of polyadenosine at its 5' end.

[0046] Step (a) - Preparation of capped RNA The methods of the present invention begin with the preparation of capped RNA. The capped RNA can be any capped RNA, either natural or synthetic. Typically, the RNA comprises a nucleotide having a base with a ribose sugar attached to the 1' position and a phosphate group that can be attached to the 5' or 3' position. The base can be adenine (A), cytosine (C), guanine (G), or uracil (U). Typically, the RNA is capped mRNA.

[0047] The cap can have any structure, natural or synthetic, that can bind to the cap-binding complex and EIF4E and function to allow translation of RNA during protein synthesis and / or protect the RNA from degradation by 5'-3' exonucleases.

[0048] In one embodiment, the cap has the structure of formula (I): [ka] (In the formula, PM is a monophosphate or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units, wherein an oxo (=O) group in any of the phosphate units is optionally replaced with a thio (=S) group; R is an end-capping moiety; B is C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R' is OH, O(C 1-4 alkyl), and halogen; The wavy line represents the rest of the RNA molecule.)

[0049] In formula (I), R can represent any group that enables the cap to perform the functions described above of binding to the cap-binding complex and EIF4E to enable translation of RNA during protein synthesis and / or protect RNA from degradation by 5'-3' exonucleases.

[0050] In one embodiment, the cap has the structure of Formula (Ia): [ka] (In the formula, PM is a monophosphate or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units, wherein an oxo (=O) group in any of the phosphate units is optionally replaced with a thio (=S) group; Nuc is a nucleoside or nucleoside analogue; B is C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R' is OH, O(C 1-4 alkyl), and halogen; The wavy line represents the rest of the RNA molecule.)

[0051] In formulas (I) and (Ia), PM is preferably a monophosphate (one phosphate unit), diphosphate (two phosphate units), triphosphate (three phosphate units), or tetraphosphate (four phosphate units), and any one or all of the oxo (=O) groups in the phosphate units (preferably only one oxo group in one phosphate unit) may be replaced with a thio (=S) group.

[0052] In one embodiment of Formula (Ia), Nuc is a nucleoside, which may be a ribonucleoside or a deoxyribonucleoside (as defined above).

[0053] In another embodiment of Formula (Ia), Nuc is a nucleoside analog as defined above. The nucleoside analog may comprise a locked nucleic acid (LNA) moiety, a peptide nucleic acid (PNA) moiety, an unlocked nucleic acid (UNA) moiety, or a morpholino moiety, as defined above.

[0054] In one embodiment of either Formula (I) or (Ia), R' is OH or OCH3.

[0055] In one embodiment, the cap has a structure of Formula (Ib) or a salt thereof: [ka] (In the formula, B is C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R1 is OH, O(C 1-4 alkyl), and halogen; R2 is H, OH, O(C 1-4 alkyl), and halogen; R3 is OH, O(C 1-4 alkyl), and halogen; R4 is H, OH, O(C 1-4 alkyl), halogen, or C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; n is 1, 2 or 3; X1, each X2, and X3 are each independently O or S; The wavy line represents the rest of the RNA molecule.)

[0056] In one embodiment of Formula (Ib), R4 is OH. In one embodiment of Formula (Ib), R4 is C 1-4 It is a nucleobase which may be alkylated on the nitrogen atom by an alkyl group.

[0057] In one embodiment, the cap has a structure of Formula (Ic) or a salt thereof: [ka] (In the formula, B and B' are each independently C 1-4 are nucleobases each optionally alkylated on the nitrogen atom by an alkyl group; R1 is OH, O(C 1-4 alkyl), and halogen; R2 is H, OH, O(C 1-4 alkyl), and halogen; R3 is OH, O(C 1-4 alkyl), and halogen; n is 1, 2 or 3; X1, each X2, and X3 are each independently O or S; The wavy line represents the rest of the RNA molecule.)

[0058] In one embodiment of Formula (Ic), B is selected from adenine (“A”), guanine (“G”), cytosine (“C”), or uracil (“U”), each of which is selected from C 1-4 It may be alkylated on the nitrogen atom by an alkyl group, for example by a methyl group. In one embodiment of formula (Ic), B is G which may be methylated on the nitrogen at the 7' position.

[0059] In one embodiment of either Formula (Ib) or (Ic), B is selected from adenine (“A”), guanine (“G”), cytosine (“C”), or uracil (“U”), each of which is selected from C 1-4 It may be alkylated on the nitrogen atom by an alkyl group, for example by a methyl group. In one embodiment of either Formula (Ib) or (Ic), B is G which may be methylated on the nitrogen at the 7' position.

[0060] In one embodiment of either Formula (Ib) or (Ic), R1 is OH or OCH3.

[0061] In one embodiment of either Formula (Ib) or (Ic), R2 is H, OH, or OCH3.

[0062] In one embodiment of either Formula (Ib) or (Ic), R3 is OH or OCH3.

[0063] In one embodiment of either formula (Ib) or (Ic), n is 1.

[0064] In one embodiment of either Formula (Ib) or (Ic), X1 is O. In one embodiment of either Formula (Ib) or (Ic), X3 is O. In one embodiment of either Formula (Ib) or (Ic), X1 is S. In one embodiment of either Formula (Ib) or (Ic), X3 is S. In one embodiment of either Formula (Ib) or (Ic), each X2 is O. In one embodiment of either Formula (Ib) or (Ic), each X2 is S.

[0065] In one embodiment of Formula (Ic), B and B' are both G, which may be methylated on the 7' nitrogen. In one embodiment of Formula (Ic), B is G and B' is 7'-methyl-G.

[0066] In one embodiment of either Formula (Ib) or (Ic), n is 1, X1 and X3 are O, and X2 is O. In one embodiment of either Formula (Ib) or (Ic), n is 1, X1 and X3 are O, and X2 is S.

[0067] In one embodiment, the cap is a naturally occurring cap structure. An example of a naturally occurring cap structure is a cap structure linked to the 5' end of the first transcribed nucleotide via a triphosphate bridge. 7 and 7-methylguanosine, resulting in a dinucleotide cap of G(5')ppp(5')N, where N is any nucleoside. This cap has the structure of formula (Ic), where B' is 7-methyl-G, n is 1, each X is O, X' is O, and R1, R2, and R3 are all OH.

[0068] In vivo, the cap is added enzymatically. It is added in the nucleus and is catalyzed by the enzyme guanylyltransferase. Addition of the cap to the 5' end of the RNA occurs immediately after the initiation of transcription. The terminal nucleoside is typically guanosine, and is in the reverse orientation relative to all other nucleotides: G(5')ppp(5')GpNpNp.

[0069] A common cap for mRNA produced by in vitro transcription is m 7 G(5')ppp(5')G, which is used as a dinucleotide cap in in vitro transcription using T7 or SP6 RNA polymerase to yield RNA with a cap structure at the 5' end. A common method for in vitro synthesis of capped mRNA uses m as the initiator of transcription. 7 A preformed dinucleotide of the form G(5')ppp(5')G ("m7 GpppG") is used.

[0070] In one embodiment, the cap structure is a synthetic cap structure. One example of a synthetic dinucleotide cap used in in vitro translation experiments is the anti-reverse cap analog ("ARCA"), a modified cap analog in which the 2' or 3' OH group is generally replaced with -OCH3. ARCA and triple methylated cap analogs are incorporated in the forward orientation. The m at either the 2' or 3' OH group of the ribose ring is replaced with -OCH3. 7 Chemical modification of G results in cap incorporation only in the forward orientation, even though the 2'OH group is not involved in the phosphodiester bond (Jemielity, J. et al., RNA, 2003, 9:1108-1122). Selective procedures for methylation at N7 of guanosine, 3'O-methylation, and 5' diphosphate synthesis have been established (Kore, A. and Parmar, G. Nucleosides, Nucleotides, and Nucleic Acids, 2006, 25, 337-340, and Kore, A.R., et al. Nucleosides, Nucleotides, and Nucleic Acids 2006, 25, 307-14).

