Quantitative Monitoring Method for mRNA Capping Efficiency

Abstract

A method for quantifying mRNA capping efficiency includes combining a sample, an enzyme mixture and an isotope standard solution in a buffer solution to generate an incubation mixture, wherein the enzyme mixture comprises a non-specific single-stranded nuclease and an acid phosphatase, and the isotope standard comprises an isotope-labeled m7G and an isotope-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] The present disclosure generally relates to the field of mass spectrometry, including methods for quantitatively monitoring mRNA capping efficiency.

Background Art

[0002] Introduction In eukaryotes, non-mitochondrial messenger RNA (mRNA) is capped at the 5' end by reverse 7-methylguanosine (m7G) (Figure 1A), which is linked to the transcript by a 5' to 5' triphosphate linkage group m7G(5')ppp(5')N called cap(0) (Figure 2A). Once added to the transcript, the first nucleotide (N) in the chain is then methylated at the 2' hydroxyl position, giving the cap(1) structure m7G(5')ppp(5')Nm (Figure 2B). For example, Figure 1B shows 2'-O-methyladenosine. Alternatively, Nm can be 2'-O-methylguanosine (Gm), 2'-O-methylcytosine Cm or 2'-O-methyluridine (Um). Capping and polyadenylation help the transcript to resist degradation by endogenous exonucleases in the cell. In addition to blocking 5' to 3' exonucleases, the 5' cap promotes transport from the nucleus to the cytoplasm and aids the translation initiation process by acting as a determinant of the ribosome docking complex.

[0003] Further changes to the transcript, called splicing events, also depend on the cap structure. Portions of the transcript called introns or intervening regions are removed, the cut regions are rejoined, and the remaining expressed coding regions (exons) remain. This coding region is flanked on both the 5' and 3' sides by untranslated regions, stretches of RNA that are not translated into protein but affect translation. The mature mRNA transcript then consists of five regions: the 5' cap, the 5' UTR, the coding region, the 3' UTR, and finally the polyadenylation tail (see Figure 3).

[0004] Due to recent events, particularly the COVID pandemic, mRNA has been widely adopted as a useful therapy, such as various mRNA-based COVID vaccines. The production of non-natural transcripts by in vitro transcription may require the addition of a modified cap structure after purification to achieve the desired therapeutic function. Modified 5’ caps can include anti-reverse cap analogs (ARCA) (U.S. Patent No. 7,074,596), TriLink Biotechnology's CleanCap analogs, or those by vaccinia capping enzyme followed by a methyltransferase reaction. The production of transcripts requires analytical tests to determine reaction results and yields, but due to the size of the transcripts, direct measurement by mass spectrometry and the like is technically challenging. Therefore, improved techniques for measuring capping efficiency and methylation efficiency are desired.

Summary of the Invention

[0005] In a first aspect, a kit for quantifying mRNA capping efficiency can include an enzyme mixture containing nuclease P1 and sweet potato acid phosphatase, and a standard mixture containing isotope-labeled m7G and isotope-labeled 2’-O-methylated nucleosides. In various embodiments, the 2’-O-methylated nucleoside can be Am, Gm, Um, or Cm.

[0006] In various embodiments of the first aspect, the kit can further include a buffer solution. In certain embodiments, the buffer solution can contain ZnCl2, such as a ZnCl2 concentration of about 0.01 mmol to about 90 mmol. In certain embodiments, the buffer solution has a divalent cation concentration of less than 100 mmol. In certain embodiments, the buffer solution can have a pH of about 2 to about 7, such as about 4 to about 6.

[0007] In various embodiments of the first aspect, the enzyme mixture can further include RNase T1.

[0008] In various embodiments of the first aspect, the enzyme mixture can be a lyophilized powder.

[0009] In various embodiments of the first aspect, the standard substance mixture can be a lyophilized powder.

[0010] In various embodiments of the first aspect, the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside include deuterium-labeled m7G (D) and deuterium-labeled 2'-O-methylated nucleoside.

[0011] In various embodiments of the first aspect, the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside are N 15 labeled or C 13 labeled.

[0012] In various embodiments of the first aspect, the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside in the standard substance mixture can be present in a molar ratio of about 2:1 to about 1:2. In certain embodiments, the standard substance mixture can include equimolar amounts of the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside.

[0013] In the second aspect, a kit for quantifying mRNA capping efficiency can include an enzyme mixture containing a non-specific single-stranded nuclease and an acid phosphatase, a standard mixture containing an isotope-labeled m7G and an isotope-labeled 2'-O-methylated nucleoside, and a buffer solution.

[0014] In various embodiments of the second aspect, the non-specific single-stranded nuclease can include nuclease P1.

[0015] In various embodiments of the second aspect, the acid phosphatase cannot be inhibited by adenosine monophosphate.

[0016] In various embodiments of the second aspect, the acidic phosphatase can include sweet potato acidic phosphatase.

[0017] In various embodiments of the second aspect, the enzyme mixture can include a site-specific single-stranded RNA endonuclease.

[0018] In various embodiments of the second aspect, the site-specific single-stranded RNA endonuclease can cleave the RNA 3’ of guanosine.

[0019] In various embodiments of the second aspect, the site-specific single-stranded RNA endonuclease can include ribonuclease T1.

[0020] In various embodiments of the second aspect, the enzyme mixture can be a lyophilized powder.

[0021] In various embodiments of the second aspect, the standard substance mixture can be a lyophilized powder.

