Method for simultaneously identifying or quantifying capping and tailing modifications of messenger RNA.

Abstract

This disclosure provides, among other things, a single-sample multi-attribute method for simultaneously identifying and / or quantifying RNA capping and tailing modifications. This disclosure provides, among other things, oligonucleotides containing RNA and DNA bases complementary to the 5' and 3' ends of RNA. Methods for producing quantified percentages of uncapped and untailed mRNA, as well as RNA for therapeutic use with specific poly-A tail lengths and polydispersity, are provided herein. Quality control assays for production lots are also provided herein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of European Patent Application No. 23305946.8, filed on 14 June 2023, and relies on the filing date thereof, the full disclosure thereof is incorporated herein by reference.

[0002] Inclusion by referencing the sequence list The contents of the ST26 sequence listing file named "Sequence Listing.xml", created on June 12, 2024, with a size of 7,194 bytes, are incorporated herein by reference in their entirety. [Background technology]

[0003] RNA (e.g., messenger RNA, or "mRNA") therapeutics are crucial for treating a variety of diseases. The rapid development and deployment of RNA vaccines revolutionized combating the coronavirus pandemic, and RNA vaccines are becoming increasingly important for treating a wide range of diseases, including infectious diseases and cancer.

[0004] mRNA vaccines induce protein production in the patient's body, thereby priming the immune system and triggering a protective immune response in the event of exposure to a pathogen. For example, mRNA therapy requires stable and effective delivery of mRNA, as well as efficient production of the mRNA-encoded protein in the patient's body.

[0005] For stable mRNA delivery and protein production in vivo, a cap is typically required at the 5' end of the mRNA, and a tail at the 3' end. Both protect mRNA from degradation and facilitate mRNA delivery for in vivo protein translation. The development of capping modifications and polyadenosine tails (poly-A) has improved mRNA stability and translation in eukaryotic cells.

[0006] Current methods for estimating capping and tailing known in the art have several drawbacks. For example, estimation methods can lead to product loss by radioactiveizing or destroying the product during the analysis process, rendering it unsuitable for therapeutic use. Furthermore, while separate quantification reactions can be performed in parallel with therapeutic synthesis reactions, such methods are inherently variable and prone to errors. To obtain accurate results, it is preferable to use representative samples or aliquots taken from the therapeutic synthesis reaction of the product itself. [Overview of the Initiative] [Means for solving the problem]

[0007] This disclosure provides, in particular, a multi-attribute method for simultaneously identifying and quantifying mRNA capping and non-tailed species using liquid chromatography with UV detection (LC-UV), liquid chromatography combined with mass spectrometry (LC-MS), or liquid chromatography combined with UV and mass spectrometry (LC-UV-MS). The method provided herein also characterizes poly(A) tail length and polydispersity by mass spectrometry in a single sample. Therefore, this disclosure is useful for characterizing mRNA as an active pharmaceutical ingredient (API) in therapeutic products.

[0008] The method described herein is used to characterize the 5' and 3' ends of RNA, including any type of therapeutic RNA, including mRNA vaccines at different stages of the manufacturing process, i.e., in vitro transcription mRNA, in-process mRNA, and mRNA vaccines at the drug substance (DS) and drug product (DP) stages after formulation. This method is an important quality control or release assay for manufacturing mRNA products. UV signaling is particularly useful in a GMP environment. Quantification of capping and tailing species provides indicators of RNA degradation at the 3' and 5' ends.

[0009] In some embodiments, a single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications is provided, comprising: (a) providing a sample comprising RNA and optionally having a 5' cap and / or a 3' poly-A tail, wherein the sample further comprises a first oligonucleotide probe complementary to the sequence in the 5' untranslated region (5'UTR) of the RNA and a second oligonucleotide probe complementary to the sequence in the 3' untranslated region (3'UTR) of the RNA; (b) annealing the RNA sample with the first oligonucleotide probe complementary to the sequence in the 5'UTR of the RNA and the second oligonucleotide probe complementary to the sequence in the 3'UTR of the RNA; (c) treating the RNA sample from step (b) with a nuclease to cleave the RNA into cap and tail fragments; and (d) (i) obtaining the results from liquid chromatography (LC-UV) using ultraviolet detection. The method includes: (ii) identifying capping species in capping fragments by measuring the retention time of peaks in mass spectra generated by LC (LC-MS) or LC-UV-MS in combination with chromatograms and / or mass spectra; (ii) identifying non-tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS; and (iii) identifying tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS, thereby simultaneously identifying RNA capping and tailing modifications in a single sample. In some embodiments, the first base of the first oligonucleotide probe is attached to the second-to-last base of the RNA or adjacent to the second-to-last base of the RNA, for example, at least 2 to 10 nucleotides from the second-to-last base of the RNA.

[0010] In some embodiments, capping species in capped fragments are identified by measuring the retention time of peaks in chromatograms generated by LC-UV. In some embodiments, capping species in capped fragments are identified by measuring the mass of capped species using mass spectra generated by MS. In some embodiments, both peak retention time and capped species mass are measured by LC-UV-MS. In some embodiments, non-tailed and tailed species in tailed fragments are identified by measuring the retention time of peaks in chromatograms generated by LC-UV. In some embodiments, non-tailed and tailed species in tailed fragments are identified by measuring the mass generated by LC-MS. In some embodiments, non-tailed and tailed species in tailed fragments are identified by measuring the mass generated by MS. In some embodiments, both peak retention time and tailed species mass are measured by LC-UV-MS.

