Methods for quantitative monitoring of mRNA capping efficiency

Abstract

This invention relates to a method for quantifying mRNA capping efficiency, the method comprising mixing a sample, an enzyme mixture, and an isotope standard solution in a buffer solution to produce an incubation mixture, the enzyme mixture comprising a nonspecific single-stranded nuclease and an acid phosphatase, and the isotope standard comprising isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside; incubating the mixture; and analyzing the mixture using liquid chromatography-mass spectrometry to determine at least one of capping efficiency and 2-O-methyltransferase efficiency.

Description

Technical Field

[0001] This disclosure generally relates to the field of mass spectrometry analysis, including methods for quantitatively monitoring mRNA capping efficiency.

[0002] introduction

[0003] In eukaryotic systems, non-mitochondrial messenger RNA (mRNA) is capped at the 5' end with inverse 7-methylguanosine (m7G). Figure 1A ), which binds to the transcript via a 5' to 5' triphosphate linker (m7G(5')ppp(5')N, referred to as Cap(0)) ( Figure 2A Once added to the transcript, the first nucleotide in chain (N) is subsequently methylated at the 2' hydroxyl position to produce the Cap(1) structure m7G(5')ppp(5')Nm( Figure 2B ).For example, Figure 1B 2'-O-methyladenosine is shown. Alternatively, Nm can be 2'-O-methylguanosine (Gm), 2'-O-methylcytosine (Cm), or 2'-O-methyluridine (Um). Capping and polyadenylation help the transcript resist degradation by innate exonucleases in the cell. In addition to blocking 5' to 3' exonucleases, the 5' cap also promotes the export from the nucleus to the cytoplasm and assists in translation initiation by acting as a determinant of the ribosome docking complex.

[0004] Further alterations to the transcript, known as splicing events, also depend on the cap structure. A portion of the transcript, called an intron or gene region, is removed, and the cleaved portion rejoins, leaving the coding region (exon) to be expressed. The 5' and 3' regions flanking the coding region are untranslated regions, segments of RNA that are not translated into protein but influence translation. The mature mRNA transcript then consists of five parts: the 5' cap, the 5' UTR, the coding region, the 3' UTR, and the final polyadenylated tail (see [link to mRNA transcript]). Figure 3 ).

[0005] Due to recent events, particularly the COVID pandemic, mRNA has been widely used as a useful therapy, such as various mRNA-based COVID vaccines. Generating non-natural transcripts via in vitro transcription may require the addition of modified cap structures after purification to achieve the desired therapeutic function. Modified 5' caps may include anti-reverse cap analogs (ARCA) (US Patent No. 7,074,596), TriLink Biotechnology's CleanCap analog, or a vaccinia capping enzyme followed by a methyltransferase reaction. Transcript production does require analytical checks to determine reaction outcomes and yields; however, direct measurements (such as using mass spectrometry) are technically challenging due to the size of the transcripts. Therefore, improved techniques for measuring capping and methylation efficiency are needed. Summary of the Invention

[0006] In a first aspect, a kit for quantifying mRNA capping efficiency may comprise: an enzyme mixture containing nuclease P1 and sweet potato acid phosphatase; and a standard mixture containing isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside. In various embodiments, the 2'-O-methylated nucleoside may be Am, Gm, Um, or Cm.

[0007] In various embodiments of the first aspect, the kit may further comprise a buffer solution. In a particular embodiment, the buffer solution may comprise ZnCl2, such as ZnCl2 with a concentration between about 0.01 mmol and about 90 mmol. In a particular embodiment, the buffer solution has a divalent cation concentration of less than 100 mmol. In a particular embodiment, the buffer solution may have a pH between about 2 and about 7, typically between about 4 and about 6.

[0008] In various embodiments of the first aspect, the enzyme mixture may also include RNase T1.

[0009] In various embodiments of the first aspect, the enzyme mixture may be a lyophilized powder.

[0010] In various embodiments of the first aspect, the standard mixture may be a lyophilized powder.

[0011] In various embodiments of the first aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside include deuterium-labeled m7G(D) and deuterium-labeled 2'-O-methylated nucleoside.

[0012] In various embodiments of the first aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside are N 15 Marked or C 13 Marked.

[0013] In various embodiments of the first aspect, the molar ratio of the isotopically labeled m7G to the isotopically labeled 2'-O-methylated nucleoside in the standard mixture may be between about 2:1 and about 1:2. In a particular embodiment, the standard mixture may comprise equimolar amounts of isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside.

[0014] In a second aspect, a kit for quantifying mRNA capping efficiency may comprise: an enzyme mixture containing a nonspecific single-stranded nuclease and an acid phosphatase; a standard mixture containing isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleosides; and a buffer solution.