[0022] In various embodiments of the first aspect, the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside include deuterium-labeled m7G(D) and deuterium-labeled 2’-O-methylated nucleoside.

[0023] In various embodiments of the first aspect, the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside are N 15 labeled or C 13 labeled.

[0024] In various embodiments of the second aspect, the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside in the standard substance mixture can be present in a molar ratio of about 2:1 to about 1:2. In certain embodiments, the standard substance mixture can include equimolar amounts of the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside.

[0025] In various embodiments of the second aspect, the buffer solution can contain ZnCl2 in a concentration range of about 0.001 mM to about 90 mM, such as about 0.05 mM to about 0.2 mM. In certain embodiments, the buffer solution can contain less than 100 mM divalent cations.

[0026] In various embodiments of the second aspect, the buffer solution has a pH of about 2 to about 7, such as about 4 to about 6.

[0027] In the third aspect, a method for quantifying mRNA capping efficiency can include the steps of combining a sample, an enzyme mixture, and an isotopic standard solution in a buffer solution to produce an incubation mixture, wherein the enzyme mixture includes a non-specific single-stranded nuclease and an acidic phosphatase, and the isotopic standard includes 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 the capping efficiency and the 2'-O-methyltransferase efficiency.

[0028] In various embodiments of the third aspect, the step of incubating the mixture can be performed at a temperature of about 30°C to about 70°C, such as about 35°C to 45°C.

[0029] In various embodiments of the third aspect, the non-specific single-stranded nuclease can include nuclease P1.

[0030] In various embodiments of the third aspect, the acidic phosphatase cannot be inhibited by adenosine monophosphate.

[0031] In various embodiments of the third aspect, the acidic phosphatase can include sweet potato acidic phosphatase.

[0032] In various embodiments of the third aspect, the enzyme mixture can further comprise a site-specific single-stranded RNA endonuclease. In certain embodiments, the site-specific single-stranded RNA endonuclease can cleave the RNA 3’ of guanosine. In certain embodiments, the site-specific single-stranded RNA endonuclease can comprise ribonuclease T1. In further embodiments, the step of incubating the mixture can be performed at a temperature of about 30°C to 50°C.

[0033] In various embodiments of the third aspect, the incubation mixture can comprise from about 0.5 unit to about 10.0 units of a site-specific single-stranded RNA endonuclease, such as from about 0.5 unit to about 2.0 units.

[0034] In various embodiments of the third aspect, the method can further comprise the step of solubilizing the lyophilized enzyme mixture in a sample buffer solution to obtain the enzyme mixture.

[0035] In various embodiments of the third aspect, the method can further comprise the step of solubilizing the lyophilized standard mixture in a sample buffer solution to obtain the standard mixture.

[0036] In various embodiments of the third aspect, the incubation mixture can comprise from about 0.5 unit to about 10.0 units of an acid phosphatase, such as from about 0.5 unit to about 2.0 units.

[0037] In various embodiments of the third aspect, the incubation mixture can comprise from about 1 unit to 100 units of a non-specific single-stranded nuclease, such as from about 5 units to about 20 units.

[0038] In various embodiments of the third aspect, the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside comprise deuterium-labeled m7G (D) and deuterium-labeled 2'-O-methylated nucleoside.

[0039] In various embodiments of the third aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside are N 15 labeled or C 13 labeled.

[0040] In various embodiments of the third aspect, the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside in the standard substance mixture are present in a molar ratio of about 2:1 to about 1:2, such as an equimolar amount of the isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside.

[0041] In various embodiments of the third aspect, the incubation mixture can contain an amount of isotopically labeled m7G that results in about 1.0 nmol to 20 nmol. In certain embodiments, the incubation mixture can contain an amount of isotopically labeled 2'-O-methylated nucleoside that results in about 1.0 nmol to about 20 nmol.

[0042] In various embodiments of the third aspect, the buffer solution can contain ZnCl2 in a concentration range of about 0.001 mM to about 90 mM. In certain embodiments, the buffer solution can contain ZnCl2 in a concentration range of about 0.05 mM to about 0.2 mM.

[0043] In various embodiments of the third aspect, the incubation mixture can contain less than 100 mM divalent cations.

[0044] In various embodiments of the third aspect, the buffer solution can have a pH of about 2 to about 7, such as about 4 to about 6.

[0045] In various embodiments of the third aspect, the step of analyzing a mixture using liquid chromatography-mass spectrometry includes separating the mixture using a chromatography column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating the peaks of m7G and isotopically labeled m7G, the 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 the capping efficiency based on the m7G:isotopically labeled m7G peak ratio, the 2'-O-methyltransferase efficiency based on the 2'-O-methylated nucleoside:isotopically labeled 2'-O-methylated nucleoside peak ratio, or both. Reference is now made to the following description, taken in conjunction with the accompanying drawings, for a more complete understanding of the principles disclosed herein and their advantages.

Brief Description of the Drawings

[0046]

Figure 1A

Figure 1B

Figure 2A

Figure 2B

Figure 3

Figure 4A

Figure 4B

Figure 5

Figure 6

Figure 7

[0047] It should be understood that the figures are not necessarily drawn to scale and that the objects within the figures are not necessarily drawn to scale in relation to each other. The figures are illustrations intended to provide clarity and understanding of the various embodiments of the apparatus, systems, and methods disclosed herein. As far as possible, the same reference numbers are used throughout the drawings to refer to the same or similar components. Further, it should be understood that the drawings are not intended to limit in any way the scope of the teachings of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0048] Embodiments of qualitative and quantitative methods for ion mobility enhancement are described herein.