[0011] In some embodiments, a single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency is provided herein, the method comprising: (a) providing a sample comprising RNA and optionally having a 5' cap and / or 3' poly-A tail; (b) annealing the RNA sample with a first oligonucleotide probe complementary to the sequence of the 5' untranslated region (5'UTR) of the RNA and a second oligonucleotide probe complementary to the sequence of the 3' untranslated region (3'UTR) of the RNA; and (c) treating the RNA sample from step (b) with a nuclease. The process includes (d) cleaving the RNA into cap and tail fragments, (e) performing LC (LC-MS) or LC-UV-MS in combination with ultraviolet detection (LC-UV) or mass spectrometry, (f) measuring the peak area of ​​each capping species in the cap fragment and each non-tail species in the tail fragment, and (g) quantifying the relative amounts of each capping species and the non-tail species to characterize the poly(A) tails in the sample from step (d), thereby simultaneously quantifying the RNA capping efficiency and tailing efficiency in the sample. In some embodiments, the first base of the first oligonucleotide probe is bound to the second-to-last base of the RNA, or adjacent to the second-to-last base of the RNA, for example, at least 2 to 10 nucleotides from the second-to-last base of the RNA.

[0012] In some embodiments, methods for characterizing poly-A tails are provided herein, comprising (i) mass measurement of the poly-A tail species, deconvolution of the mass spectrometry signal, thereby providing a mass distribution of the tail species, and (ii) determining the minimum length, maximum length, average length, and polydispersity of the poly-A tail based on the corresponding mass distribution.

[0013] In some embodiments, the RNA is in vitro transcribed mRNA.

[0014] In some embodiments, RNA is obtained from the manufacturing process.

[0015] In some embodiments, RNA is obtained from the active pharmaceutical ingredient (DS) in the final step of the manufacturing process.

[0016] In some embodiments, RNA is obtained from a deformulated drug product (DP).

[0017] In some embodiments, the RNA is unmodified. In some embodiments, the RNA does not contain modified nucleotides.

[0018] In some embodiments, the RNA is modified.

[0019] In some embodiments, the nuclease is RNAse H.

[0020] In some embodiments, the masses of the cap and tail fragments are measured by LC-MS.

[0021] In some embodiments, methods provided herein are provided for identifying capping species, non-tail species, and tail species from a UV signal by comparing the retention time of the obtained peak with the retention time of a reference standard.

[0022] In some embodiments, methods are provided herein in which the capping species is cap 1, cap 0, cap G, or uncapped. In some embodiments, the capping species is cap 1. In some embodiments, the capping species is cap 0. In some embodiments, the capping species is cap G. In some embodiments, the capping species is uncapped.

[0023] In some embodiments, the capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, GGACA, or pppGGACA. In some embodiments, the capping species is m7Gpppm7GGACA. In some embodiments, the capping species is m7GpppGGACA. In some embodiments, the capping species is GpppGGACA. In some embodiments, the capping species is GGACA. In some embodiments, the capping species is pppGGACA.

[0024] In some embodiments, the uncapped species is GGACA or pppGGACA.

[0025] In some embodiments, the non-tail species is UGCAUC, where U is unmodified uridine.

[0026] In some embodiments, the non-tail species is U*GCAU*C, where U* is N1-methylpseudolidine.

[0027] In some embodiments, the relative amount of each capping species is a percentage of the total amount of capping species in the sample, calculated by dividing the area under the peak of the capping species of interest by the sum of the total areas under the peak representing cap 1, cap 0, cap G, and uncapped species, and multiplying by 100.

[0028] In some embodiments, the relative amount of non-tail species is a percentage calculated by dividing the area under the peak of non-tail species by the sum of the areas under the peak of cap 1, cap 0, cap G, and uncapped species, and multiplying by 100.

[0029] In some embodiments, the oligonucleotide is about 10 to 40 nucleotides long. In some embodiments, the oligonucleotide is about 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 35 to 40 nucleotides long, including all distinct mediating amounts. In some embodiments, the oligonucleotide is 10 nucleotides long. In some embodiments, the oligonucleotide is 11 nucleotides long. In some embodiments, the oligonucleotide is 12 nucleotides long. In some embodiments, the oligonucleotide is 13 nucleotides long. In some embodiments, the oligonucleotide is 14 nucleotides long. In some embodiments, the oligonucleotide is 15 nucleotides long. In some embodiments, the oligonucleotide is 16 nucleotides long. In some embodiments, the oligonucleotide is 17 nucleotides long. In some embodiments, the oligonucleotide is 18 nucleotides long. In some embodiments, the oligonucleotide is 19 nucleotides long. In some embodiments, the oligonucleotide is 20 nucleotides long. In some embodiments, the oligonucleotide is 21 nucleotides long. In some embodiments, the oligonucleotide is 22 nucleotides long. In some embodiments, the oligonucleotide is 23 nucleotides long. In some embodiments, the oligonucleotide is 24 nucleotides long. In some embodiments, the oligonucleotide is 25 nucleotides long. In some embodiments, the oligonucleotide is 26 nucleotides long. In some embodiments, the oligonucleotide is 27 nucleotides long. In some embodiments, the oligonucleotide is 28 nucleotides long. In some embodiments, the oligonucleotide is 29 nucleotides long. In some embodiments, the oligonucleotide is 30 nucleotides long. In some embodiments, the oligonucleotide is 31 nucleotides long. In some embodiments, the oligonucleotide is 32 nucleotides long. In some embodiments, the oligonucleotide is 33 nucleotides long.In some embodiments, the oligonucleotide is 34 nucleotides long. In some embodiments, the oligonucleotide is 35 nucleotides long. In some embodiments, the oligonucleotide is 36 nucleotides long. In some embodiments, the oligonucleotide is 37 nucleotides long. In some embodiments, the oligonucleotide is 38 nucleotides long. In some embodiments, the oligonucleotide is 39 nucleotides long. In some embodiments, the oligonucleotide is 40 nucleotides long.