[0015] In various embodiments of the second aspect, the nonspecific single-stranded nuclease may include nuclease P1.

[0016] In various implementations of the second aspect, the acid phosphatase cannot be inhibited by adenosine monophosphate.

[0017] In various embodiments of the second aspect, the acid phosphatase may include sweet potato acid phosphatase.

[0018] In various embodiments of the second aspect, the enzyme mixture may include a site-specific single-stranded RNA endonuclease.

[0019] In various embodiments of the second aspect, the site-specific single-stranded RNA endonuclease can cleave the RNA 3' of guanosine.

[0020] In various embodiments of the second aspect, the site-specific single-stranded RNA endonuclease may include ribonuclease T1.

[0021] In various embodiments of the second aspect, the enzyme mixture may be a lyophilized powder.

[0022] In various embodiments of the second aspect, the standard mixture may be a lyophilized powder.

[0023] In various embodiments of the first aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside include deuterium-labeled m7G(D) and deuterium-labeled 2'-O-methylated nucleoside.

[0024] In various embodiments of the first aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside are N 15 Marked or C 13 Marked.

[0025] In various embodiments of the second aspect, the molar ratio of the isotopically labeled m7G to the isotopically labeled 2'-O-methylated nucleoside in the standard mixture can be between about 2:1 and about 1:2. In a particular embodiment, the standard mixture may comprise equimolar amounts of isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside.

[0026] In various embodiments of the second aspect, the buffer solution may include ZnCl2 with a concentration ranging from about 0.001 mM to about 90 mM, such as between about 0.05 mM and about 0.2 mM. In a particular embodiment, the buffer solution may include less than 100 mM of divalent cations.

[0027] In various embodiments of the second aspect, the buffer solution has a pH between about 2 and about 7, such as between about 4 and about 6.

[0028] In a third aspect, a method for quantifying mRNA capping efficiency may include combining a sample, an enzyme mixture, and an isotope standard solution in a buffer solution to produce an incubation mixture, the enzyme mixture comprising a nonspecific single-stranded nuclease and an acid phosphatase, and the isotope standard comprising an isotopically labeled m7G and an isotopically labeled 2'-O-methylated nucleoside; incubating the mixture; and analyzing the mixture using liquid chromatography-mass spectrometry to determine at least one of capping efficiency and 2'-O-methyltransferase efficiency.

[0029] In various embodiments of the third aspect, the mixture may be incubated at a temperature between about 30°C and 70°C, such as between about 35°C and 45°C.

[0030] In various implementations of the third aspect, the nonspecific single-stranded nuclease may include nuclease P1.

[0031] In various implementation schemes of the third aspect, acid phosphatase cannot be inhibited by adenosine monophosphate.

[0032] In various embodiments of the third aspect, the acid phosphatase may include sweet potato acid phosphatase.

[0033] In various embodiments of the third aspect, the enzyme mixture may further include a site-specific single-stranded RNA endonuclease. In a particular embodiment, the site-specific single-stranded RNA endonuclease is capable of cleaving the RNA 3' of guanosine. In a particular embodiment, the site-specific single-stranded RNA endonuclease may include ribonuclease T1. In another embodiment, the mixture may be incubated at a temperature between about 30°C and 50°C.

[0034] In various embodiments of the third aspect, incubating the mixture may include a site-specific single-stranded RNA endonuclease of about 0.5 units to about 10.0 units, such as between about 0.5 units and about 2.0 units.

[0035] In various embodiments of the third aspect, the method may further include dissolving the lyophilized enzyme mixture in a sample buffer to obtain the enzyme mixture.

[0036] In various embodiments of the third aspect, the method may further include dissolving the lyophilized mixture of standards with a sample buffer to obtain the mixture of standards.

[0037] In various embodiments of the third aspect, the incubation mixture may include an acid phosphatase between about 0.5 units and about 10.0 units, such as between about 0.5 units and about 2.0 units.

[0038] In various embodiments of the third aspect, the incubation mixture may include a nonspecific single-stranded nuclease ranging from about 1 unit to 100 units, such as between about 5 units and about 20 units.

[0039] In various embodiments of the third aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside include deuterium-labeled m7G(D) and deuterium-labeled 2'-O-methylated nucleoside.

[0040] In various embodiments of the third aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside are N 15 Marked or C 13 Marked.

[0041] In various embodiments of the third aspect, the molar ratio of the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside in the standard mixture is between about 2:1 and about 1:2, such as equimolar amounts of isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside.

[0042] In various embodiments of the third aspect, the incubation mixture may include an amount of isotopically labeled m7G between about 1.0 nmol and 20 nmol. In a particular embodiment, the incubation mixture may include an amount of isotopically labeled 2'-O-methylated nucleoside between about 1.0 nmol and about 20 nmol.