[0049] The section headings used herein are for organizational purposes only and should not be construed as in any way limiting the subject matter described.

[0050] In this detailed description of various embodiments, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. However, one of ordinary skill in the art will understand that these various embodiments may be practiced without these specific details. In other instances, structures and devices are shown in block diagram form. Further, the specific order in which methods are presented and executed is exemplary, and it is contemplated that the order may be changed and still remain within the spirit and scope of the various embodiments disclosed herein, which can be readily understood by one of ordinary skill in the art.

[0051] All documents and similar materials cited in this application, including but not limited to patents, patent applications, articles, books, papers, and Internet web pages, are hereby expressly incorporated by reference in their entirety for any purpose. Unless otherwise specified, all technical and scientific terms used in this specification are meant to have the meaning commonly understood by one of ordinary skill in the art to which the various embodiments described herein belong.

[0052] There is an implicit "about" in front of temperatures, concentrations, times, pressures, flow rates, cross-sectional areas, etc. discussed in this disclosure, and thus it will be understood that very small and slight deviations are within the scope of this disclosure. In this application, the use of the singular form includes the plural unless specifically stated otherwise. Similarly, the use of "comprise", "comprises", "comprising", "contain", "contains", "containing", "include", "includes", and "including" is not intended to be limiting. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and are not restrictive of the disclosure.

[0053] As used herein, "a" or "an" may mean "at least one" or "one or more". Also, the use of "or" is inclusive, such that the phrase "A or B" applies when A applies, when B applies, or when both A and B apply. Further, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include singulars.

[0054] "System" refers to a set of components, whether physical or abstract, where each component interacts with or is related to at least one other component within the whole.

[0055] The production of transcripts may require analytical tests to determine reaction results and yields. However, due to the size of transcripts, direct measurement by mass spectrometry, etc. is technically challenging. Two published analytical methods have been developed that use enzymes to digest transcripts into a more manageable size range. The first, named CapMap, uses the nuclease P1, a digestive enzyme, to reduce intact transcripts to their individual 5'-phosphorylated nucleotides and is reported to also generate cap dinucleotides. Next, the digestion mixture is spiked with ARCA dinucleotides, separated on a porous graphite-based carbon-packed chromatography column, and analyzed by mass spectrometry. Two problems have been observed with this method. The first is the control of the digestive enzyme, and the second is the chromatography platform. It is well known that the enzyme nuclease P1, which the authors use as the only digestive system, results in the production of 5'-monophosphates. All cap structures are linked 5' to 5', and when exposed to nuclease P1, the m7G nucleotide and the first nucleotide in the chain, usually 2'-O-methyladenosine, are cleaved, although other 2'-O-methylated nucleosides are possible. Second, the porous graphite-based carbon-packed chromatography column is known to degrade upon injection and needs to be conditioned again. In summary, the CAPMAP method may result in inaccurate measurements, either due to partial digestion or integration of poor chromatography peaks.

[0056] The second method uses RNase H, a digestive enzyme. RNase H cleaves the RNA strand at Watson-Crick RNA:DNA base pairs in an RNA:DNA double-stranded hybrid. This 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 the design of a biotinylated molecular probe approximately 25 nt in length that is complementary to the 5’ end of the transcript. The probe is annealed to the 5’ end of the mRNA and then complexed with magnetic beads, which are then subjected to RNase H digestion. The cleaved products are separated by a magnet, washed, eluted from the beads and probe with hot methanol-water, dried, and then analyzed by LC-MS. The results will indicate whether capping has occurred. However, the authors of this method acknowledge that its recovery rate was less than 60%. Furthermore, the published data on the commercially available enzyme RNase H1, which is referenced in Beverly's paper, does not cleave at single-stranded RNA:DNA base pairs but instead produces multiple cleavage products in the strand downstream of the chimeric base pair. When replicating the digest, it is possible to detect multiple different oligonucleotides with 3’ cleavage ends of the downstream strand that are 1-2 nucleotides away from the RNA:DNA base pair. Additionally, due to the sequence homology between different nucleotide regions at the 5’ end of the mRNA, off-target RNase H cleavage products can also be detected in high abundance.

[0057] Methods for determining capping efficiency and methylation efficiency Methods for the direct measurement of both 5’ capping efficiency and 2’-O-methyltransferase efficiency are described herein. A defined amount of mRNA is spiked with a known amount of an isotope-labeled m such as 2’-O-methyladenosine (Am), 2’-O-methylguanosine (Gm), 2’-O-methylcytosine Cm, or 2’-O-methyluridine (Um). 7G and isotope-labeled 2'-O-methylated nucleosides can be spiked into the nucleoside. 7 Isotopically labeled m in G(D) with three deuterium atoms 7 Figure 4B shows isotopically labeled Am, which is labeled with three deuterium atoms at the methyl group Am (D). A single enzymatic digest can then be performed to reduce the mRNA to its individual nucleosides. The spiked digests can then be separated chromatographically using a single ammonium-based buffer system and analyzed by mass spectrometry. Accurate quantification can be performed by comparison of the peak areas of heavy vs. light m7G / 2'O-methylated nucleosides.