[0071] In one embodiment, the cap structure is a β-S-ARCA cap structure, which is a structure of formula (Ic) where B is G, B' is 7'-methyl-G, n is 1, X1 and X3 are O, X2 is S, R1 is OH, R2 is OCH3, and R3 is OH.

[0072] In one embodiment, the cap structure is that of CleanCap® 413, which has the structure of formula (Ic) where B is A, B' is 7'-methyl-G, n is 1, X1, X2, and X3 are all O, R1 is OH, R2 is OH, and R3 is OCH3, and the structure is connected to G via a monophosphate intersubunit bond with a wavy line. CleanCap® 413 is commercially available from TriLink Biotechnologies.

[0073] In one embodiment, the cap structure is CleanCap® AU, which has the structure of formula (Ic) where B is A, B' is 7'-methyl-G, n is 1, X1, X2, and X3 are all O, R1 is OH, R2 is OH, and R3 is OCH3, and the structure is connected to U via a monophosphate intersubunit bond with a wavy line. CleanCap® AU is commercially available from TriLink Biotechnologies.

[0074] Preparation of capped RNA Capped RNA can be produced by any means known in the art. Typically, the RNA is produced by transcription of the corresponding DNA sequence.

[0075] In some embodiments, capped RNA is produced by in vitro transcription, a method originally developed by Krieg and Melton (Methods Enzymol., 1987, 155:397-415) for the synthesis of RNA using RNA phage polymerases. Typically, these reactions include at least a phage RNA polymerase (e.g., T7, T3, or SP6), a DNA template containing a phage polymerase promoter, nucleotides (particularly nucleoside triphosphates such as ATP, CTP, GTP, and UTP, or modified nucleotides such as N1-Me-Pseudo-UTP), and a buffer containing a salt (particularly a magnesium salt).

[0076] RNA synthesis yields can be optimized by increasing nucleotide concentrations, adjusting magnesium concentrations, and including inorganic pyrophosphatase (U.S. Pat. No. 5,256,555; Gurevich, et al., Anal. Biochem. 1991, 195 207-213; Sampson, JR and Uhlenbeck, OC, Proc. Natl. Acad. Sci. USA. 1988, 85, 1033-1037; Wyatt, JR, et al., Biotechniques, 1991, 11, 764-769). Some embodiments utilize commercially available kits for large-scale synthesis of in vitro transcripts (e.g., MEGAscript®, Ambion). RNA synthesized in these reactions is typically characterized by a 5'-terminal nucleotide bearing a triphosphate at the 5' position of the ribose. Typically, this nucleotide is guanosine, but may also be adenosine, depending on the combination of RNA polymerase and promoter used (see, e.g., Coleman, TM, et al., Nucleic Acids Res., 2004, 32, e14).

[0077] To synthesize capped RNA by in vitro transcription, use a cap analog (e.g., N-7 methyl GpppG; i.e., m 7 A cap analog (GpppG) is included in the transcription reaction. In some embodiments, RNA polymerase incorporates the cap analog as readily as any other nucleotide, i.e., there is no bias for the cap analog. In some embodiments, the cap analog is incorporated at the 5' end by the enzyme guanylyltransferase.

[0078] In some embodiments using T7, T3 and SP6 RNA polymerases, the +1 nucleotide of their respective promoters is typically a G residue, with GTP and m 7When both GpppGs are present at equal concentrations in a transcription reaction, they each have an equal chance of being incorporated at the +1 position. 7 GpppG is present in these reactions at concentrations several times higher than GTP to increase the likelihood that transcripts will have a 5' cap. In some embodiments, the mMESSAGE mMACHINE® kit (Cat. No. 1344, Ambion) is used according to the manufacturer's instructions, and a cap-to-GTP ratio of 4:1 (6 mM:1.5 mM) is recommended. In some embodiments, as the ratio of cap analog to GTP increases during the reaction, the ratio of capped to uncapped RNA increases proportionally.

[0079] Capping efficiency considerations must be balanced with yield considerations. Increasing the ratio of cap analog to GTP in the transcription reaction reduces the yield of total RNA because the concentration of GTP becomes limiting when the total concentration of cap and GTP is held constant. Thus, the final RNA yield depends on the concentration of GTP required for transcript elongation. Other nucleotides (ATP, CTP, UTP) are present in excess.

[0080] In another embodiment, mRNA is synthesized by in vitro transcription from a plasmid DNA template encoding a selected gene.

[0081] In one embodiment, the method includes purifying the capped RNA. The capped RNA can be purified by any means known in the art.

[0082] In one embodiment, the capped RNA is purified using magnetic beads. As known to those skilled in the art, magnetic separation of nucleic acids involves introducing magnetic beads into a solution containing RNA (typically with a binding buffer), followed by applying a magnetic field (e.g., by using a permanent magnet) to separate the beads with bound RNA. The supernatant containing impurities can then be washed away, and the RNA can be eluted from the beads. Such techniques are generally described in S. Berensmeier, Appl. Microbiol. Biotech., 2006, 73, 495-504.

[0083] In one embodiment, capped RNA is purified using tangential flow filtration (TFF). As known to those skilled in the art, tangential flow filtration (also known as cross-flow filtration) typically operates by passing a feed across (tangentially) a filter membrane at positive pressure relative to the permeate side. The portion of material smaller than the membrane pore size passes through the membrane as permeate or filtrate, while everything else is retained on the feed side of the membrane as retentate. The tangential movement of the fluid mass across the membrane scrapes off particles trapped on the filter surface.

[0084] Step (b)—Enzymatic hydrolysis of capped RNA According to step (b) of the method of the present invention, the capped RNA is hydrolyzed to produce hydrolysis products that include a capped product that includes a dinucleotide and a non-capped product that includes a nucleotide.

[0085] The capped RNA is hydrolyzed by contacting it with a nuclease. The exact nature of the nuclease is not limited, as long as it is capable of hydrolyzing the capped RNA to produce a capped product containing a dinucleotide and an uncapped product containing a nucleotide. Typically, the nuclease is a protein.

[0086] In one embodiment, the nuclease is unable to hydrolyze nucleotides to nucleosides.

[0087] In step (b), the nuclease may be present as part of an enzyme composition containing additional enzymes. In this embodiment, typically the nuclease constitutes more than 50% by weight of the total weight of the uncapped hydrolysate, such as more than 60% by weight, for example more than 70% by weight, such as more than 80% by weight, for example more than 90% by weight, such as more than 95% by weight, for example more than 96% by weight, such as more than 97% by weight, for example more than 98% by weight, such as more than 99% by weight, for example more than 99.5% by weight, such as more than 99.7% by weight, for example more than 99.9% by weight, such as more than 99.99% by weight.

[0088] Step (b) is carried out in the absence of a nucleic acid having a base sequence complementary to the sequence of the capped RNA. In one embodiment, step (b) is carried out in the absence of a DNA having a base sequence complementary to the sequence of the capped RNA. In contrast to the method of WO 2014 / 152659, which uses a nuclease in combination with a complementary oligonucleotide probe to hydrolyze RNA, the use of a nuclease as the sole hydrolysis agent avoids the need to prepare and test specific complementary probes for each different RNA to be quantified by the assay. This makes the method easier to use and applicable to any RNA sequence.

[0089] In one embodiment, a nuclease is the only agent that hydrolyzes RNA. In contrast to the methods used by Muthmann et al. and WO 2017 / 149139, which use a nuclease in combination with alkaline phosphatase and phosphodiesterase to completely hydrolyze RNA into nucleosides, using a nuclease as the only hydrolyzing agent, thereby limiting the scope of hydrolysis to nucleotides, allows the method to be performed using a single enzyme rather than multiple enzymes, thereby reducing the number of enzymatic steps and simplifying the method by reducing or eliminating the risk of cross-reactivity between enzymes.