[0043] In various embodiments of the third aspect, the buffer solution may comprise ZnCl2 at a concentration ranging from about 0.001 mM to about 90 mM. In a particular embodiment, the buffer solution may comprise ZnCl2 at a concentration ranging from about 0.05 mM to about 0.2 mM.

[0044] In various embodiments of the third aspect, the incubation mixture may include divalent cations of less than 100 mM.

[0045] In various embodiments of the third aspect, the buffer solution may have a pH between about 2 and about 7, such as between about 4 and about 6.

[0046] In various embodiments of the third aspect, analysis of the mixture using liquid chromatography-mass spectrometry further includes separating the mixture using a chromatographic column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating peaks of m7G and isotopically labeled m7G, peaks of 2'-O-methylated nucleosides and isotopically labeled 2'-O-methylated nucleosides, or both; calculating the m7G:isotopically labeled m7G peak ratio, the 2'-O-methylated nucleoside:isotopically labeled 2'-O-methylated nucleoside peak ratio, or both; and determining capping efficiency based on the m7G:isotopically labeled m7G peak ratio, determining 2-O-methyltransferase efficiency based on the 2'-O-methylated nucleoside:isotopically labeled 2'-O-methylated nucleoside peak ratio, or both. Attached Figure Description

[0047] To more fully understand the principles and advantages disclosed herein, please now refer to the following description in conjunction with the accompanying drawings, wherein:

[0048] Figure 1A and Figure 1B These are diagrams showing the chemical structures of 7-methylguanosine and 2'-O-methyladenosine, respectively.

[0049] Figure 2A and Figure 2B These are diagrams showing the chemical structures of Cap(0) and Cap(1), respectively.

[0050] Figure 3 This is a diagram showing the structure of mRNA.

[0051] Figure 4A and Figure 4B These are diagrams showing the chemical structures of deuterated 7-methylguanosine and deuterated 2'-O-methyladenosine according to various embodiments.

[0052] Figure 5 This is a flowchart of a method for determining capping efficiency and 2'-O-methyltransferase efficiency based on various implementation schemes.

[0053] Figure 6 This is a block diagram of an exemplary chromatography system according to various implementation schemes.

[0054] Figure 7 This is a block diagram of an example mass spectrometry analysis system based on various implementation schemes.

[0055] It should be understood that the accompanying drawings are not necessarily drawn to scale, nor are the objects depicted in them. The drawings are intended to clearly illustrate various embodiments of the devices, systems, and methods disclosed herein. Where possible, the same reference numerals will be used throughout the drawings to refer to the same or similar parts. Furthermore, it should be understood that the drawings are not intended to limit the scope of this teaching in any way. Detailed Implementation

[0056] This article describes the implementation scheme of qualitative and quantitative methods for enhancing ion mobility.

[0057] The chapter headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter in any way.

[0058] In the detailed descriptions of the various embodiments, numerous specific details are set forth for illustrative purposes to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will appreciate that these various embodiments can be practiced with or without these specific details. In other instances, structures and apparatus are shown in block diagram form. Furthermore, those skilled in the art will readily recognize that the specific order in which the methods are presented and performed is exemplary and is expected to vary while remaining within the substance and scope of the various embodiments disclosed herein.

[0059] All literature and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are expressly incorporated in their entirety by reference for any purpose. Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which the various embodiments described herein pertain.

[0060] It should be understood that the terms "approximately" are implied before terms such as temperature, concentration, time, pressure, flow rate, and cross-sectional area discussed in this teaching, resulting in slight and non-substantial deviations within the scope of this teaching. In this application, the singular is used to include the plural unless otherwise expressly stated. Furthermore, the use of "comprising," "including," and "containing" is not intended to be restrictive. It should be understood that both the foregoing general description and the following detailed description are exemplary and illustrative only, and not limiting of this teaching.

[0061] As used herein, “a” or “one” can also mean “at least one” or “one or more”. Furthermore, the use of “or” is inclusive, meaning that the phrase “A or B” is true when “A” is true, “B” is true, or both “A” and “B” are true. Additionally, unless the context otherwise requires, singular terms should include plural forms and plural terms should include singular forms.

[0062] A “system” describes a set of real or abstract components, including the whole, in which each component interacts with or relates to at least one other component within the whole.