[0058] In another embodiment, the isotopically labeled standard is an isotopically labeled 5' phosphate-m 7 G and an isotopically labeled 5'-phosphate-2'-O-methylated nucleotide. In this scenario, the phosphatase can cleave the 5' phosphate from the isotopically labeled standard to an isotopically labeled methylated nucleoside.

[0059] Mass spectrometry has proven to be a robust and reliable application for the direct measurement of biomolecules. By transitioning biomolecules from a liquid or solid matrix to the gas phase as ions, the mass of these ions can be detected and measured. One method of generating ions is electrospray ionization (ESI). Quantification of molecules generated by ESI can be performed using a standard curve, in which case a known amount of a synthetic standard is required, the resulting data is plotted, and the equation of the slope is used to find the concentration of the target in the sample. The challenges posed by the generation of a standard curve are mainly finding / generating a synthetic standard and matching the matrix of the target sample. Matrix matching is important due to the nature of ESI where all polar molecules in the sample compete for surface charge. Such charge competition can change the measured responsiveness in the sample from that of the standard curve, causing measurement error. More accurate measurement of molecules is done by spiking the sample with an isotope-labeled standard. In this method, a synthetic standard of the target molecule is generated, where in the case of carbon, one or more atoms are replaced with its stable isotope, C 13 , in the case of nitrogen, N 15 , and in the case of hydrogen, deuterium (D). A known amount of a stable isotope-labeled standard (SILS) is spiked into the sample and the sample is processed and obtained. Since the SILS is structurally identical to the target molecule, it has the same elution profile as the molecule under consideration, the difference being only a mass shift. The spiked SILS will undergo the same ionization charge competition as the target molecule and will avoid any measurement error that might occur when trying to match the matrix. The amount of the target molecule is quantified by employing the area ratio of the heavy and light peaks. In the case of RNA nucleoside analysis, the main hindrance to this approach was the availability of SILS. The method described herein uses only the post-purification heavy-labeled nucleoside molecule as an internal standard without performing cell culture, RNA purification, and sample dilution, resulting in an accurate measurement of the target molecule.

[0060] Figure 5 illustrates a method 500 for measuring 5'-capping efficiency and / or 2'-O-methyltransferase efficiency. At 502, the mRNA sample can be spiked with an isotopically labeled standard such as isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside. In various embodiments, the isotopically labeled m7G and 2'-O-methylated nucleoside can be deuterium-labeled such as m7G(D) and Am(D), or 15 labeled by N 13 or C. In various embodiments, the isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside can be present in a molar ratio of about 2:1 to about 1:2, such as an equimolar amount. The concentration of the standard can be such that the amount of isotopically labeled m7G in the incubation mixture can be about 1.0 nmol to 20 nmol, and the amount of isotopically labeled 2'-O-methylated nucleoside in the incubation mixture can be about 1.0 nmol to about 20 nmol.

[0061] At 504, an enzyme mixture can be added to the sample. The enzyme mixture can include a non-specific single-stranded nuclease such as nuclease P1, and an acidic phosphatase such as sweet potato acid phosphatase. An important feature of the acidic phosphatase is that the digestion of mRNA to monophosphate by nuclease P1 results in the production of adenosine monophosphate, which can inhibit some acidic phosphatases, so the acidic phosphatase may not be inhibited by adenosine monophosphate. Optionally, the enzyme mixture can include a site-specific single-stranded RNA endonuclease such as an endonuclease that cleaves the 3' of guanosine RNA. Examples of site-specific single-stranded RNA endonucleases that cleave the 3' of guanosine RNA include RNase T1, RNase N1, RNase Sa, Barnase, and other similar endonucleases.

[0062] In various embodiments, the enzyme mixture can contain sufficient non-specific single-stranded nuclease such that a non-specific single-stranded nuclease of about 1 unit to about 100 units, such as about 5 units to about 20 units, is added to the mRNA sample. In various embodiments, the enzyme mixture can contain sufficient acid phosphatase such that an acid phosphatase of about 0.5 units to about 10.0 units, such as about 0.5 units to about 2.0 units, is added to the mRNA sample. In various embodiments, the enzyme mixture can contain sufficient site-specific single-stranded RNA endonuclease (if present) such that a site-specific single-stranded RNA endonuclease of about 0.5 units to about 10.0 units, such as about 0.5 units to about 2.0 units, is added to the mRNA sample. Increasing the amount of enzyme can shorten the time to completion, and those skilled in the art know that they can specify the desired incubation period and the appropriate amount of enzyme.

[0063] In various embodiments, the mRNA sample, the isotope-labeled standard, and / or the enzyme mixture can be in a dry state and can be reconstituted by adding a buffer solution. For example, the mRNA sample can be precipitated, and the supernatant can be removed, such as removing salts and solvents from mRNA synthesis that can interfere with enzyme digestion. In a further example, the isotope-labeled standard and the enzyme mixture can be supplied as a lyophilized powder, such as to extend the shelf life. Alternatively, the standard or the enzyme mixture can be a frozen solution that is thawed as needed. In various embodiments, the precipitated mRNA can be reconstituted using a specified amount of buffer solution, and the enzyme mixture and the isotope-labeled standard can be added in specific amounts. Alternatively, a specific amount of the enzyme mixture and the isotope-labeled standard can be added to the precipitated mRNA, and the mRNA can be reconstituted by mixing or the like.