[0090] In one embodiment, step (b) is performed in the absence of PDE1. In one embodiment, step (b) is performed in the absence of snake venom phosphodiesterase. In one embodiment, step (b) is performed in the absence of phosphatase. In one embodiment, step (b) is performed in the absence of alkaline phosphatase.

[0091] In one embodiment, the nuclease used in step (b) is nuclease P1 or nuclease S1. In one embodiment, the nuclease used in step (b) is nuclease P1.

[0092] In one embodiment, step (b) is carried out at a nuclease concentration of 10 to 70 μmol / L, preferably 20 to 35 μmol / L.

[0093] In one embodiment, step (b) is performed at a temperature between room temperature and 60°C. In one embodiment, step (b) is performed at a temperature between 30 and 55°C. In one embodiment, step (b) is performed at a temperature of 37°C. In one embodiment, step (b) is performed at a temperature of 50°C.

[0094] In one embodiment, step (b) is carried out for a time period of 30 minutes to 48 hours. In one embodiment, step (b) is carried out for a time period of 1 hour to 36 hours. In one embodiment, step (b) is carried out for a time period of 2 hours to 30 hours. In one embodiment, step (b) is carried out for a time period of 3 hours to 24 hours.

[0095] In one embodiment, step (b) is carried out at a pH of 4 to 6. In one embodiment, step (b) is carried out at a pH of 4.3 to 5.5. In one embodiment, step (b) is carried out at a pH of 4.5. In one embodiment, step (b) is carried out at a pH of 5.3.

[0096] Capped hydrolysis products Hydrolysis of RNA by the methods of the present invention yields a capped RNA product.

[0097] In one embodiment, the capped hydrolysis product comprises a dinucleotide. In one embodiment, the capped hydrolysis product consists essentially of a dinucleotide. In one embodiment, the capped hydrolysis product is a dinucleotide. In one embodiment, the capped hydrolysis product consists of a dinucleotide. Dinucleotides are as defined and exemplified above.

[0098] In one embodiment, the dinucleotide constitutes more than 50% by weight of the total weight of the capped hydrolysate, such as more than 60% by weight, for example more than 70% by weight, such as more than 80% by weight, for example more than 90% by weight, such as more than 95% by weight, for example more than 96% by weight, such as more than 97% by weight, for example more than 98% by weight, such as more than 99% by weight, for example more than 99.5% by weight, such as more than 99.7% by weight, for example more than 99.9% by weight, such as more than 99.99% by weight.

[0099] In one embodiment, the capped hydrolysis product has the structure of formula (II): [ka] (In the formula, PM is a monophosphate or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units, wherein an oxo (=O) group in any of the phosphate units is optionally replaced with a thio (=S) group; R is an end-capping moiety; B is C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R' is OH, O(C 1-4 alkyl), and halogen)

[0100] In formula (II), R can represent any group that enables the cap to perform the functions described above of binding to the cap-binding complex and EIF4E to enable translation of RNA during protein synthesis and / or protect RNA from degradation by 5'-3' exonucleases.

[0101] In one embodiment, the capped hydrolysis product has the structure of Formula (IIa): [ka] (In the formula, PM is a monophosphate or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units, wherein an oxo (=O) group in any of the phosphate units is optionally replaced with a thio (=S) group; Nuc is a nucleoside or nucleoside analogue; B is C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R' is OH, O(C 1-4 alkyl), and halogen)

[0102] In formulas (II) and (IIa), PM is preferably a monophosphate (one phosphate unit), diphosphate (two phosphate units), triphosphate (three phosphate units), or tetraphosphate (four phosphate units), and any one or all of the oxo (=O) groups in the phosphate units (preferably only one oxo group in one phosphate unit) may be replaced with a thio (=S) group.

[0103] In one embodiment of Formula (IIa), Nuc is a nucleoside, which may be a ribonucleoside or a deoxyribonucleoside (as defined above).

[0104] In another embodiment of Formula (IIa), Nuc is a nucleoside analog as defined above. The nucleoside analog may comprise a locked nucleic acid (LNA) moiety, a peptide nucleic acid (PNA) moiety, a non-locked nucleic acid (UNA) moiety, or a morpholino moiety, as defined above.

[0105] In one embodiment, the capped hydrolysis product has the structure of Formula (IIb) or a salt thereof: [ka] (In the formula, B is C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R1 is OH, O(C 1-4 alkyl), and halogen; R2 is H, OH, O(C 1-4 alkyl), and halogen; R3 is OH, O(C 1-4 alkyl), and halogen; R4 is H, OH, O(C 1-4 alkyl), halogen, or C 1-4 a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; n is 1, 2 or 3; X1, each X2, and X3 are each independently O or S.

[0106] In one embodiment of Formula (IIb), R4 is OH. In one embodiment of Formula (IIb), R4 is C 1-4 It is a nucleobase which may be alkylated on the nitrogen atom by an alkyl group.

[0107] In one embodiment, the capped hydrolysis product has the structure of formula (IIC) or a salt thereof. [ka] (In the formula, B and B' are each independently C 1-4 are nucleobases each optionally alkylated on the nitrogen atom by an alkyl group; R1 is OH, O(C 1-4 alkyl), and halogen; R2 is H, OH, O(C 1-4 alkyl), and halogen; R3 is OH, O(C 1-4 alkyl), and halogen; n is 1, 2 or 3; each X and X' is independently O or S;

[0108] In one embodiment of Formula (IIc), B is selected from adenine (“A”), guanine (“G”), cytosine (“C”), or uracil (“U”), each of which is selected from C 1-4 It may be alkylated on the nitrogen atom by an alkyl group, for example by a methyl group. In one embodiment of formula (IIc), B is G which may be methylated on the nitrogen at the 7' position.

[0109] In one embodiment of either Formula (IIb) or (IIc), B is selected from adenine (“A”), guanine (“G”), cytosine (“C”), or uracil (“U”), each of which is selected from C 1-4It may be alkylated on the nitrogen atom by an alkyl group, for example by a methyl group. In one embodiment of either Formula (IIa) or (IIb), B is G which may be methylated on the nitrogen at the 7' position.

[0110] In one embodiment of either Formula (IIb) or (IIc), R1 is OH or OCH3.

[0111] In one embodiment of either Formula (IIb) or (IIc), R2 is H, OH, or OCH3.

[0112] In one embodiment of either Formula (IIb) or (IIc), R3 is OH or OCH3.

[0113] In one embodiment of either formula (IIb) or (IIc), n is 1.

[0114] In one embodiment of either Formula (IIb) or (IIc), X1 is O. In one embodiment of either Formula (IIb) or (IIc), X3 is O. In one embodiment of either Formula (IIb) or (IIc), X1 is S. In one embodiment of either Formula (IIb) or (IIc), X3 is S. In one embodiment of either Formula (IIb) or (IIc), each X2 is O. In one embodiment of either Formula (IIb) or (IIc), each X2 is S.

[0115] In one embodiment of Formula (IIc), B and B' are both G, which may be methylated on the 7' nitrogen. In one embodiment of Formula (IIc), B is G and B' is 7'-methyl-G.

[0116] In one embodiment of either Formula (IIb) or (IIc), n is 1, X1 and X3 are O, and X2 is O. In one embodiment of either Formula (IIb) or (IIc), n is 1, X1 and X3 are O, and X2 is S.

[0117] In one embodiment, the capped hydrolysis product is m 7 G(5')ppp(5')N,m 7 G(5')ppp(5')-(cap0), m 7 G(5')ppp(5')Nm-(cap1), m 7 It is selected from the group consisting of G(5')ppp(5')G, ARCA, and β-S-ARCA, where G is guanosine, p is a phosphate residue, N is any nucleoside, and Nm is a nucleoside having a 2'-methyl group.

[0118] In one embodiment, the capped hydrolysis product is a dinucleotide resulting from the hydrolysis of a β-S-ARCA-capped RNA, i.e., a structure of formula (IIc) where B is G, B' is 7'-methyl-G, n is 1, X1 and X3 are O, X2 is S, R1 is OH, R2 is OCH3, and R3 is OH.