[0063] The production of transcripts may require analytical testing to determine reaction outcomes and yields. However, direct measurement (such as using mass spectrometry) is technically challenging due to the size of the transcripts. Two publicly available analytical methods have been developed that use enzymes to digest transcripts into a more manageable size range. The first (named CapMap) uses the digestive enzyme nuclease P1 to reduce the intact transcript to its individual 5' phosphorylated nucleotides, and has also reportedly produced cap dinucleotides. The digestion mixture, doped with ARCA dinucleotides, is then separated on a porous graphite carbon-packed column and analyzed by mass spectrometry. Two challenges were observed with this method: first, control of the digestive enzyme, and second, the chromatographic platform. Nuclease P1, the enzyme used by the authors as the sole digestion system, is known to result in the production of 5' monophosphates. All cap structures are connate at 5' to 5', and when subjected to nuclease P1, this leads to the cleavage of the m7G nucleotide and the first nucleotide in the chain (typically 2'-O-methyladenosine), although other 2'-O-methylated nucleotides are possible. Secondly, porous graphite carbon-packed columns are known to degrade with injection and require remediation. In summary, the CAPMAP method may produce incorrect measurements due to partial digestion or poor chromatographic peak integration.

[0064] The second method uses the digestive enzyme RNase H. RNase H cleaves the RNA strand in the RNA:DNA double-stranded hybrid at Watson-Crick RNA:DNA base pairs. A method published by Beverly (Beverly, M.; Dell, A.; Parmar, P.; Houghton, L. Label-Free Analysis of mRNA Capping Efficiency Using RNase H Probes and LC-MS. Anal. Bioanal. Chem. 2016, 408(18), 5021-5030) involves designing biotinylated molecular probes approximately 25 nt in length complementary to the 5' end of the transcript. The probes are annealed to the 5' end of the mRNA, then complexed with magnetic beads, and subsequently digested with RNase H. The lysate is separated by magnetization, washed, eluted from the beads and probe with hot methanol-water, dried, and then analyzed by LC-MS. The results will show that capping occurred. However, the authors of this method noted that their recovery was less than 60%. Furthermore, publicly available data on RNase H1 (the commercially available enzyme cited in the Beverly paper) show that instead of cleavage at a single RNA:DNA base pair, it produces multiple cleavage products along the downstream strand from chimeric base pairings. During replication digestion, it is possible to detect multiple distinct oligonucleotides from the RNA:DNA base pair, with 1 to 2 nucleotides cleaved from the 3' end of the downstream strand. Additionally, off-target RNase H cleavage products can also be detected in high abundance due to sequence homology between different nucleotide portions at the 5' end of the mRNA.

[0065] Methods for determining capping efficiency and methylation efficiency

[0066] This article describes a method for directly measuring 5' capping efficiency and 2'-O-methyltransferase efficiency. Aliquots of mRNA can be doped with a known amount of isotopically labeled mRNA. 7 G and isotopically labeled 2'-O-methylated nucleosides, such as 2'-O-methyladenosine (Am), 2'-O-methylguanosine (Gm), 2'-O-methylcytosine (Cm), or 2'-O-methyluridine (Um). Figure 4A Shown in methyl m 7 m is labeled with an isotope containing three deuterium atoms at G(D). 7 G. Similarly, Figure 4BThe image shows the isotopically labeled Am with three deuterium atoms at the methyl Am(D) position. A single-enzyme digestion can then be performed to reduce the mRNA to its individual nucleosides. The doped digest can then be separated chromatographically using a simple ammonium-based buffer system and analyzed by mass spectrometry. Precise quantification can be achieved by comparing the peak areas of the heavy m7G / 2'O-methylated nucleosides with those of the light m7G / 2'O-methylated nucleosides.

[0067] In other embodiments, isotope-labeled standards may include isotope-labeled 5'-phosphate-m 7 G and isotopically labeled 5'-phosphate-2'-O-methylated nucleotides. In this case, phosphatases can cleave the 5' phosphate from the isotopically labeled standard to produce isotopically labeled methylated nucleosides.

[0068] Mass spectrometry has proven to be a robust and reliable application for the direct measurement of biomolecules. By converting biomolecules as ions from a liquid or solid matrix into the gas phase, the mass of the ions can be detected and measured. One method for generating ions is electrospray ionization (ESI). Quantification of molecules generated by ESI can be performed using a standard curve, where a known amount of a synthetic standard is obtained and the results are plotted, and the concentration of the target in the sample can be found using a slope equation. The main challenges in generating a standard curve are discovering / generating synthetic standards and matching the matrix to the target sample. Due to the nature of ESI, matrix matching is important, where each polar molecule in the sample competes for surface charge. This charge competition can cause the reaction measured in the sample to differ from the reaction on the standard curve, leading to measurement errors. More precise molecular measurements can be achieved by doping the sample with isotopically labeled standards. In this method, a synthetic standard for the target molecule is generated, in which one or more atoms are replaced by their stable isotope, C0. 13 Replaced by carbon, N 15 Nitrogen is substituted for deuterium (D), and hydrogen is substituted for deuterium (D). A known amount of a stable isotope-labeled standard (SILS) is incorporated into the sample, and the sample is processed and acquired. Since the SILS is structurally identical to the target molecule, it will have the same elution curve as the molecule under study, the only difference being the mass variation. The incorporated SILS will undergo the same ionization charge competition as the target molecule, thus avoiding any measurement errors that may occur when attempting to match the matrix. The amount of the target molecule is quantified by obtaining the ratio of the heavy peak area to the light peak area. For RNA nucleoside analysis, the main obstacle of this method is the availability of SILS. The method described in this paper uses only purified, heavily labeled nucleoside molecules as internal standards, eliminating the need for cell culture, RNA purification, and sample dilution, and resulting in accurate measurement of the target molecule.