[0064] In various embodiments, the buffer solution can contain ZnCl2 in a concentration range of about 0.001 mM to about 90 mM, such as a concentration range of about 0.05 mM to about 0.2 mM. Further, the incubation mixture contains less than 100 mM divalent cations. In various embodiments, the buffer solution can have a pH of about 2 to about 7, such as a pH of about 4 to about 6.

[0065] At 506, enzymatic digestion of mRNA can be performed. In various embodiments, an incubation mixture containing mRNA, an isotope-labeled standard, and an enzyme mixture can be incubated at a temperature of about 30°C to about 70°C, such as about 30°C to about 50°C, and further such as about 35°C to about 45°C. Enzymatic digestion can be carried out for a sufficient time with respect to nucleases that digest mRNA into nucleotides, which are the constituent components, and with respect to acid phosphatase that converts nucleotides to nucleosides by removing phosphate from the nucleotides. In various embodiments, enzymatic digestion can be carried out 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 about overnight (about 12 - 16 hours). Generally, enzymatic digestion can be less than about 1 day.

[0066] At 508, the digested sample can be separated by chromatography, and the sample separated by chromatography at 510 can be analyzed by mass spectrometry. This can include obtaining m / z and intensity data using a mass spectrometer, identifying and integrating the peaks of m7G and isotope-labeled m7G, and / or the peaks of 2'-O-methylated nucleosides and isotope-labeled 2'-O-methylated nucleosides. Further, at 512, the capping efficiency can be determined from the peak ratio of m7G:isotope-labeled m7G, and the 2-O-methyltransferase efficiency can be determined based on the peak ratio of 2'-O-methylated nucleoside:isotope-labeled 2'-O-methylated nucleoside.

[0067] Enzymatic digestion kit In various embodiments, a kit for quantifying mRNA capping efficiency can include an enzyme mixture and an isotope-labeled standard mixture. The enzyme mixture and the isotope-labeled standard mixture can be provided as lyophilized powders that can be reconstituted by the addition of a buffer solution. In other embodiments, the enzyme mixture and the isotope-labeled standard mixture can be provided as solutions such as frozen solutions. The enzyme mixture and the isotope-labeled standard mixture can be advantageous in that they can be provided in small amounts, such as in single-use tubes, sufficient for one or a few reactions rather than a bulk solution for use in a large number of reactions.

[0068] The enzyme mixture can include a non-specific single-stranded nuclease and an acid phosphatase. The non-specific single-stranded nuclease can include nuclease P1. The acid phosphatase can be an acid phosphatase that is not inhibited by adenosine monophosphate, such as sweet potato acid phosphatase. The enzyme mixture can include a site-specific single-stranded RNA endonuclease, such as a site-specific single-stranded RNA endonuclease that cleaves the 3’ of guanosine of RNA. In various embodiments, the site-specific single-stranded RNA endonuclease can include RNase T1, RNase N1, RNase Sa, Barnase, and other similar endonucleases.

[0069] The isotope-labeled standard mixture can include isotope-labeled m7G and 2’-O-methylated nucleosides. Generally, the isotope-labeled standard mixture can include heavy stable isotopes incorporated into the compounds of the standards. The use of heavy stable isotopes can shift the m / z charge ratio without modifying the chemical properties of the compounds, enabling their use as internal standards in mass spectrometry. In various embodiments, the isotope-labeled standard mixture can include isotope-labeled m7G and 2’-O-methylated nucleosides. The isotope-labeled m7G and 2’-O-methylated nucleosides can be deuterium-labeled, N 15 labeled, C 13 labeled, O 17 labeled, O 18It can be labeled or can be any combination thereof. The isotopically labeled standard substance mixture can contain isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside in a molar ratio of about 2:1 to about 1:2, such as an equimolar amount of isotopically labeled m7G and isotopically labeled 2'-O-methylated nucleoside.

[0070] In other embodiments, the isotopically labeled standard substance mixture can contain isotopically labeled 5'-phosphate-m 7 G and isotopically labeled 5'-phosphate-2'-O-methylated nucleotide. In this scenario, the phosphatase can cleave the 5'-phosphate from the isotopically labeled standard substance to form an isotopically labeled methylated nucleoside.

[0071] The kit can also contain a buffer solution. The buffer solution can contain ZnCl2 at a concentration such as about 0.01 mmol to about 90 mmol. The buffer solution can have divalent cations at a concentration of less than 100 mmol. The buffer solution has a pH of about 2 to about 7, such as about 4 to about 6.

[0072] Chromatography platform Figure 6 illustrates a liquid chromatography system 600 according to one aspect of the present invention. The liquid chromatography system 600 includes an analytical pump 602 for pumping a solvent by the system 600. The system 600 includes a sample reservoir 604 containing the sample to be analyzed. The system 600 further includes a separation column 606 and a detector 608. The system 600 also includes a control device 610.

[0073] The liquid chromatography system 600 is configured to recover the sample from the sample reservoir 604. Next, the sample can be introduced into the system.

[0074] The liquid chromatography system 1000 is further configured to introduce the sample into the separation column 606.

[0075] System 600 is also adapted to inject a sample into separation column 606 by an analysis flow. This can be done by guiding the sample by analysis pump 602. Separation column 606 can separate the sample into component species based on the 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, a refractive index detector, a fluorescence detector, etc. In other embodiments, detector 608 can be a conductivity detector or an electrochemical detector. In still other embodiments, detector 608 can be a mass spectrometer.