[0030] In some embodiments, the oligonucleotide is about 10 to 40 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is about 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 35 to 40 nucleotides long, including all distinct intermediaries, and contains 4 DNA bases. In some embodiments, the oligonucleotide is 10 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 11 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 12 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 13 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 14 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 15 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 16 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 17 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 18 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 19 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 20 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 21 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 22 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 23 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 24 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 25 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 26 nucleotides long and contains 4 DNA bases.In some embodiments, the oligonucleotide is 27 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 28 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 29 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 30 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 31 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 32 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 33 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 34 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 35 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 36 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 37 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 38 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 39 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 40 nucleotides long and contains 4 DNA bases.

[0031] In some embodiments, the oligonucleotide includes RNA and DNA bases.

[0032] In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 10:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 9:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 8:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 7:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 6:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 5:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 4:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 3:1.

[0033] In some embodiments, the oligonucleotide is 10 to 40 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide, including all distinct intermediaries, is 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 35 to 40 nucleotides long and contains 4 DNA bases.

[0034] In some embodiments, oligonucleotides complementary to the sequence of the 5' untranslated region of RNA are used. [ka] Therefore, the italicized CTGT represents a DNA base.

[0035] In some embodiments, oligonucleotides complementary to the sequence of the 3' untranslated region of RNA are: [ka] Therefore, the italicized TTCA represents a DNA base.

[0036] In some embodiments, capping species and non-tailed species are quantified by single LC-UV, LC-MS, or LC-UV-MS analysis. In some embodiments, capping species and non-tailed species are quantified by single LC-UV analysis. In some embodiments, capping species and non-tailed species are quantified by single LC-MS analysis. In some embodiments, capping species and non-tailed species are quantified by single LC-UV-MS analysis. In some embodiments, capping species and non-tailed species are measured by ultrafast liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS).

[0037] In some embodiments, the tail species is characterized by single LC-MS or LC-UV-MS analysis. In some embodiments, the tail species is characterized by single LC-MS analysis. In some embodiments, the tail species is characterized by single LC-UV-MS analysis.

[0038] In some embodiments, one or more processes are automated. In some embodiments, one process is automated. In some embodiments, two or more processes are automated. In some embodiments, all processes are automated.

[0039] In some embodiments, oligonucleotides complementary to the 5'UTR or 3'UTR sequence of RNA are provided herein, wherein the oligonucleotide is about 10 to 40 nucleotides long and contains RNA bases and DNA bases. In some embodiments, oligonucleotides complementary to the 5'UTR sequence of RNA are provided herein, wherein the oligonucleotide is about 10 to 40 nucleotides long and contains RNA bases and DNA bases. In some embodiments, oligonucleotides complementary to the 3'UTR sequence of RNA are provided herein, wherein the oligonucleotide is about 10 to 40 nucleotides long and contains RNA bases and DNA bases.

[0040] In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 10:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 9:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 8:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 7:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 6:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 5:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 4:1. In some embodiments, the oligonucleotide contains RNA bases and DNA bases in a ratio of about 3:1.

[0041] In some embodiments, the oligonucleotide is about 10 to 40 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is about 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, or 35 to 40 nucleotides long and contains 4 DNA bases.

[0042] In some embodiments, oligonucleotides complementary to the sequence within the 5'UTR of RNA are used. [ka] Therefore, the italicized CTGT represents a DNA base.

[0043] In some embodiments, oligonucleotides complementary to the sequence within the 3'UTR of RNA are used. [ka] Therefore, the italicized TTCA represents a DNA base.

[0044] In some embodiments, methods for producing RNA having quantified percentage capped and untailed mRNA are provided herein, comprising: (a) providing in vitro synthesized RNA; (b) annealing the in vitro synthesized RNA with a first oligonucleotide complementary to the sequence in the 5'UTR of the RNA and a second oligonucleotide complementary to the sequence in the 3'UTR of the RNA; (c) treating the RNA with a nuclease to cleave the RNA into capped and tailed fragments; and (d) identifying capped and untailed species by liquid chromatography with UV detection (LC-UV), and / or The method includes (e) identifying and measuring the mass of capping species and non-tailed species by liquid chromatography combined with mass spectrometry (LC-MS) and / or UV detection, and characterizing the poly(A) tail; and (e) quantifying the relative amount of each capping species, quantifying the relative amount of non-tailed species using LC-UV or LC-MS or LC-UV-MS, and characterizing the poly(A) tail using LC-MS or LC-UV-MS simultaneously in a single sample, thereby producing RNA containing the quantified percentage of capped mRNA and non-tailed mRNA. In some embodiments, the first base of the first oligonucleotide is attached to the second-to-last base of the RNA, or adjacent to the second-to-last base of the RNA, for example, at least 2 to 10 nucleotides from the second-to-last base of the RNA.