[0069] Figure 5A method 500 for measuring 5' capping efficiency and / or 2'-O-methyltransferase efficiency is shown. At 502, the mRNA sample may be doped with isotopically labeled standards, such as isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleosides. In various embodiments, the isotopically labeled m7G and 2'-O-methylated nucleosides may be deuterium-labeled, such as m7G(D) and Am(D), or may be N-labeled. 15 Or C 13 Labeling. In various embodiments, the molar ratio of isotopically labeled m7G to isotopically labeled 2'-O-methylated nucleoside can be between about 2:1 and about 1:2, such as equimolar amounts. The concentration of the standard can be such that the amount of isotopically labeled m7G in the incubation mixture is between about 1.0 nmol and 20 nmol, and the amount of isotopically labeled 2'-O-methylated nucleoside in the incubation mixture is between about 1.0 nmol and about 20 nmol.

[0070] At position 504, an enzyme mixture can be added to the sample. The enzyme mixture may include nonspecific single-stranded nucleases, such as nuclease P1, and acid phosphatases, such as sweet potato acid phosphatase. A notable characteristic of acid phosphatases may be that they are not inhibited by adenosine monophosphate (ATP), as nuclease P1 digests mRNA into monophosphate, leading to the production of ATP, which may inhibit some acid phosphatases. Optionally, the enzyme mixture may include site-specific single-stranded RNA endonucleases, such as guanosine-lysine 3'-RNA endonucleases. Examples of guanosine-lysine 3'-RNA site-specific single-stranded RNA endonucleases include RNase T1, RNase N1, RNase Sa, Bacillus RNase (Barnase), and other similar endonucleases.

[0071] In various embodiments, the enzyme mixture may include sufficient nonspecific single-stranded nuclease such that between about 1 unit and about 100 units of nonspecific single-stranded nuclease are added to the mRNA sample, such as between about 5 units and about 20 units. In various embodiments, the enzyme mixture may include sufficient acid phosphatase such that between about 0.5 units and about 10.0 units of acid phosphatase are added to the mRNA sample, such as between about 0.5 units and about 2.0 units. In various embodiments, the enzyme mixture may include sufficient site-specific single-stranded RNA endonuclease (when present) such that between about 0.5 units and about 10.0 units of site-specific single-stranded RNA endonuclease are added to the mRNA sample, such as between about 0.5 units and about 2.0 units. Increasing the amount of enzyme is known to shorten the completion time, and those skilled in the art can determine the appropriate amount of enzyme for the desired incubation period.

[0072] In various embodiments, the mRNA sample, isotope-labeled standards, and / or enzyme mixture can be dried and reconstructed by adding a buffer solution. For example, the mRNA sample can be precipitated and the supernatant can be removed, such as by removing salts and solvents that could interfere with enzymatic digestion from the synthesis of mRNA. In another example, the isotope-labeled standards and enzyme mixture can be provided as a lyophilized powder, such as to extend shelf life. Alternatively, the standards or enzyme mixture can be a frozen solution that is thawed as needed. In various embodiments, the precipitated mRNA can be reconstructed with a specific amount of buffer solution, and the enzyme mixture and isotope-labeled standards can be added in specific amounts. Alternatively, specific amounts of the enzyme mixture and isotope-labeled standards can be added to the precipitated mRNA, and the mRNA can be reconstructed, for example, by mixing.

[0073] In various embodiments, the buffer solution may comprise ZnCl2 with a concentration ranging from about 0.001 mM to about 90 mM, such as from about 0.05 mM to about 0.2 mM. Furthermore, the incubation mixture contains less than 100 mM of divalent cations. In various embodiments, the buffer solution may have a pH between about 2 and about 7, such as between about 4 and about 6.