[0119] In one embodiment, the capped hydrolysis product is a dinucleotide resulting from the hydrolysis of RNA capped with CleanCap® 413, i.e., a structure of formula (IIc) where B is A, B' is 7'-methyl-G, n is 1, X1, X2, and X3 are all O, R1 is OH, R2 is OH, and R3 is OCH3.

[0120] In one embodiment, the capped hydrolysis product is a dinucleotide resulting from the hydrolysis of a CleanCap® AU-capped RNA, i.e., a structure of formula (IIc) where B is A, B' is 7'-methyl-G, n is 1, X1, X2, and X3 are all O, R1 is OH, R2 is OH, and R3 is OCH3.

[0121] Uncapped hydrolysis products Additionally, hydrolysis of RNA by the methods of the present invention results in an uncapped RNA product.

[0122] In one embodiment, the uncapped hydrolysis product comprises a nucleotide. In one embodiment, the uncapped hydrolysis product consists essentially of a nucleotide. In one embodiment, the uncapped hydrolysis product is a dinucleotide. In one embodiment, the uncapped hydrolysis product consists of a dinucleotide.

[0123] Nucleotides are as defined and exemplified above, i.e., phosphate esters of nucleosides. The phosphate group can be located at any oxygen on the sugar moiety of the nucleotide. Typically, the phosphate group is located at the 3' or 5' position, preferably the 5' position. The phosphate group can contain any number of phosphate units, typically 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units. Preferably, the phosphate group is monophosphate (one phosphate unit), diphosphate (two phosphate units), or triphosphate (three phosphate units).

[0124] In one embodiment, the nucleotides constitute more than 50% by weight of the total weight of the uncapped hydrolysate, such as more than 60% by weight, for example more than 70% by weight, such as more than 80% by weight, for example more than 90% by weight, such as more than 95% by weight, for example more than 96% by weight, such as more than 97% by weight, for example more than 98% by weight, such as more than 99% by weight, for example more than 99.5% by weight, such as more than 99.7% by weight, for example more than 99.9% by weight, such as more than 99.99% by weight.

[0125] In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside 5'-monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside 3'-monophosphate.

[0126] In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside 5'-diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside 3'-diphosphate.

[0127] In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside 5'-triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of a nucleoside 3'-triphosphate.

[0128] In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine 5'-monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine 3'-monophosphate.

[0129] In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine 5'-diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine 3'-diphosphate.

[0130] In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine 5'-triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of guanosine 3'-triphosphate.

[0131] In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine 5'-monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine 3'-monophosphate.

[0132] In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine 5'-diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine 3'-diphosphate.

[0133] In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine 5'-triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of adenosine 3'-triphosphate.

[0134] In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine 5'-monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine 3'-monophosphate.

[0135] In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine 5'-diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine 3'-diphosphate.

[0136] In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine 5'-triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of uridine 3'-triphosphate.

[0137] In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine 5'-monophosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine 3'-monophosphate.

[0138] In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine 5'-diphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine 3'-diphosphate.

[0139] In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine adenosine triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine 5'-triphosphate. In one embodiment, the nucleotide comprises, consists essentially of, or consists of cytidine 3'-triphosphate.

[0140] In one embodiment, the uncapped hydrolysis product comprises guanosine 5'-triphosphate (GTP), and the method comprises determining the amount of GTP.

[0141] In one embodiment, the uncapped hydrolysis product comprises adenosine 5'-triphosphate (ATP), and the method comprises determining the amount of ATP.

[0142] Step (c)—Chromatographic Separation Step (c) of the method of the invention involves separating the hydrolysis products by chromatography. In general, the term "chromatography" refers to a technique for the separation of mixtures, typically where the mixture is dissolved in a fluid called the "mobile phase" or "eluent," which carries the mixture through a structure that holds another material called the "stationary phase."

[0143] Chromatography can be carried out according to a wide range of possible techniques, generally known to those skilled in the art. Any chromatographic method can be used, as long as it can be coupled to the mass spectrometry method used in step (d) below.

[0144] Chromatographic techniques can be classified by the physical state of the mobile phase. In one embodiment, the chromatographic method used in step (c) is liquid chromatography (i.e., the mobile phase is a liquid). In one embodiment, the chromatographic method used in step (c) is gas chromatography (i.e., the mobile phase is a gas).

[0145] If the chromatographic method used in step (c) is liquid chromatography, then a method in which the stationary phase is more polar than the mobile phase (e.g., toluene as the mobile phase and silica as the stationary phase) is called normal phase liquid chromatography (NPLC), and the reverse (e.g., water-methanol mixture as the mobile phase and C18 (octadecylsilyl) as the stationary phase) is called reversed phase liquid chromatography (RPLC).

[0146] When the chromatographic method used in step (c) is liquid chromatography, in one embodiment, the chromatography used in step (c) is high-performance liquid chromatography (HPLC). As known to those skilled in the art, HPLC is a technique in analytical chemistry used to separate, identify, and quantify components in a mixture. It relies on a pump to pass a pressurized liquid solvent containing the sample mixture through a column packed with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, resulting in different flow rates for different components, resulting in separation of the components as they exit the column. It is typically performed at a pressure of about 50-600 bar. It is typically performed in a column with a diameter of 1-10 mm, preferably 2-5 mm. It is typically performed in a column with a length of 10-500 mm, preferably 30-250 mm in diameter. It is typically performed using adsorbent particles with an average particle size of 1-100 μm, preferably 2-50 μm.

[0147] In one embodiment, the chromatography used in step (c) is ultra-high performance liquid chromatography. Typically, it is performed at a pressure of about 400 to 1200 bar. Typically, it is performed in a column having a diameter of 0.5 to 5 mm, preferably 1 to 4 mm. Typically, it is performed in a column having a length of 5 to 300 mm, preferably 10 to 250 mm. Typically, it is performed using adsorbent particles having an average particle size of 0.1 to 10 μm, preferably 0.5 to 3 μm.

[0148] Chromatography techniques can also be classified by the separation mechanism. In one embodiment, the chromatography used in step (c) is hydrophilic interaction chromatography. In one embodiment, the chromatography used in step (c) is ion exchange chromatography. In one embodiment, the chromatography used in step (c) is reversed phase chromatography. In one embodiment, the chromatography used in step (c) is size exclusion chromatography.

[0149] In one embodiment, the chromatographic technique used in step (c) is reverse-phase liquid chromatography (RPC). As known to those skilled in the art, the term "reverse-phase chromatography" refers to any liquid chromatographic procedure in which the mobile phase is significantly more polar than the stationary phase.

[0150] In one embodiment, the chromatographic technique used in step (c) is ion exchange chromatography. As known to those skilled in the art, the term "ion exchange chromatography" (or "ion chromatography") refers to a chromatographic technique that separates ions and polar molecules based on their affinity for an ion exchanger. Ion exchange chromatography separates molecules based on their respective charged groups. Ion exchange chromatography retains analyte molecules on a column based on Coulombic (ionic) interactions. The matrix of ion exchange chromatography consists of positively and negatively charged ions. Molecules undergo electrostatic interactions with opposite charges on the stationary phase matrix. The stationary phase consists of a stationary matrix containing charged ionizable functional groups or ligands. The stationary phase surface exhibits ionizable functional groups (RX) that interact with analyte ions of opposite charge. To achieve electroneutrality, these inert charges bind with exchangeable counterions in solution. The ionizable molecules to be purified compete with the exchangeable counterions for binding to the immobilized charges on the stationary phase. These ionizable molecules are retained or eluted based on their charge. First, molecules that do not bind or bind weakly to the stationary phase are washed away first. Elution of molecules that bind to the stationary phase requires a change in conditions. The concentration of exchangeable counterions that compete with the molecules for binding can be increased, or the pH can be changed. The change in pH affects the charge on certain molecules, thus altering binding. Molecules then begin to elute based on the change in their charge due to the adjustment. Furthermore, such adjustments can be used to release proteins of interest. Furthermore, the concentration of counterions can be gradually changed to separate ionized molecules. This type of elution is called gradient elution. Alternatively, step elution can be used, in which the concentration of counterions is changed in one step.