[0045] In some embodiments, a method is provided herein in which a 5' cap is enzymatically added to the RNA from step (a) after transcription.

[0046] In some embodiments, the RNA does not contain a 5' cap.

[0047] In some embodiments, the 3' tail is encoded in the plasmid during in vitro synthesis.

[0048] In some embodiments, a 3' tail is enzymatically added to the RNA from step (a) after transcription.

[0049] In some embodiments, the RNA does not contain a 3' tail.

[0050] In some embodiments, RNA is not spliced.

[0051] In some embodiments, RNA is spliced.

[0052] In some embodiments, a manufacturing lot quality control assay is provided herein, which includes (a) providing a manufactured RNA sample containing a 5' cap and / or 3' tail, (b) quantifying the RNA capping efficiency and tailing efficiency in the sample by a method provided herein, and (c) comparing the capping and tailing efficiency in step (b) with the capping and tailing efficiency in a reference sample, wherein a decrease in the amount of capping species compared to the reference sample indicates 5' degradation, and an increase in the amount of non-tailed species and / or different poly-A tail lengths or polydispersity indicates 3' degradation, thereby simultaneously monitoring 5' and 3' degradation of the RNA product and determining the product quality of the manufactured lot.

[0053] In some embodiments, quality control assays are provided herein in which the reference sample is a stable RNA sample having more than 75% integrity. In some embodiments, quality control assays are provided herein in which the reference sample is a stable RNA sample having 75% to 100% integrity. In some embodiments, quality control assays are provided herein in which the reference sample is a stable RNA sample having 75% to 99% integrity. In some embodiments, quality control assays are provided herein in which the reference sample is a stable RNA sample having 75% to 95% integrity. In some embodiments, quality control assays are provided herein in which the reference sample is a stable RNA sample having 75% to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100% integrity, including all intervening amounts.

[0054] In some embodiments, the RNA product is therapeutic mRNA.

[0055] In some embodiments, the RNA product is an mRNA vaccine.

[0056] The drawings are for illustrative purposes only and are not intended to be limiting. [Brief explanation of the drawing]

[0057] [Figure 1] This figure shows a flowchart illustrating the steps of the method of this disclosure, which includes annealing a hybrid RNA oligonucleotide containing four DNA bases to the 5' and 3' ends of an mRNA sample in an annealing step, followed by the addition of ribonuclease H (RNAse H) enzyme and shrimp alkaline phosphatase (rSAP) to digest the mRNA of the capped and uncapped RNA:DNA hybrid analytes, and subsequent analysis. Two exemplary hybrid oligonucleotides are as follows: HO2.16 is a 16nts custom-designed hybrid oligonucleotide. [ka] The bold CTGT near the 3' end represents a DNA base; HO2.27 is a 27-nt custom-designed hybrid oligonucleotide. [ka] The bolded TTCA near the 5' end represents a DNA base. [Figure 2] This diagram shows a flowchart of the annealing process of this method. RNA from exemplary samples and exemplary custom hybrid oligonucleotides is heated to 75°C to induce denaturation and strand separation. As the temperature decreases from 75°C to 23°C, hydrogen bonds are formed between the complementary DNA sequence and the RNA sequence. Complementary base pairing between the hybrid oligonucleotide and mRNA in specific regions results in RNA:DNA hybrid formation at the end of the annealing process. [Figure 3] This diagram shows a flowchart of the digestion process in this method. Digestion of RNA:DNA hybrids is performed by incubating RNAse H and alkaline phosphatase (rSAP) at 37°C. The RNAse H enzyme cleaves the phosphodiester bonds between the double-stranded RNA:DNA hybrids generated in the annealing step. Alkaline phosphatase (rSAP) catalyzes terminal dephosphorylation to prevent self-ligation. [Figure 4A]Figure 4A shows an exemplary extracted ion chromatogram (EIC) illustrating the peaks generated for capped species (cap 0, cap 1, cap G), uncapped species, and untailed species. A quantification method is also provided. The percentage of any particular capped species (cap 0, cap 1, cap G, or uncapped) is calculated by dividing the area under the curve of the capped species by the sum of the areas under the curves of all capped peaks (cap 0, cap 1, cap G) and uncapped peaks, and multiplying by 100. Similarly, the percentage of untailed species is calculated by dividing the area under the curve of the untailed species by the sum of the total areas under the curves of all capped peaks (cap 0, cap 1, cap G) and uncapped peaks, and multiplying by 100. In the exemplary EIC shown in Figure 4A, the quantification results demonstrate the presence of 6.8% untailed, 4.2% uncapped, 0.4% cap G, 0.0% cap 0, and 95.4% cap 1 species. In some embodiments, EIC is generated by, for example, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-ultraviolet spectroscopy / mass spectrometry (LC-UV / MS), ultrafast liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS), or other mass spectrometry methods. [Figure 4B] This is a schematic flowchart illustrating the synthesis of exemplary mRNA capped and uncapped structures. Uncapped mRNA (e.g., in vitro transcribed mRNA, pppG-mRNA) loses one phosphate group upon dephosphorylation. Subsequent treatment with guanylyltransferase synthesizes capped G (GpppG-mRNA). In the presence of S-(5'-adenosyl)-L-methionine p-toluenesulfonate (SAM-TOS) and N7-methyltransferase, the capped G structure is converted to capped O structure (m7GpppG-mRNA). In the presence of 2'-O-ribose-methyltransferase and SAM-TOS, the capped O structure is converted to capped O structure (m7GpppmG-mRNA). [Figure 5A]For an exemplary drug substance, the mean deconvolution mass spectrum of an exemplary mRNA used to study poly-A tailing is shown. In the graph, the mass spectrometry signal intensity is plotted against m / z (mass-to-charge ratio). [Figure 5B] This graph shows the percentage of mass spectrometry signal intensity against poly(A) tail length. The results indicate the average poly(A) tail length and poly(A) polydispersity in a representative mRNA sample of the active pharmaceutical ingredient.