[0074] At 506, enzymatic digestion of mRNA can be performed. In various embodiments, the incubation mixture, including mRNA, isotopically labeled standards, and enzyme mixture, can be incubated at a temperature between about 30°C and about 70°C, such as between about 30°C and about 50°C, or even between about 35°C and about 45°C. Enzymatic digestion can proceed for a sufficient time to allow nucleases to digest the mRNA into component nucleotides and acid phosphatases to convert nucleotides into nucleosides by removing phosphate from the nucleotides. In various embodiments, enzymatic digestion can proceed for at least about 30 minutes, such as at least about 45 minutes, such as at least about 1 hour, such as at least about 4 hours, such as at least overnight (about 12 to 16 hours). Typically, enzymatic digestion can be performed in less than about 1 day.

[0075] At 508, the digested sample can be chromatographically separated, and at 510, the chromatographically separated sample can be analyzed by mass spectrometry. This may include obtaining m / z and intensity data using a mass spectrometer, identifying and integrating the peaks of m7G and isotopically labeled m7G and / or the peaks of 2'-O-methylated nucleosides and isotopically labeled 2'-O-methylated nucleosides. Furthermore, at 512, the capping efficiency can be determined based on the m7G:isotopically labeled m7G peak ratio, and the 2'-O-methyltransferase efficiency can be determined based on the 2'-O-methylated nucleoside:isotopically labeled 2'-O-methylated nucleoside peak ratio.

[0076] Enzyme digestion kit

[0077] In various embodiments, kits for quantifying mRNA capping efficiency may include an enzyme mixture and a mixture of isotope-labeled standards. The enzyme mixture and isotope-labeled standards mixture may be a lyophilized powder that can be reconstituted by adding buffer. In other embodiments, the enzyme mixture and isotope-labeled standards mixture may be a solution, such as a frozen solution. It may be advantageous for the enzyme mixture and isotope-labeled standards mixture to be in small quantities, such as disposable tubes sufficient for one or a few reactions, rather than large solutions that can be used for dozens of reactions.

[0078] The enzyme mixture may include nonspecific single-stranded nucleases and acid phosphatases. Nonspecific single-stranded nucleases may include nuclease P1. Acid phosphatases may be acid phosphatases not inhibited by adenosine monophosphate, such as sweet potato acid phosphatase. The enzyme mixture may include site-specific single-stranded RNA endonucleases, such as site-specific single-stranded RNA endonucleases that cleave guanosine at the RNA 3' site. In various embodiments, the site-specific single-stranded RNA endonucleases may include RNase T1, RNase N1, RNase Sa, Bacillus RNase, and other similar endonucleases.

[0079] Isotope-labeled standard mixtures may include isotope-labeled m7G and 2'-O-methylated nucleosides. Typically, isotope-labeled standard mixtures may include re-stable isotopes incorporated into the compounds used as standards. The use of re-stable isotopes can alter the m / z charge ratio without changing the chemical properties of the compounds, thus allowing them to be used as internal standards in mass spectrometry. In various embodiments, isotope-labeled standard mixtures may include isotope-labeled m7G and 2'-O-methylated nucleosides. The isotope-labeled m7G and 2'-O-methylated nucleosides may be deuterium-labeled, N... 15 Marked, C 13 Marked, O 17 Marked, O 18 Labeled or any combination thereof. Mixtures of isotopically labeled standards may include isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleosides in a molar ratio between about 2:1 and about 1:2, such as equimolar amounts of isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleosides.

[0080] In other embodiments, the isotope-labeled standard mixture may include isotope-labeled 5'-phosphate-m 7 G and isotopically labeled 5'-phosphate-2'-O-methylated nucleotides. In this case, phosphatases can cleave the 5' phosphate from the isotopically labeled standard to produce isotopically labeled methylated nucleosides.

[0081] The kit may also include a buffer solution. The buffer solution may include ZnCl2, such as at a concentration between about 0.01 mmol and about 90 mmol. The buffer solution may have a divalent cation concentration of less than 100 mmol. The pH of the buffer solution is between about 2 and about 7, such as between about 4 and about 6.

[0082] Chromatography platform

[0083] Figure 6 A liquid chromatography system 600 is depicted according to one aspect of the present invention. The liquid chromatography system 600 includes an analytical pump 602 to pump solvent through the system 600. The system 600 includes a sample reservoir 604 containing a sample to be analyzed. The system 600 also includes a separation column 606 and a detector 608. The system 600 also includes a controller 610.

[0084] The liquid chromatography system 600 is adapted to extract samples from the sample reservoir 604. The samples can then be introduced into the system.

[0085] The liquid chromatography system 1000 is also suitable for introducing samples into the separation column 606.