[0151] Ion exchange chromatography can be further subdivided into cation exchange chromatography and anion exchange chromatography. Positively charged molecules bind to cation exchange resins, and negatively charged molecules bind to anion exchange resins. The cationic species M + and anion species B - Ionic compounds consisting of cations and cations can be retained on the stationary phase. Cation exchange chromatography retains positively charged cations because the stationary phase exhibits negatively charged functional groups. Anion exchange chromatography retains anions using positively charged functional groups.

[0152] In one embodiment, the chromatographic technique used in step (c) is hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC). As known to those skilled in the art, the term "hydrophilic interaction chromatography" refers to a technique in which the mobile phase is hydrophobic and the stationary phase is hydrophilic, such that the order of elution is typically the opposite of that obtained with reversed-phase chromatography—see AJ Alpert, J. Chromatography A, 1990, 499, 177-196. As mentioned above, analyte determination using HILIC in combination with mass spectrometry using a triple quadrupole mass spectrometer is particularly advantageous for the quantification of nucleotides, as HILIC allows for the retention and separation of very less polar analytes such as nucleotides. The stationary phase may be unbonded silica, a silanol- or diol-bonded phase; an amino- or anion-bonded phase; an amide-bonded phase; a cation-bonded phase; or a zwitterion-bonded phase. Preferably, the stationary phase is an amide-bonded phase.

[0153] In one embodiment, the chromatographic technique used in step (c) is ion interaction chromatography (also known as ion pair chromatography). As known to those skilled in the art, this term refers to a reversed-phase technique in which a charged substance is mixed with ion pairing reagents (IPR) added to the mobile phase, and the analyte typically binds to its mutual ion in the IPR. This pairing affects the pair's interaction with the mobile and stationary phases of the column, thereby allowing for the separation of different ion pairs.

[0154] When the chromatography used in step (c) is liquid chromatography, the mobile phase (eluent) may be any suitable liquid known in the art. Suitable examples include water, C1-4 alcohols such as methanol, ethanol, and isopropanol, C1-4 halogenated alcohols such as hexafluoroisopropanol, aprotic solvents miscible with water (e.g., nitriles such as acetonitrile, and ethers, particularly cyclic ethers such as tetrahydrofuran and 1,4-dioxane), and any mixtures thereof. In one embodiment, the mobile phase used in liquid chromatography is a mixture of water and acetonitrile. In one embodiment, the mobile phase used in liquid chromatography is hexafluoroisopropanol.

[0155] In one embodiment, the mobile phase used in liquid chromatography comprises a buffer. As known to those skilled in the art, a buffer solution is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. The pH of a buffer solution changes little when a small amount of a strong acid or strong base is added to it. The buffer may be any suitable buffer known in the art. Suitable examples include citric acid / citrate buffer, acetic acid / acetate buffer, phosphate buffer, borate buffer, ammonia / ammonium salt buffer, carbonate / bicarbonate-based buffer, bicine (2-(bis(2-hydroxyethyl)amino)acetic acid), tris(tris(hydroxylmethyl)aminomethane, or 2-amino-2-(hydroxylmethyl)propane-1,3-diol), tricine (N-[tris(hydroxylmethyl)methyl]glycine), TAPSO (3-[N-trimethyl-2-hydroxyethyl]aminomethane, or 2-amino-2-(hydroxylmethyl)propane-1,3-diol), tricine (N-[tris(hydroxylmethyl)methyl]glycine), glycine-containing sorbitol (PEG-10 ... Examples of suitable buffers include ammonium carbonate (N,N'-bis(2-hydroxypropanesulfonic acid)), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TES (2-[[1,3-dihydroxy-2-(hydroxylmethyl)propan-2-yl]amino]ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), and MES (2-(N-morpholino)ethanesulfonic acid). In one embodiment, the buffer is ammonium carbonate.

[0156] When the chromatography used in step (c) is liquid chromatography, in one embodiment, step (c) is carried out at a temperature between room temperature and 90° C. In one embodiment, step (c) is carried out at a temperature between 30 and 70° C. In one embodiment, step (c) is carried out at a temperature of 55° C.

[0157] When the chromatography used in step (c) is liquid chromatography, in one embodiment, step (c) is carried out using a stationary phase which may be any finely divided adsorbent solid, typical examples being silica and alumina.

[0158] When the chromatography used in step (c) is liquid chromatography, in one embodiment, step (c) is carried out at a pH of 2-11, preferably 8-10.

[0159] When the chromatography used in step (c) is liquid chromatography, in one embodiment, step (c) is carried out at a flow rate of 0.1 to 2 ml / min, preferably 0.3 to 1.0 ml / min.

[0160] Step (d) - Mass analysis Step (d) of the method of the present invention involves determining the concentration of the hydrolysis products by mass spectrometry, thereby quantifying the RNA capping efficiency.

[0161] As known to those skilled in the art, a mass spectrometer typically consists of three components: an ion source, a mass analyzer, and a detector. The ionizer converts a portion of the sample into ions. As detailed below, a wide variety of ionization techniques exist, depending on the phase of the sample (solid, liquid, gas) and the efficiency of various ionization mechanisms for unknown species. Mass spectrometers also typically include an extraction system that removes ions from the sample, which are then directed through a mass analyzer to the detector. Differences in the mass-to-charge (m / z) of the fragments allow the mass analyzer to classify ions by their mass-to-charge ratio. Finally, the detector measures an index value, providing data for calculating the abundance of each ion present.

[0162] In a typical mass spectrometry method, the first step involves ionizing the sample. In one embodiment, ionization involves electron ionization (EI), which involves bombarding the sample with electrons. In another embodiment, ionization involves chemical ionization (CI), in which ions are generated through collisions between the analyte and ions of a reagent gas present in the ion source (examples of suitable reagent gases include methane, ammonia, and isobutane). In another embodiment, ionization involves atmospheric pressure chemical ionization (APCI). In another embodiment, ionization involves atmospheric pressure photon ionization (APPI).

[0163] If the ionization is electron ionization, then typically the molecule will have the same mass (M) as the parent molecule but a charge (M + or M - When the ionization is chemical ionization, mass ions are typically produced that have the mass of the parent molecule and the chemical species used to ionize the molecule, a well-known example being [M+H] + , [MH] - , [M+NH4] + , and [M+Na] + Such molecular ions are also referred to as "quasi-molecular ions" in this specification.

[0164] In another embodiment, ionization includes electrospray ionization (ESI), in which a liquid containing the analyte of interest is dispersed into a fine aerosol by electrospray. In another embodiment, ionization includes matrix-assisted laser desorption / ionization (MALDI), which typically involves a three-step process: (1) mixing the sample with a suitable matrix material and applying it to a surface, typically a metal plate; (2) irradiating the sample, typically with a pulsed laser, thereby inducing ablation and desorption of the sample and matrix material; and (3) accelerating the ions in the jet of hot gas produced by ablation into a mass spectrometer, where they are protonated or deprotonated to ionize and analyze the analyte molecules. These ionization techniques are well known to those skilled in the art. Ionization, particularly electron ionization, can break down some of the sample's molecules into charged fragments.

[0165] Following ionization, the ions produced in the first step are separated according to their mass-to-charge (m / z) ratio in a mass analyzer. This is typically done by one or more of the following mass-to-charge separation techniques: quadrupole fields used in quadrupole mass analyzers, ion trap quadrupole fields used in ion trap mass analyzers, longitudinal ion transit time used in time-of-flight mass analyzers, and electric and / or magnetic field deflection conventionally used in electric and magnetic sector mass analyzers. This last technique involves accelerating ions and exposing them to an electric or magnetic field, which deflects the ions. Ions of the same mass-to-charge ratio experience the same degree of deflection.