[0058] definition To facilitate understanding of this disclosure, certain terms are defined first. Further definitions of the following terms and other terms are provided throughout this specification.

[0059] Affinity: As is known in the art, "affinity" is a measure of the tightness to which a particular ligand binds (e.g., non-covalently associates) and / or the rate or frequency of dissociation from its partner. As is known in the art, affinity can be determined using any of the following techniques. In many embodiments, affinity represents a measure of specific binding.

[0060] Annealing or Hybridization: As used herein, the terms “annealing,” “hybridization,” and their grammatical equivalents refer to the formation of a complex (also called a double helix or hybrid) between nucleotide sequences that are sufficiently complementary to form a complex via Watson-Crick base pairing or non-standard base pairing. It will be understood that the annealing or hybridizing sequences do not need to have perfect complementarity to provide a stable hybrid. In many situations, a stable hybrid will be formed with less than about 10% of the bases being mismatched. Thus, as used herein, the term “complementary” generally refers to nucleic acid molecules that, under certain conditions, have about 90% or more homology (e.g., about 95% or more, about 98% or more, or about 99% or more homology) to form a stable double helix with their complement. Those skilled in the art will understand how to estimate and adjust the stringency of hybridization conditions so that sequences with at least a desired level of complementarity stably hybridize, but sequences with lower levels of complementarity do not. For examples of hybridization conditions and parameters, see, for example, Sambrook et al., "Molecular Cloning: A Laboratory Manual", 1989, Second Edition, Cold Spring Harbor Press: Plainview, NY and Ausubel, "Current Protocols in Molecular Biology", 1994, John Wiley & Sons: Secaucus, NJ. Complementarity between two nucleic acid molecules is said to be "complete," "whole," or "perfect" if all the nucleic acid bases match, and "partial" if they do not.

[0061] Approximately: As used herein, the terms “approximately” or “about” applied to one or more values ​​of interest refer to values ​​that are similar to the stated reference values. In certain embodiments, unless otherwise specified or evident from the context (except where such numbers exceed 100% of the possible values), the terms “approximately” or “about” refer to a range of values ​​that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less (including all values ​​and subranges in between) in either direction (greater than or less than) the stated reference values.

[0062] Chromatography: As used herein, the term “chromatography” refers to a technique for separating mixtures. Typically, a mixture is dissolved in a fluid called a “mobile phase” that carries it through a structure that holds another material called a “stationary phase.” Column chromatography is a separation technique in which the fixed bed is located in a tube, or column.

[0063] Compounds and Drugs: The terms “compound” and “drug” are used interchangeably herein. They refer to any natural or non-natural (i.e., synthetic or recombinant) molecules, such as biological macromolecules (e.g., nucleic acids, polypeptides, or proteins), organic or inorganic molecules, or extracts made from biological materials such as cells or tissues of bacteria, plants, fungi, or animals (especially mammals, including humans). Compounds may be a single molecule or a mixture or complex of at least two molecules.

[0064] Control, Standard, or Reference: Where used herein, the terms “control,” “standard,” or “reference,” used interchangeably, have the meanings understood in the art of which a result is a standard of a known value or quantity compared to which a result is compared. Typically, a control is used to enhance the integrity of an experiment by isolating the variable in order to draw conclusions about such a variable. In some embodiments, a control is a reaction or assay performed concurrently with the test reaction or assay to provide a comparison. In one experiment, the “test” (i.e., the variable being tested) is applied. In a second experiment, the “control,” the variable being tested, is not applied. In some embodiments, the control is a historical control (i.e., a previously performed test or assay, or a previously known quantity or result). In some embodiments, the control is or includes a printed or otherwise preserved record. A control may be a positive control or a negative control. Extracted Ion Chromatogram: An extracted ion chromatogram (EIC) is created by plotting the intensity of the signal observed at a selected mass-charge value or a set of values ​​within a series of mass spectra recorded as a function of retention time.