[0086] System 600 is also adapted to inject a sample into separation column 606 via an analytical stream. This can be accomplished by guiding the sample using an analytical pump 602. Separation column 606 can separate the sample into component substances based on retention time within separation column 606. After separation of the sample by separation column 606, the separated components can be detected by detector 608. In some embodiments, detector 608 can be an optical detector, such as an absorption detector, refractive index detector, fluorescence detector, etc. In other embodiments, detector 608 can be a conductivity detector or an electrochemical detector. In other embodiments, detector 608 can be a mass spectrometer.

[0087] In various implementations, the separation column 606 typically consists of a tube filled with a stationary phase medium. The stationary phase medium can affect the time it takes for a compound to travel through the column (retention time). This effect can vary for different compounds, allowing individual components of the sample to be separated based on their respective retention times. Various stationary phase media exist, including porous materials, ionic materials, polar materials, and nonpolar materials. Porous materials can affect retention time based on molecular size and the ability of molecules to enter the porous material. Ionic materials can affect retention time based on the charge attraction or repulsion between the ionic material and the compound. Polar and nonpolar materials can affect retention time based on the hydrophobicity or hydrophilicity of the compound.

[0088] In various embodiments, reversed-phase separation can be used to separate nucleotides and nucleosides, wherein a hydrophobic, nonpolar stationary phase material is used with a mobile phase having different hydrophobicities depending on the ratio of polar solvent to organic solvent. For example, a C18 column can be used with an ammonium acetate or ammonium formate buffer system to separate nucleosides. In a particular embodiment, an aqueous mobile phase of 5 mM ammonium acetate at a pH of about 5 can be used, and a gradient of increasing concentrations of acetonitrile (up to about 40%) or methanol (up to about 50%) can be used to separate nucleosides. Suitable column and buffer systems will be apparent to those skilled in the art and are within the scope of this disclosure.

[0089] In other embodiments, hydrophilic interaction liquid chromatography (HILIC) can be used to separate nucleotides and nucleosides, wherein a hydrophilic stationary phase material and a hydrophobic mobile phase, such as acetonitrile, are used. Suitable column and buffer systems will be apparent to those skilled in the art and are within the scope of this disclosure.

[0090] Mass spectrometry platform

[0091] Various implementation schemes of the mass spectrometry platform 700 may include, for example Figure 7 The components are shown in the block diagram. In various embodiments, the mass spectrometry platform 700 can operate as the detector 608 of the system 600. In various embodiments, Figure 7 The components can be incorporated into the mass spectrometry platform 700. According to various embodiments, the mass spectrometer 700 may include an ion source 702, a mass analyzer 704, an ion detector 706, and a controller 708.

[0092] In various implementation schemes, ion source 702 generates multiple ions from the sample. Ion source may include, but is not limited to, matrix-assisted laser desorption / ionization (MALDI) source, electrospray ionization (ESI) source, atmospheric pressure chemical ionization (APCI) source, atmospheric pressure photoionization (APPI) source, inductively coupled plasma (ICP) source, electron beam ionization source, chemical ionization source, photoionization source, glow discharge ionization source, thermal spray ionization source, etc.

[0093] In various embodiments, the mass analyzer 704 can separate ions based on their mass-to-charge ratio. For example, the mass analyzer 704 may include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., an orbital trap) mass analyzer, a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, etc. In various embodiments, the mass analyzer 704 may also be configured to fragment ions using collision-induced dissociation (CID), electron transfer dissociation (ETD), electron capture dissociation (ECD), photoinduced dissociation (PID), surface-induced dissociation (SID), etc., and further separate fragment ions based on their mass-to-charge ratio.

[0094] In various embodiments, the ion detector 706 can detect ions. For example, the ion detector 706 may include an electron multiplier, a Faraday cup, etc. Ions leaving the mass analyzer can be detected by the ion detector. In various embodiments, the ion detector can be quantitative, allowing for the determination of an accurate ion count.

[0095] In various implementations, the controller 708 can communicate with the ion source 702, the mass analyzer 704, and the ion detector 706. For example, the controller 708 can configure the ion source or enable / disable the ion source. Additionally, the controller 708 can configure the mass analyzer 704 to select a specific mass range to be detected. Furthermore, the controller 708 can adjust the sensitivity of the ion detector 706, such as by adjusting the gain. Additionally, the controller 708 can adjust the polarity of the ion detector 706 based on the polarity of the detected ions. For example, the ion detector 706 can be configured to detect positive ions or to detect negative ions.

[0096] While this teaching is described in conjunction with various implementation schemes, it is not intended to limit this teaching to such implementations. Rather, this teaching encompasses various alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0097] Furthermore, in describing various embodiments, the specification may present the method and / or process as a specific sequence of steps. However, the method or process should not be limited to the specific sequence of steps described herein, as long as the method or process is not dependent on the specific order of the steps set forth herein. Other sequences of steps are also possible, as will be understood by those skilled in the art. Therefore, the specific order of steps set forth in the specification should not be construed as a limitation of the claims. Additionally, claims relating to the method and / or process should not be limited to performing the steps in the written order, and those skilled in the art will readily understand that the sequence can be varied while still remaining within the spirit and scope of the various embodiments.