[0166] After separation, the ions are detected. Typically, a detector records either the charge induced or the current generated as the ions pass through or strike a surface. In scanning instruments, the signal generated by the detector during the scan, relative to the position of the instrument during the scan, produces a mass spectrum, which is a record of ions as a function of m / z.

[0167] According to the present invention, mass spectrometry step (d) is used in parallel with the chromatographic separation technique in step (c). In one embodiment, the chromatographic technique is gas chromatography, and this combined technique is known as gas chromatography-mass spectrometry (GC / MS, GCMS, or GC-MS). As known to those skilled in the art, this technique uses a gas chromatograph to separate different compounds. This separated compound stream is fed to a mass spectrometer for ionization, mass analysis, and detection, as described above.

[0168] In one embodiment, the chromatographic technique is liquid chromatography, and this combined technique is known as liquid chromatography-mass spectrometry (LC / MS, LCMS, or LC-MS). As described with respect to step (c) above and generally known to those skilled in the art, this technique separates compounds chromatographically using a liquid mobile phase. Typically, the liquid phase is a mixture of water and an organic solvent. The separated compound stream is then fed to a mass spectrometer for ionization, mass analysis, and detection, as described above.

[0169] In one embodiment, the mass spectrometry is direct sampling mass spectrometry. As known to those skilled in the art, this technique involves introducing a sampling probe containing the sample to be analyzed directly into the ionization chamber of a mass spectrometer. The sample may be solid, liquid, or gaseous, and is preferably solid.

[0170] In one embodiment, the mass spectrometry is infusion sampling mass spectrometry, a technique known to those skilled in the art that involves introducing the sample to be analyzed into a mass spectrometer by spraying a liquid containing the sample into the mass spectrometer.

[0171] The mass spectrometry used in step (d) is tandem mass spectrometry. MS / MS, MS 2 or MS n Tandem mass spectrometry, also known as tandem mass spectrometry (where n is at least 2, preferably 2-10, more preferably 2-5, even more preferably 2 or 3, and most preferably 2), involves multiple steps of mass spectrometry selection, with some form of fragmentation occurring during these steps. Tandem mass spectrometry is particularly preferred as a mass spectrometry method of the present invention because it can determine analytes with high selectivity, flexibility, and sensitivity, especially, but not limited to, when coupled with liquid chromatography.

[0172] Typically, tandem mass spectrometry involves the following steps: (i) Ionization of the sample to produce ions. Ionization is typically performed by any of the ionization techniques mentioned above, particularly electron impact (EI), electrospray ionization (ESI), secondary electrospray ionization (SESI), desorption electrospray ionization (DESI), easy ambient sonic spray ionization (EASI), extractive electrospray ionization (EESI), neutral desorption electrospray ionization (ND-ESI), jet desorption electrospray ionization (JEDI), liquid extraction surface analysis (LESA), surface activated chemical ionization (SACI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photon ionization (APPI), and direct analysis in real time (DART). This can be done using ELISA (Analysis in Real Time) or matrix-assisted laser desorption / ionization (MALDI). (ii) separating the ions according to their mass-to-charge ratio to produce one or more precursor ions, the separation being generally as described above. (iii) fragmenting one or more separated precursor ions to obtain product ions. A wide variety of methods can be used to fragment ions, resulting in different types of fragmentation and, consequently, different information about the structure and composition of the molecule. In one embodiment, the fragmentation method includes collision-induced dissociation. Typically, this method involves collision of ions with neutral atoms or molecules in the gas phase, followed by dissociation of the ions. In one embodiment, the fragmentation method includes electron impact capture and / or transfer methods. Typically, these methods use energy released when electrons are transferred to or captured by multiply charged ions to induce fragmentation. Examples of electron capture and / or transfer methods used to induce fragmentation include electron capture dissociation, electron transfer dissociation, negative electron transfer dissociation, electron detachment dissociation, and charge transfer dissociation. In one embodiment, the fragmentation method includes photodissociation. Typically, in this method, the energy required for dissociation can be applied by photon absorption. Examples of photodissociation methods include infrared multiphoton dissociation, blackbody infrared radiation dissociation, or surface-induced dissociation. In another embodiment, fragmentation techniques include in-source fragmentation (i.e., fragmentation within the ionization chamber), where the ionization process is sufficiently violent that the resulting ions retain sufficient internal energy to fragment within the mass spectrometer (e.g., by electron impact, chemical ionization, or "accelerated ion dissociation"). All of these techniques are well known to those skilled in the art. (iv) separating the product ions resulting from the fragmentation process according to their mass-to-charge ratio, the separation typically being carried out as generally described above. (v) detecting the separated ions, which is typically performed as generally described above.

[0173] In one embodiment, the tandem mass spectrometry is ion trap mass spectrometry. As known to those skilled in the art, a quadrupole ion trap is a type of ion trap that uses a dynamic electric field to trap charged particles.

[0174] The tandem mass spectrometry used in step (d) is triple quadrupole mass spectrometry (TQMS). As known to those skilled in the art, a triple quadrupole mass spectrometer is a tandem mass spectrometer consisting of two quadrupole mass analyzers in series, with a (non-mass resolving) radio-frequency-only quadrupole between them that serves as a cell for collision-induced dissociation. TQMS allows for detection with higher sensitivity than other tandem mass spectrometry methods.

[0175] In one embodiment, the triple quadrupole mass analyzer used in step (d) employs multiple reaction monitoring (β) techniques. MRM allows for detection with higher sensitivity and selectivity than other tandem mass spectrometry techniques. As known to those skilled in the art, triple quadrupole mass analyzers can be used in different scan modes. Full scan mode is a single-stage scan type that provides a complete mass spectrum of each analyte. The mass analyzer scans from the low mass end to the high mass end of a user-defined mass range. Product ion scans involve selecting ions of a single mass-to-charge ratio (parent ions). These ions then collide with a collision gas in the collision cell. Collisions of the parent ions fragment and produce product ions. Precursor ion scans precursor ions in Q1 and selects specific fragment ions in Q3. All collision-induced dissociation was performed in Q2. Neutral loss scans scan all ions in Q1 and selects ions with neutral loss in Q3. Selected ion monitoring (SIM) is a single-stage technique in which a desired ion or set of ions is monitored. Selected reaction monitoring (SRM) or multiple reaction monitoring (MRM) is a two-stage (MS / MS) technique in which parent and product ion pairs are monitored. In MRM mode, analytes can be measured in complex mixtures because the sample matrix (other sample components) is largely removed through this two-stage filtration mechanism.

[0176] In one embodiment, the tandem mass spectrometry is quadrupole time-of-flight mass spectrometry. As known to those skilled in the art, a quadrupole time-of-flight mass spectrometer is a triple quadrupole mass spectrometer, as described above, with the last quadrupole replaced by a time-of-flight device. As known to those skilled in the art, time-of-flight mass spectrometry (TOFMS) is a mass spectrometry method in which the mass-to-charge ratio (m / z) of an ion is determined by time measurement. This technique involves accelerating ions through an electric field of known strength. This acceleration results in ions with the same kinetic energy as other ions with the same charge. The velocity of the ion depends on the mass-to-charge ratio. The time it takes for the particle to reach a detector at a known distance is then measured. This time depends on the mass-to-charge ratio of the particle, with heavier particles reaching lower velocities. From this time and known experimental parameters, the user can determine the mass-to-charge ratio of the ion.

[0177] In one embodiment, the tandem mass spectrometry is quadrupole ion trap mass spectrometry. In one embodiment, the tandem mass spectrometry is quadrupole time-of-flight mass spectrometry. In one embodiment, the tandem mass spectrometry is ion mobility-quadrupole ion trap-time-of-flight mass spectrometry. In one embodiment, the tandem mass spectrometry is quadrupole-Orbitrap mass spectrometry. In one embodiment, the tandem mass spectrometry is quadrupole ion trap mass spectrometry. In one embodiment, the tandem mass spectrometry is ion mobility spectrometer-quadrupole ion trap mass spectrometry. In one embodiment, the tandem mass spectrometry is quadrupole-Orbitrap mass spectrometry. In one embodiment, the tandem mass spectrometry is triple quadrupole-Orbitrap mass spectrometry. In one embodiment, the tandem mass spectrometry is quadrupole ion trap-Orbitrap mass spectrometry. In one embodiment, the tandem mass spectrometry is time-of-flight ion trap-Fourier transform mass spectrometry. Details of these techniques are known to those skilled in the art.