[0065] Kit: As used herein, the term “kit” refers to any delivery system for delivering materials. Such a delivery system may include a system that enables the storage, transport, or delivery of various diagnostic or therapeutic reagents (e.g., oligonucleotides, antibodies, enzymes, etc. in appropriate containers) and / or supporting materials (e.g., buffers, written instructions for performing assays, etc.) from one place to another. For example, a kit includes one or more enclosures (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term “fragmented kit” refers to a delivery system comprising two or more separate containers, each containing a small portion of the entire kit components. The containers may be delivered together or separately to intended recipients. For example, the first container may contain an enzyme for use in an assay, and the second container may contain an oligonucleotide. The term “fragmented kit” is intended to include, but is not limited to, kits containing analysis-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system comprising two or more separate containers, each containing a portion of the entire kit components, falls under the term “fragmented kit.” In contrast, a “combined kit” refers to a delivery system that contains all components in a single container (for example, in a single box containing each of the desired components). The term “kit” includes both fragmented kits and combined kits.

[0066] Nucleoside: The terms “nucleoside” or “nucleic acid base” as used herein refer to adenine ("A"), guanine ("G"), cytosine ("C"), uracil ("U"), thymine ("T"), and their analogues linked to a carbohydrate, e.g., D-ribose (in RNA) or 2'-deoxy-D-ribose (in DNA), via an N-glycosidic bond between the anomeric carbon (the 1'-carbon atom of the carbohydrate) and the nucleic acid base. When the nucleic acid base is a purine, e.g., A or G, the ribose sugar is generally attached to the N9 position of the purine heterocycle. When the nucleic acid base is a pyrimidine, e.g., C, T, or U, the sugar is generally attached to the N1 position of the heterocycle. Carbohydrates may or may not be substituted. Substituted ribose sugars include, but are not limited to, those in which one or more carbon atoms, for example, a 2'-carbon atom, are substituted with one or more identical or different Cl, F, -R, --OR, --NR2, or halogen groups, and each R can independently be H, C1-C6 alkyl, or C5-C 14 These are aryl riboses. Examples of riboses include ribose, 2'-deoxyribose, 2',3'-dideoxyribose, 2'-halolibose, 2'-fluororibose, 2'-chlororibose, and 2'-alkylriboses, e.g., 2'-O-methyl, 4'-alpha-anomeric nucleotides, 1'-alpha-anomeric nucleotides (Asseline et al., Nucleic Acids Research, 19:4067-74

[1991] ), 2'-4'- and 3'-4'-links and other "locked" or "LNAs", bicyclic sugar modifications (International Publication No. 98 / 22489; International Publication No. 98 / 39352; International Publication No. 99 / 14226).

[0067] Nucleotides: As used herein, the term “nucleotide” refers to a phosphorylated nucleoside (a phosphate ester of a nucleoside), a monomer unit, or a polynucleotide polymer. “5'-triphosphate nucleotide” refers to a nucleotide having a triphosphate ester group at the 5' position, and is sometimes called “NTP,” “dNTP,” or “ddNTP,” particularly highlighting the structural features of the ribose sugar. The triphosphate ester group may contain various oxygen moieties, such as sulfur substitutions to α-thionucleotide 5'-triphosphate. Nucleotides can exist in mono-, di-, or tri-phosphorylated forms. The carbon atoms of ribose present in nucleotides are denoted by a prime symbol (') to distinguish them from the skeletal numbering in bases. For a review of the chemistry of polynucleotides and nucleic acids, see Shabarova, Z. and Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids, VCH, New York, 1994.

[0068] Nucleic acids: The terms “nucleic acid,” “nucleic acid molecule,” “polynucleotide,” or “oligonucleotide” may be used interchangeably herein. They refer to polymers of nucleotide monomers or analogues, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and combinations thereof. Nucleotides may be of genomic, synthetic, or semi-synthetic origin. Unless otherwise specified, this term encompasses nucleic acid-like structures with synthetic backbones, as well as amplification products. As will be understood by those skilled in the art, the length of these polymers (i.e., the number of nucleotides they contain) can often vary considerably depending on their intended function or use. Polynucleotides are also H + NH4 + , trialkylammonium, Mg + kaNa + They have associated counterions such as the following. Polynucleotides may consist entirely of deoxyribonucleotides, entirely of ribonucleotides, or as a chimeric mixture thereof. Polynucleotides may consist of internucleotide nucleic acid bases and sugar analogs.

[0069] Oligonucleotides: The term “oligonucleotide” is used herein to refer to polynucleotides containing about 10 to about 40 nucleotides, about 15 to about 40 nucleotides, or about 15 to about 30 nucleotides. Throughout this specification, whenever an oligonucleotide is represented by a sequence of letters (for example, selected from four base letters representing adenosine, cytidine, guanosine, and thymidine, respectively: A, C, G, and T), the nucleotides are represented in a 5' to 3' order from left to right.

[0070] Polynucleotide: The term "polynucleotide sequence" refers to a sequence of nucleotide monomers containing a polymer. Unless otherwise indicated, whenever a polynucleotide sequence is expressed, the nucleotides should be understood to be oriented from left to right, 5' to 3'.