[0076] In various embodiments, separation column 606 generally consists of a tube filled with a stationary phase medium. The stationary phase medium can affect the time (retention time) it takes for a compound to travel through the column. This effect can be different for different compounds such that the individual components of the sample can be separated based on their individual retention times. There are a number of stationary phase media including porous materials, ionic materials, polar materials, nonpolar materials, etc. Porous materials can affect the retention time based on the size of the molecule and the ability of the molecule to enter into the porous material. Ionic materials can affect the retention time based on the charge attraction or repulsion between the ionic material and the compound. Polar and nonpolar materials can affect the retention time based on the hydrophobicity or hydrophilicity of the compound.

[0077] In various embodiments, nucleotides and nucleosides can be separated using reverse phase separation, where a hydrophobic nonpolar stationary phase material is used with a mobile phase having various hydrophobicities depending on the ratio of polarity to an organic solvent. For example, a C18 column can be used with an ammonium acetate or ammonium formate buffer system to separate nucleosides. In certain embodiments, an aqueous mobile phase of 5 mM ammonium acetate at a pH of about 5 can be used, and a gradient with an increased concentration of acetonitrile (up to about 40%) or methanol (up to about 50%) can be used to separate the nucleosides. Suitable columns and buffer systems will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0078] In other embodiments, nucleotides and nucleosides can be separated using hydrophilic interaction liquid chromatography (HILIC), where a hydrophilic stationary phase material is used with a hydrophobic mobile phase such as acetonitrile. Suitable columns and buffer systems will be apparent to those skilled in the art and are within the scope of the present disclosure.

[0079] Mass spectrometry platform Various embodiments of the mass spectrometry platform 700 can include the components shown in the block diagram of FIG. 7. In various embodiments, the mass spectrometry platform 700 can operate as the detector 608 of the system 600. In various embodiments, the elements of FIG. 7 can be incorporated into the mass spectrometry platform 700. According to various embodiments, the mass spectrometer 700 can include an ion source 702, a mass analyzer 704, an ion detector 706, and a controller 708.

[0080] In various embodiments, the ion source 702 generates a plurality of ions from a sample. The ion source can include, but is not limited to, a matrix-assisted laser desorption / ionization (MALDI) source, an electrospray ionization (ESI) source, an atmospheric pressure chemical ionization (APCI) source, an atmospheric pressure photoionization source (APPI), an inductively coupled plasma (ICP) source, an electron ionization source, a chemical ionization source, a photoionization source, a glow discharge ionization source, a thermospray ionization source, and the like.

[0081] In various embodiments, the mass analyzer 704 can separate ions based on their mass-to-charge ratio. For example, the mass analyzer 704 can include a quadrupole mass filter analyzer, a quadrupole ion trap analyzer, a time-of-flight (TOF) analyzer, an electrostatic trap (e.g., an orbitrap) mass analyzer, a Fourier transform ion cyclotron resonance (FT-ICR) mass analyzer, and the like. In various embodiments, the mass analyzer 704 can 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), and the like, and further separate the fragmented ions based on their mass-to-charge ratio.

[0082] In various embodiments, the ion detector 706 can detect ions. For example, the ion detector 706 can include an electron multiplier, a Faraday cup, and the like. The ions exiting the mass spectrometer can be detected by the ion detector. In various embodiments, the ion detector can be quantitative, and thus the exact amount of ions can be determined.

[0083] In various embodiments, the control device 708 can communicate with the ion source 702, the mass spectrometer 704, and the ion detector 706. For example, the control device 708 can configure the ion source or enable / disable the ion source. Further, the control device 708 can configure the mass spectrometer 704 to select a specific mass range to detect. Further, the control device 708 can adjust the sensitivity of the ion detector 706, such as by adjusting the gain. Further, the control device 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 can be configured to detect negative ions.

[0084] Although the present teachings are described in conjunction with various embodiments, it is not intended to limit the present teachings to such embodiments. Rather, the present teachings include various alternatives, modifications, and equivalents, as will be understood by those skilled in the art.