[0178] In one embodiment, the tandem mass spectrometry is secondary electrospray ionization (SESI) mass spectrometry. SESI is an electrospray ionization technique performed at atmospheric pressure. The term "SESI" generally encompasses a range of modified ESI techniques in which the hot gases of electrospray ionization ionize material in the immediate region of the hot gases of electrospray. Typically, SESI techniques are performed in an ionization chamber located before the skimmer inlet of an atmospheric pressure ionization mass spectrometer.

[0179] In one embodiment, the mass analysis is secondary ion mass spectrometry (SIMS). As known to those skilled in the art, SIMS is an MS technique typically performed on solid targets and thin films. The ionization step typically involves sputtering the solid target surface with a primary ion beam, typically generated by a primary ion gun. A primary ion column can also be used to accelerate and focus the primary ion beam onto the target. When the ion beam strikes the target, it ejects secondary ions from the target's surface. These secondary ions are then subjected to mass analysis using a mass analyzer and detector, generally as described above. The mass analyzer may be an electrostatic analyzer, a quadrupole mass analyzer, or a time-of-flight mass analyzer. The detector may be a Faraday cup, an electron multiplier, or a microchannel plate detector.

[0180] In one embodiment, mass analysis is performed in full scan monitoring mode, which, as known to those skilled in the art, involves scanning a range of masses from the lowest to the highest expected ions (as compared to selected ion monitoring mode, in which data is collected only for selected masses of interest).

[0181] In a preferred embodiment, the method used in steps (c) and (d) is liquid chromatography coupled to tandem mass spectrometry. Tandem mass spectrometry coupled to liquid chromatography can be used to determine analytes with high selectivity, flexibility and sensitivity.

[0182] In a particularly preferred embodiment, the chromatographic separation method used in step (c) is hydrophilic interaction liquid chromatography and the mass spectrometry method used in step (d) is triple quadrupole mass spectrometry. As noted above, the combination of HILIC and triple quadrupole mass spectrometry (particularly with a mass spectrometer operating in MRM mode) allows for highly selective, sensitive, and accurate determination of nucleotides in complex matrices.

[0183] Capping efficiency calculation Based on the concentration of the analyte measured using step (d), the RNA capping efficiency can be calculated.

[0184] In general, RNA capping efficiency can be measured according to the formula: [concentration of dinucleotide] / [concentration of dinucleotide+concentration of nucleotide].

[0185] If the uncapped hydrolysis product is GTP, the RNA capping efficiency is measured according to the formula: [concentration of dinucleotide] / [concentration of dinucleotide + concentration of GTP].

[0186] If the uncapped hydrolysis products contain both GTP and ATP, the RNA capping efficiency is measured according to the formula: [concentration of dinucleotide] / [concentration of dinucleotide + concentration of GTP + concentration of ATP]. [Example] [Example]

[0187] enzymatic hydrolysis In this experiment, RNA was enzymatically hydrolyzed to obtain single nucleotides (mononucleotide monophosphates and 5'-terminal mononucleotide triphosphates or 5'-dinucleotides of the 5'-cap), which were used for further analysis using LC-MS / MS (see Example 2).

[0188] Enzymatic hydrolysis of RNA RNA was completely hydrolyzed by the action of one hydrolase. Nuclease P1 (NP1) from Penicillium citrinum was obtained as a dry powder from Sigma-Aldrich. NP1 was dissolved in 1 ml of water to a concentration of approximately 1 mg / ml and stored at -20°C.

[0189] RNA was filtered using an Amicon Ultra 0.5 ml MWCO 30 kDa filter. Depending on the length of the RNA, 200–400 μg of RNA was hydrolyzed by adding 15 μl of NH4OAc buffer (100 mM, pH 4.5) and 15 μl of NP1 solution and incubating on a thermomixer at 37°C and 450 rpm for 3 hours. 1 μl of the resulting solution was analyzed by LC-MS / MS (see Example 2). [Example]

[0190] LC-MS / MS analysis of hydrolyzed RNA In this experiment, nucleotides obtained by enzymatic RNA hydrolysis (see Example 1) were analyzed by LC-MS / MS to determine capping efficiency. The method of the present invention can be used as a quality control for in vitro transcribed RNA.

[0191] 1. HPLC Analysis of Nucleotides After enzymatic hydrolysis (see Example 1), RNA samples were separated using a commercially available HPLC setup. For chromatographic separation of nucleotides, an amide column (Waters XBridge Premier BEH Amide VanGuard FIT Column, pore size: 130 Å, particle size: 2.5 μm, dimensions (h × ID) 2.1 mm × 50 mm, Waters) was used, with a linear HILIC gradient from 75% Buffer B (acetonitrile + 0.1% water) to 55% Buffer A (100 mM ammonium carbonate, pH 8.9) at a column temperature of 55°C. Commercially available or commissioned standards were obtained for calibration. Nucleotides were detected by mass spectrometry. Exemplary chromatograms for hydrolyzed β-S-ARCA-capped RNA, hydrolyzed CleanCap® 413-capped RNA, and the respective standards are shown in Figures 1, 2, and 3.

[0192] 2. Mass Analysis of Nucleotides Chromatographic peak detection was performed by a commercial (Shimadzu 8050) triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. Table 1 shows the mass transitions of the nucleotides and caps used for quantification. [Table 1] JPEG2025539043000010.jpg75170

[0193] 3. Quantification of Nucleotide Concentration Nucleotide concentrations were calculated using isotope-labeled internal standards. Mononucleotide standards were commercially available, and dinucleotide standards were synthesized by a contract manufacturer (Hongene Biotech).

[0194] The area ratio between the analyte area and the internal standard area is used to calculate a calibration curve by applying an appropriately weighted linear or quadratic regression curve to the areas of the calibration samples by least-squares analysis using the quantitation software of the LC-MS / MS / MS system.

[0195] 4. Calculation of Capping Efficiency The amount of dinucleotides from the capping structure ((Cap) concentration) in the RNA hydrolysates, as well as the amount of GTP ((GTP) concentration) and ATP ((ATP) concentration), representing uncapped RNA, were determined. The percentage of capped RNA (% capping efficiency) can be determined in each sample using one of the following formulas depending on the 5' cap incorporated:

number

number

[0196] The 5' cap of RNA is an essential structure for protein-coding RNA, since uncapped RNA cannot be translated into protein. Therefore, determining the capping efficiency of in vitro transcribed RNA is an important quality control of in vitro transcribed RNA.

[0197] Figure 4 shows the capping efficiency of various CleanCap® 413 amounts during in vitro transcription, and Figure 5 shows the capping efficiency of RNA with two different cap structures, i.e., β-S-ARCA and CleanCap® 413. Figures 4 and 5 demonstrate that the methods of the present invention are particularly suitable for measuring RNA quality attributes, such as RNA capping efficiency for various amounts of cap during in vitro transcription (Figure 4) and capping structures that exhibited various levels of capping efficiency (Figure 5). Capping is an important characteristic of RNA, as uncapped RNA cannot be translated into protein.

[0198] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in chemistry, biochemistry, molecular biology, biotechnology or related fields are intended to be within the scope of the following claims.

Claims

1. A method for quantifying RNA capping efficiency, comprising the following steps (a) to (d): (a) providing a sample of capped RNA; (b) contacting the capped RNA with a protein nuclease, thereby hydrolyzing the RNA to produce hydrolysis products that include capped products that include dinucleotides and uncapped products that include nucleotides, wherein step (b) is performed in the absence of a nucleic acid having a base sequence complementary to that of the capped RNA; (c) separating the hydrolysis products by chromatography; and (d) determining the concentration of said hydrolysis products by triple quadrupole mass spectrometry, thereby quantifying the RNA capping efficiency;

2. 2. The method of claim 1, wherein in step (b), the nuclease is the only enzyme that hydrolyzes RNA.