[0071] Modified nucleotides: Nucleic acids, polynucleotides, and oligonucleotides may consist of standard nucleotide bases or may be substituted with nucleotide isoform analogs, including but not limited to iso-C and iso-G bases, which may hybridize more or less acceptablely than standard bases and preferentially hybridize with complementary isoform analog bases. Many such isoform bases are described, for example, by Benner et al., (1987) Cold Spring Harb. Symp. Quant. Biol. 52, 53-63. Naturally occurring analogues of nucleotide monomers include, for example, 7-deazaadenine, 7-deazaguanine, 7-deaza-8-azaguanine, 7-deaza-8-azaadenine, 7-methylguanine, inosine, nebularine, nitropyrrole (Bergstrom, J. Amer. Chem. Soc., 117:1201-1209

[1995] ), nitroindole, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, pseudouridine, pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine, isoguanine (Seela, U.S. Patent No. 6,147,199), 7- Deazaguanine (Seela, U.S. Patent No. 5,990,303), 2-Azapurine (Seela, International Publication No. 01 / 16149), 2-Thiopyrimidine, 6-Thioguanine, 4-Thiothymine, 4-Thiouracil, 0-6-Methylguanine, N-6-Methyladenine, O-4-Methylthymine, 5,6-Dihydrothymine, 5,6-Dihydrouracil, 4-Methylindole, Pyrazolo[3,4-D]pyrimidine, "PPG" (Meyer, U.S. Patent Nos. 6,143,877 and 6,127,121; Gall, International Publication No. 01 / 38584), and Etenoadenine (Fasman (1989) One example is "Practical Handbook of Biochemistry and Molecular Biology," pp. 385-394, CRC Press, Boca Raton, Fla.

[0072] "3' end" and "3' terminus": As used herein, these terms refer to the end of a nucleic acid containing a free hydroxyl group bonded to the 3' carbon of the terminal pentose sugar.

[0073] "5' end" and "5' terminus": As used herein, these terms refer to the end of a nucleic acid molecule that contains a free hydroxyl group or phosphate group bonded to the 5' carbon of a terminal pentose sugar.

[0074] Target: As used herein, the term "target" refers to the molecule of interest. [Modes for carrying out the invention]

[0075] The increasing importance of RNA therapeutics and RNA vaccines for treating various cancers and infectious diseases necessitates accurate determination of product quality and stability. One of the biggest challenges in RNA therapeutics and RNA vaccines is stability, as single-stranded RNA is highly susceptible to degradation. The 5' cap and 3' tail protect RNA, such as mRNA, from exonucleases, thereby enhancing mRNA stability.

[0076] To ensure high stability standards and batch-to-batch consistency, the integrity of mRNA products must be accurately assessed, and quality control, for example, for releasing manufacturing lots, must ensure that degradation is minimized or eliminated entirely. mRNA stability also needs to be assessed after various periods of storage and transport, and after thawing frozen drugs or mRNA vaccines stored at -70°C, -20°C, to higher temperatures, such as 4°C or room temperature. Furthermore, accurately determining mRNA integrity and stability is crucial for administration. Accurate identification, characterization, and quantification of mRNA capping and tailing are important for quality control in determining the in vivo safety and efficacy of mRNA therapeutics. Identification, characterization, and quantification of mRNA capping and tailing provide a measure of mRNA quality and stability.

[0077] As described in detail below, this disclosure is in part based on a simultaneous qualitative identification and quantification method of capping and tailing by chromatography. Accordingly, this disclosure provides a simple, reliable, and efficient approach for simultaneously evaluating mRNA capping and tailing efficiency in the same sample, and in particular provides a method for producing RNA with quantified capping and tailing efficiency and a quality control method for mRNA therapy.

[0078] This disclosure provides, in particular, a multi-attribute method for simultaneously characterizing or quantifying mRNA capping and tailing efficiency from a single sample preparation. The method for identifying and quantifying mRNA capping and tailing is especially important for evaluating the quality and stability of mRNA in mRNA therapies, including mRNA vaccines.

[0079] This disclosure provides, in particular, a single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications, the method comprising: (a) providing a sample comprising RNA, optionally having a 5' cap and / or a 3' poly-A tail, the sample further comprising a first oligonucleotide probe complementary to the sequence in the 5' untranslated region (5'UTR) of the RNA and a second oligonucleotide probe complementary to the sequence in the 3' untranslated region (3'UTR) of the RNA; (b) annealing the RNA sample with the first oligonucleotide probe complementary to the sequence in the 5'UTR of the RNA and the second oligonucleotide probe complementary to the sequence in the 3'UTR of the RNA; (b) treating the RNA sample from step (b) with a nuclease to cleave the RNA into cap and tail fragments; and (c) (i) a chromatogram generated from LC-UV and / or LC-MS or The method includes: (ii) identifying capping species in capping fragments by measuring the retention time of peaks in mass spectra generated by LC-UV-MS; (ii) identifying non-tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS; and (iii) identifying tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS, using the sample from step (c), thereby simultaneously identifying RNA capping and tailing modifications in a single sample. In some embodiments, the first base of the first oligonucleotide probe is bound to the second-to-last base of the RNA, or adjacent to the second-to-last base of the RNA, for example, at least 2 to 10 nucleotides from the second-to-last base of the RNA.

[0080] In some embodiments, capping species in capped fragments are identified by measuring the retention time of peaks in chromatograms generated by LC-UV. In some embodiments, capping species in capped fragments are identified by measuring the mass of capped species using mass spectra generated by MS. In some embodiments, both peak retention time and capped species mass are measured by LC-UV-MS. In some embodiments, non-tailed and tailed species in tailed fragments are identified by measuring the retention time of peaks in chromatograms generated by LC-UV. In some embodiments, non-tailed and tailed species in tailed fragments are identified by measuring the mass of capped species using mass spectra generated by MS. In some embodiments, both peak retention time and tailed species mass are measured by LC-UV-MS.