[0085] Further, in the description of various embodiments, this specification may present methods and / or processes as a particular sequence of steps. However, to the extent that the method or process does not depend on a particular sequence of the steps described herein, the method or process should not be limited to the particular sequence of steps described. As will be understood by those skilled in the art, other sequences of steps may be possible. Accordingly, the particular sequence of steps described herein should not be construed as a limitation in the claims. In addition, the claims directed to the method and / or process should not be limited to the execution of those steps in the written order, and it can be readily understood by those skilled in the art that the order may be changed and still remain within the spirit and scope of the various embodiments. Another aspect of the present invention may be as follows. 〔1〕An enzyme mixture comprising nuclease P1 and sweet potato acid phosphatase, A standard substance mixture comprising isotope-labeled m7G and isotope-labeled 2'-O-methylated nucleoside (Am, Gm, Cm or Um), A kit for quantifying mRNA capping efficiency, comprising: 〔2〕The kit according to 〔1〕above, further comprising a buffer solution. 〔3〕The kit according to 〔2〕above, wherein the buffer solution contains ZnCl 2 〔4〕The kit according to 〔3〕above, wherein the buffer solution has a ZnCl 2 concentration of about 0.01 mmol to about 90 mmol. 〔5〕The kit according to 〔3〕above, wherein the buffer solution has a divalent cation concentration of less than about 100 mmol. 〔6〕The kit according to 〔2〕above, wherein the buffer solution has a pH of about 2 to about 7. 〔7〕The kit according to 〔6〕above, wherein the buffer solution has a pH of about 4 to about 6. 〔8〕The kit according to 〔1〕above, wherein the enzyme mixture further comprises RNase T1. 〔9〕The kit according to 〔1〕above, wherein the enzyme mixture is a lyophilized powder. 〔10〕The kit according to 〔1〕above, wherein the standard substance mixture is a lyophilized powder. 〔11〕The kit according to 〔1〕above, wherein the isotope-labeled m7G and 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)). 〔12〕The kit according to 〔1〕above, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside are labeled with N 15 or labeled with C 13 〔13〕The kit according to 〔1〕above, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside in the standard substance mixture are present in a molar ratio of about 2:1 to about 1:2. 〔14〕The kit according to 〔13〕above, wherein the standard substance mixture comprises equimolar amounts of isotope-labeled m7G and isotope-labeled 2'-O-methylated nucleoside. 〔15〕An enzyme mixture comprising a non-specific single-stranded nuclease and acid phosphatase, A standard substance mixture comprising isotope-labeled m7G and isotope-labeled 2'-O-methylated nucleoside (Am, Gm, Cm or Um), A buffer solution, A kit for quantifying mRNA capping efficiency, comprising 〔16〕The kit according to 〔15〕, wherein the non-specific single-stranded nuclease comprises nuclease P1. 〔17〕The kit according to 〔15〕, wherein the acid phosphatase is not inhibited by adenosine monophosphate. 〔18〕The kit according to 〔15〕, wherein the acid phosphatase comprises sweet potato acid phosphatase. 〔19〕The kit according to 〔15〕, wherein the enzyme mixture comprises a site-specific single-stranded RNA endonuclease. 〔20〕The kit according to 〔19〕, wherein the site-specific single-stranded RNA endonuclease cleaves the RNA 3’ of guanosine. 〔21〕The kit according to 〔19〕, wherein the site-specific single-stranded RNA endonuclease comprises ribonuclease T1. 〔22〕The kit according to 〔15〕, wherein the enzyme mixture is a lyophilized powder. 〔23〕The kit according to 〔15〕, wherein the standard substance mixture is a lyophilized powder. 〔24〕The kit according to 〔15〕, wherein the isotope-labeled m7G and the isotope-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)). 〔25〕The kit according to 〔15〕, wherein the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside are N 15 labeled or C 13 labeled. 〔26〕The kit according to 〔15〕, wherein the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside in the standard substance mixture are present in a molar ratio of about 2:1 to about 1:2. 〔27〕The kit according to 〔26〕, wherein the standard substance mixture comprises equimolar amounts of the isotope-labeled m7G and the isotope-labeled 2’-O-methylated nucleoside. 〔28〕The kit according to 〔15〕, wherein the buffer solution comprises ZnCl in a concentration range of about 0.001 mM to about 90 mM 2 . 〔29〕The kit according to 〔28〕, wherein the buffer solution comprises ZnCl in a concentration range of about 0.05 mM to about 0.2 mM 2 . 〔30〕The kit according to 〔28〕, wherein the buffer solution comprises divalent cations of less than about 100 mM. 〔31〕The kit according to 〔15〕, wherein the buffer solution has a pH of about 2 to about 7. 〔32〕The kit according to 〔31〕, wherein the buffer solution has a pH of about 4 to about 6. Step of combining a sample, an enzyme mixture, and an isotope standard substance solution in a buffer solution to produce an incubation mixture, wherein the enzyme mixture contains a non-specific single-stranded nuclease and an acid phosphatase, and the isotope standard substance contains an isotope-labeled m7G and an isotope-labeled 2'-O-methylated nucleoside (Am, Gm, Cm, or Um). Step of incubating the mixture. Step of analyzing the mixture using liquid chromatography-mass spectrometry to determine at least one of the capping efficiency and the 2'-O-methyltransferase efficiency. A method for quantifying mRNA capping efficiency, comprising the above steps. The method according to

[33] above, wherein the incubation of the mixture is carried out at a temperature of about 30 °C to 70 °C. The method according to

[34] above, wherein the incubation of the mixture is carried out at a temperature of about 35 °C to 45 °C. The method according to

[33] above, wherein the non-specific single-stranded nuclease contains nuclease P1. The method according to

[33] above, wherein the acid phosphatase is not inhibited by adenosine monophosphate. The method according to

[33] above, wherein the acid phosphatase contains sweet potato acid phosphatase. The method according to

[33] above, wherein the enzyme mixture further contains a site-specific single-stranded RNA endonuclease. The method according to

[39] above, wherein the site-specific single-stranded RNA endonuclease cleaves the RNA 3' of guanosine. The method according to

[39] above, wherein the site-specific single-stranded RNA endonuclease contains ribonuclease T1. The method according to

[41] above, wherein the incubation of the mixture is carried out at a temperature of about 30 °C to 50 °C. The method according to

[33] above, further comprising the step of dissolving a lyophilized enzyme mixture in the sample buffer solution to obtain the enzyme mixture. The method according to

[33] above, further comprising the step of dissolving a lyophilized standard substance mixture in the sample buffer solution to obtain the standard substance mixture. The method according to