3. 3. The method of claim 1 or 2, wherein the nuclease used in step (b) is nuclease P1 or nuclease S1.

4. 4. The method of claim 3, wherein the nuclease used in step (b) is nuclease P1.

5. The method according to any one of claims 1 to 4, wherein the chromatography used in step (c) is liquid chromatography.

6. 6. The method of claim 5, wherein the chromatography used in step (c) is selected from the group consisting of high performance liquid chromatography and ultra high performance liquid chromatography.

7. 6. The method of claim 5, wherein the chromatography used in step (c) is selected from the group consisting of hydrophilic interaction chromatography and ion exchange chromatography.

8. 6. The method of claim 5, wherein the chromatography used in step (c) is hydrophilic interaction liquid chromatography.

9. 2. The method of claim 1, wherein the triple quadrupole mass spectrometry used in step (d) uses multiple reaction monitoring (MRM) techniques.

10. The method according to any one of claims 1 to 9, wherein the method used in steps (c) and (d) is liquid chromatography coupled to triple quadrupole mass spectrometry.

11. 11. The method of claim 10, wherein the method used in steps (c) and (d) is hydrophilic interaction liquid chromatography coupled to triple quadrupole mass spectrometry.

12. The method according to any one of claims 1 to 11, wherein the RNA is mRNA.

13. The method of any one of claims 1 to 12, wherein the capped hydrolysis product is a dinucleotide.

14. 14. The method of claim 13, wherein the capped hydrolysis product consists of a dinucleotide.

15. 15. The method of claim 1, 13 or 14, wherein the dinucleotide comprises two nucleosides linked by a 5',5'-triphosphate bridge.

16. 10. The method of claim 1, wherein the capped hydrolysis product has the structure of formula (II): 【Chemistry 1】 (In the formula, PM is a monophosphate or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units, wherein an oxo (=O) group in any of the phosphate units is optionally replaced with a thio (=S) group; R is an end-capping moiety; B is C １－４ a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R' is OH, O(C １－４ alkyl), and halogen

17. 10. The method of claim 1, wherein the capped hydrolysis product has the structure of formula (IIa): 【Chemistry 2】 (In the formula, PM is a monophosphate or polyphosphate moiety containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate units, wherein an oxo (=O) group in any of the phosphate units is optionally replaced with a thio (=S) group; Nuc is a nucleoside or nucleoside analog; B is C １－４ a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R' is OH, O(C １－４ alkyl), and halogen

18. 17. The method of claim 16, wherein the capped hydrolysis product has the structure of formula (IIb) or a salt thereof: 【Transformation 3】 (In the formula, B is C １－４ a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; R １ is OH, O(C １－４ alkyl), and halogen; R ２ is H, OH, O(C １－４ alkyl), and halogen; R ３ is OH, O(C １－４ alkyl), and halogen; R ４ is H, OH, O(C １－４ alkyl), halogen, or C １－４ a nucleobase optionally alkylated on the nitrogen atom by an alkyl group; n is 1, 2 or 3; X １ , each X ２ , and X ３ are each independently O or S.

19. 20. The method of claim 18, wherein the capped hydrolysis product has the structure of formula (IIc) or a salt thereof: 【Chemistry 4】 (In the formula, B and B' are each independently C １－４ are nucleobases each optionally alkylated on the nitrogen atom by an alkyl group; R １ is OH, O(C １－４ alkyl), and halogen; R ２ is H, OH, O(C １－４ alkyl), and halogen; R ３ is OH, O(C １－４ alkyl), and halogen; n is 1, 2 or 3; Each X and X' is independently O or S.

20. 20. The method of any one of claims 1 to 19, wherein the capped hydrolysis product is selected from the group consisting of m7G(5')ppp(5')N, m7G(5')ppp(5')-(cap0), m7G(5')ppp(5')Nm-(cap1), m7G(5')ppp(5')G, ARCA, and β-S-ARCA, wherein G is guanosine, p is a phosphate residue, N is any nucleoside, and Nm is a nucleoside having a 2'-methyl group.

21. 21. The method of any preceding claim, wherein the dinucleotides constitute more than 50% by weight of the total weight of the capped hydrolysate, such as more than 60% by weight, for example more than 70% by weight, such as more than 80% by weight, for example more than 90% by weight, such as more than 95% by weight, for example more than 96% by weight, such as more than 97% by weight, for example more than 98% by weight, such as more than 99% by weight, for example more than 99.5% by weight, such as more than 99.7% by weight, for example more than 99.9% by weight, such as more than 99.99% by weight.

22. 22. The method of any preceding claim, wherein the nucleotides constitute more than 50% by weight of the total weight of the uncapped hydrolysate, such as more than 60% by weight, for example more than 70% by weight, such as more than 80% by weight, for example more than 90% by weight, such as more than 95% by weight, for example more than 96% by weight, such as more than 97% by weight, for example more than 98% by weight, such as more than 99% by weight, for example more than 99.5% by weight, such as more than 99.7% by weight, for example more than 99.9% by weight, such as more than 99.99% by weight.

23. 2. The method of claim 1, wherein the uncapped hydrolysis product comprises guanosine triphosphate (GTP), the method comprising determining the amount of GTP.

24. 24. The method of claim 23, wherein the RNA capping efficiency is measured according to the formula: [dinucleotide concentration] / [dinucleotide concentration + GTP concentration].

25. 2. The method of claim 1, wherein the uncapped hydrolysis products comprise adenosine triphosphate (ATP), the method comprising determining the amount of ATP.

26. 26. The method of claim 25, wherein the RNA capping efficiency is measured according to the formula: [dinucleotide concentration] / [dinucleotide concentration + GTP concentration + ATP concentration].

27. The method of any one of claims 1 to 26, wherein the amount of hydrolysis product is determined using an external standard.

28. 28. The method of any one of claims 1 to 27, carried out in the absence of snake venom phosphodiesterase.

29. The method of any one of claims 1 to 28, which is carried out in the absence of phosphatase.

30. 30. The method of any one of claims 1 to 29, wherein the capped RNA is purified prior to step (b).

31. 31. The method of claim 30, wherein the capped RNA is purified by filtration prior to step (b).

32. 32. The method of claim 30 or 31, wherein the capped RNA is purified by high performance liquid chromatography prior to step (b).

33. The method of any one of claims 1 to 32, wherein step (b) is carried out at a nuclease concentration of 10 to 70 µmol / L.

34. 34. The method of any one of claims 1 to 33, wherein step (b) is carried out at a temperature between room temperature and 90°C.

35. 35. The method of any preceding claim, wherein step (b) is carried out for a period of from 30 minutes to 48 hours.

36. 36. The method of any one of claims 1 to 35, wherein step (b) is carried out at a pH of 4 to 6.

37. The method according to any one of claims 4 to 36, wherein in step (c), the mobile phase used in the liquid chromatography is a mixture of water and acetonitrile.

38. The method according to any one of claims 4 to 36, wherein the mobile phase used in the liquid chromatography is hexafluoroisopropanol.

39. The method of any one of claims 4 to 38, wherein the mobile phase used in the liquid chromatography comprises a buffer.

40. 40. The method of claim 39, wherein the buffer is ammonium carbonate.

41. 40. The method of any one of claims 4 to 39, wherein step (c) is carried out at a pH of 2 to 11.

42. 42. The method of claim 41, wherein step (c) is carried out at a pH of 8 to 10.

43. 40. The method of any one of claims 4 to 39, wherein step (c) is carried out at a flow rate of 0.1 to 2 ml / min.

44. 44. The method of claim 43, wherein step (c) is carried out at a flow rate of 0.3 to 1.0 ml / min.

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