Claims

1. A method for quantifying mRNA capping efficiency, the method comprising: combining an mRNA sample, an enzyme mixture, and an isotopic standard solution in a buffer solution to produce an incubation mixture, the enzyme mixture comprising a nuclease P1 and a sweet potato acid phosphatase, wherein the acid phosphatase is not inhibited by adenosine monophosphate, and the isotopic standard comprises an isotopically labeled m7G and an isotopically labeled 2’-O-methylated nucleoside (Am, Gm, Cm, or Um); incubating the mixture; and analyzing the mixture using liquid chromatography-mass spectrometry to determine at least one of capping efficiency and 2’-O-methyltransferase efficiency.

2. The method of claim 1, wherein incubating the mixture is performed at a temperature between 30 °C and 70 °C.

3. The method of claim 1, wherein incubating the mixture is performed at a temperature between 35 °C and 45 °C.

4. The method of claim 1, further comprising dissolving a lyophilized enzyme mixture with a sample buffer to obtain the enzyme mixture.

5. The method of claim 1, further comprising dissolving a lyophilized standard mixture with a sample buffer to obtain the standard mixture.

6. The method of claim 1, wherein the isotopically labeled m7G and the isotopically labeled 2’-O-methylated nucleoside comprise deuterium-labeled m7G (D) and deuterium-labeled 2’-O-methylated nucleoside Am(D), Gm(D), Cm(D), or Um(D).

7. The method of claim 1, wherein the isotopically labeled m7G and the isotopically labeled 2'-0-methylated nucleoside are N 15 labeled or C 13 labeled.

8. The method of claim 1, wherein the molar ratio of the isotopically labeled m7G and the isotopically labeled 2’-O-methylated nucleoside in the standard mixture is between 2: 1 to 1:

2.

9. The method of claim 8, wherein the standard mixture comprises equimolar amounts of isotopically labeled m7G and isotopically labeled 2’-O-methylated nucleoside.

10. The method of claim 1, wherein the incubation mixture comprises an amount of isotopically labeled m7G between 1.0 nmol and 20 nmol.

11. The method of claim 1, wherein the incubation mixture comprises an amount of isotopically labeled 2’-O-methylated nucleoside between 1.0 nmol and 20 nmol.

12. The method of claim 1, wherein the buffer comprises ZnCl2 at a concentration ranging from 0.001 mM to 90 mM.

13. The method of claim 1, wherein the buffer comprises ZnCl2 at a concentration ranging from 0.05 mM to 0.2 mM.

14. The method of claim 1, wherein the incubation mixture comprises less than 100 mM divalent cations.

15. The method of claim 1, wherein the buffer has a pH between 2 to 7.

16. The method of claim 1, wherein the buffer has a pH between 4 to 6.

17. The method of claim 1, wherein analyzing the mixture using liquid chromatography-mass spectrometry further comprises separating the mixture using a chromatographic column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating peaks of m7G and isotopically labeled m7G, peaks of 2'-0-methylated nucleosides and isotopically labeled 2'-0-methylated nucleosides, or both; calculating m7G: isotopically labeled m7G peak ratios, 2'-0-methylated nucleoside: isotopically labeled 2'-0-methylated nucleoside peak ratios, or both; and determining the capping efficiency based on the m7G: isotopically labeled m7G peak ratios, the 2'-0-methyltransferase efficiency based on the 2'-0-methylated nucleoside: isotopically labeled 2'-0-methylated nucleoside peak ratios, or both.

18. The method of claim 1, wherein analyzing the mixture using liquid chromatography-mass spectrometry further comprises separating the mixture using a chromatographic column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating peaks of m7G and isotopically labeled m7G, peaks of 2'-0-methylated nucleosides and isotopically labeled 2'-0-methylated nucleosides, or both; calculating m7G: isotopically labeled m7G peak ratios, 2'-0-methylated nucleoside: isotopically labeled 2'-0-methylated nucleoside peak ratios, or both; and determining the capping efficiency based on the m7G: isotopically labeled m7G peak ratios, the 2'-0-methyltransferase efficiency based on the 2'-0-methylated nucleoside: isotopically labeled 2'-0-methylated nucleoside peak ratios, or both.

19. The method of claim 1, wherein analyzing the mixture using liquid chromatography-mass spectrometry further comprises separating the mixture using a chromatographic column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating peaks of m7G and isotopically labeled m7G, peaks of 2'-0

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