[33] above, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside contain deuterium-labeled m7G (D) and deuterium-labeled 2'-O-methylated nucleoside (Am(D), Gm(D), Cm(D), or Um(D)). 〔46〕The method according to 〔33〕, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside are N 15 labeled or C 13 labeled. 〔47〕The method according to 〔33〕, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside in the standard substance mixture are present in a molar ratio of about 2:1 to about 1:2. 〔48〕The method according to 〔47〕, wherein the standard substance mixture contains equimolar amounts of the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside. 〔49〕The method according to 〔33〕, wherein the incubation mixture contains an amount of the isotope-labeled m7G that results in about 1.0 nmol to 20 nmol. 〔50〕The method according to 〔49〕, wherein the incubation mixture contains an amount of the isotope-labeled 2'-O-methylated nucleoside that results in about 1.0 nmol to about 20 nmol. 〔51〕The method according to 〔33〕, wherein the buffer solution contains ZnCl in a concentration range of about 0.001 mM to about 90 mM 2 . 〔52〕The method according to 〔51〕, wherein the buffer solution contains ZnCl in a concentration range of about 0.05 mM to about 0.2 mM 2 . 〔53〕The method according to 〔33〕, wherein the incubation mixture contains less than 100 mM of divalent cations. 〔54〕The method according to 〔33〕, wherein the buffer solution has a pH of about 2 to about 7. 〔55〕The method according to 〔54〕, wherein the buffer solution has a pH of about 4 to about 6. 〔56〕The step of analyzing the mixture using liquid chromatography-mass spectrometry includes separating the mixture using a chromatography column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating the peaks of m7G and isotope-labeled m7G, the peaks of 2'-O-methylated nucleoside and isotope-labeled 2'-O-methylated nucleoside, or both, calculating the m7G:isotope-labeled m7G peak ratio, the 2'-O-methylated nucleoside:isotope-labeled 2'-O-methylated nucleoside peak ratio, or both, and further determining the capping efficiency based on the m7G:isotope-labeled m7G peak ratio, the 2-O-methyltransferase efficiency based on the 2'-O-methylated nucleoside:isotope-labeled 2'-O-methylated nucleoside peak ratio, or both. The method according to 〔33〕.

Claims

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

2. The method according to claim 1, wherein the incubation of the incubation mixture is carried out at a temperature of 30°C to 70°C.

3. The method according to claim 2, wherein the incubation of the incubation mixture is carried out at a temperature of 35°C to 45°C.

4. The method according to claim 1, wherein the acid phosphatase includes sweet potato acid phosphatase.

5. The method according to claim 1, wherein the enzyme mixture further contains a site-specific single-stranded RNA endonuclease.

6. The method according to claim 5, wherein the site-specific single-stranded RNA endonuclease cleaves the 3' of guanosine in RNA.

7. The method according to claim 5, wherein the site-specific single-stranded RNA endonuclease includes ribonuclease T1.

8. The method according to claim 7, wherein the incubation of the incubation mixture is carried out at a temperature of 30°C to 50°C.

9. The method according to claim 1, further comprising dissolving a lyophilized enzyme mixture in a sample buffer solution to obtain the enzyme mixture.

10. The method according to claim 1, further comprising dissolving a lyophilized standard mixture in a sample buffer solution to obtain the isotope standard solution.

11. The method according to claim 1, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside include deuterium-labeled m7G (D) and deuterium-labeled 2'-O-methylated nucleoside (Am(D), Gm(D), Cm(D), or Um(D)).

12. The isotopically labeled m7G and the isotopically labeled 2'-O-methylated nucleoside are 15 Labeled or C 13 The method of claim 1 which is labeled.

13. The method according to claim 1, wherein the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside in the isotope standard substance solution are present in a molar ratio of 2:1 to 1:

2.

14. The method according to claim 13, wherein the isotope standard substance solution contains equimolar amounts of the isotope-labeled m7G and the isotope-labeled 2'-O-methylated nucleoside.

15. The method according to claim 1, wherein the incubate mixture contains an amount of the isotope-labeled m7G that results in 1.0 nmol to 20 nmol.

16. The method according to claim 15, wherein the incubate mixture contains an amount of the isotope-labeled 2'-O-methylated nucleoside that results in 1.0 nmol to 20 nmol.

17. The buffer solution contains ZnCl in a concentration range of 0.001 mM to 90 mM 2 The method according to claim 1, comprising

18. The buffer solution contains ZnCl in a concentration range of 0.05 mM to 0.2 mM 2 The method according to claim 17, comprising this.

19. The method according to claim 1, wherein the incubate mixture contains divalent cations of less than 100 mM.

20. The method according to claim 1, wherein the buffer solution has a pH of 2 to 7.

21. The method according to claim 20, wherein the buffer solution has a pH of 4 to 6.

22. The step of analyzing the incubate mixture using liquid chromatography-mass spectrometry includes separating the incubate mixture using a chromatography column, obtaining m / z and intensity data using a mass spectrometer, identifying and integrating the peaks of m7G and the isotope-labeled m7G, the peaks of the 2'-O-methylated nucleoside and the isotope-labeled 2'-O-methylated nucleoside, or both, calculating the m7G:isotope-labeled m7G peak ratio, the 2'-O-methylated nucleoside:isotope-labeled 2'-O-methylated nucleoside peak ratio, or both, and further determining the capping efficiency based on the m7G:isotope-labeled m7G peak ratio, the 2-O-methyltransferase efficiency based on the 2'-O-methylated nucleoside:isotope-labeled 2'-O-methylated nucleoside peak ratio, or both. The method according to claim 1.

Images