[0081] A single-sample method for simultaneously quantifying RNA capping and tailing efficiency is provided herein, comprising: (a) providing a sample containing RNA and optionally having a 5' cap and / or 3' poly-A tail; (b) annealing the RNA sample with a first oligonucleotide probe complementary to the sequence of the 5' untranslated region (5'UTR) of the RNA and a second oligonucleotide probe complementary to the sequence of the 3' untranslated region (3'UTR) of the RNA; and (c) treating the RNA sample from step (b) with a nuclease to cap and tail the RNA. The process includes (d) cutting into tail fragments, (e) performing LC (LC-MS) or LC-UV-MS in combination with liquid chromatography-UV (LC-UV) or mass spectrometry using UV detection, (f) measuring the peak area of ​​each capping species in the cap fragment and each non-tail and tail species in the tail fragment, and (g) quantifying the relative amount of each capping species and the relative amount of the non-tail species to characterize the poly(A) tail in the sample from step (d), thereby simultaneously quantifying the RNA capping efficiency and tailing efficiency in the sample. In some embodiments, the first base of the first oligonucleotide probe is bound to the second-to-last base of the RNA, or adjacent to the second-to-last base of the RNA, for example, at least 2 to 10 nucleotides from the second-to-last base of the RNA.

[0082] Various embodiments of this disclosure are useful for identifying and quantifying capping and tailing modifications during in vitro mRNA synthesis and other stages of mRNA product development and release, such as in-process mRNA during manufacturing, active pharmaceutical ingredients, and post-formulation formulations, for use in quality control or release assays. Therefore, this disclosure provides an important quality control approach for manufacturing mRNA, particularly for evaluating the safety, efficacy, and commercial feasibility of mRNA with therapeutic applications. The use of the methods in release assays provides lot-to-lot or batch-to-batch consistency, ensuring product quality of mRNA therapeutics, such as mRNA vaccines.

[0083] Various aspects of this disclosure are described in detail in the following sections. The use of sections is not intended to limit this disclosure. Each section may apply to any aspect of this disclosure. In this application, the use of "or" means "and / or" unless otherwise stated.

[0084] As used in this application, the terms “about” and “approximately” are to be used interchangeably. Any figures used in this application are intended to cover any normal variation understood by those skilled in the art, either approximately or without approximately.

[0085] Other features, purposes, and advantages of this disclosure will become apparent in the following detailed description. However, it should be understood that the detailed description illustrates, but is not limiting to, embodiments of this disclosure. Various changes and modifications within the scope of this disclosure will become apparent to those skilled in the art from the detailed description.

[0086] Hybrid oligonucleotides The methods of the present disclosure rely, in particular, on the design and synthesis of hybrid oligonucleotides specific to targets complementary to the 5' or 3' end of RNA, thereby annealing to the complementary sequence at the 5' or 3' end, respectively. The hybrid oligonucleotides of the present disclosure comprise RNA and DNA bases and provide specificity for the subsequent digestion of RNA.

[0087] Typically, oligonucleotides are about 10–40 nucleotides long and contain RNA and DNA bases. In some embodiments, oligonucleotides are about 10–40 nucleotides long. In some embodiments, oligonucleotides are about 10–15, 15–20, 20–25, 25–30, or 35–40 nucleotides long, including all distinct mediating amounts.

[0088] In some embodiments, the oligonucleotide is about 10 to 40 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is about 10 to 40 nucleotides long and contains more than 4 DNA bases. In some embodiments, the oligonucleotide is about 10 to 40 nucleotides long and contains 4 to 10 DNA bases. In some embodiments, the oligonucleotide is about 15 to 30 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is about 15 to 30 nucleotides long and contains more than 4 DNA bases. In some embodiments, the oligonucleotide is about 15 to 30 nucleotides long and contains 4 to 10 DNA bases. In some embodiments, the oligonucleotide is 15 to 18 nucleotides, 18 to 22 nucleotides, 22 to 25 nucleotides, or 25 to 30 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 15 to 18 nucleotides, 18 to 22 nucleotides, 22 to 25 nucleotides, or 25 to 30 nucleotides long and contains more than 4 DNA bases. In some embodiments, the oligonucleotide is 15-18 nucleotides, 18-22 nucleotides, 22-25 nucleotides, or 25-30 nucleotides long and contains 4-10 DNA bases. In some embodiments, the oligonucleotide is 10-40 nucleotides long and contains 4 DNA bases. In some embodiments, the oligonucleotide is 10-40 nucleotides long and contains more than 4 DNA bases. In some embodiments, the oligonucleotide is 10-40 nucleotides long and contains 4-10 DNA bases. In some embodiments, including all distinct intermediaries, the oligonucleotide is 10-15, 15-20, 20-25, 25-30, or 35-40 nucleotides long and contains 4 DNA bases. In some embodiments, including all distinct intermediaries, the oligonucleotide is 10-15, 15-20, 20-25, 25-30, or 35-40 nucleotides long and contains more than 4 DNA bases.In some embodiments, the oligonucleotides, including all distinct intermediaries, are 10-15, 15-20, 20-25, 25-30, or 35-40 nucleotides long and contain 4-10 DNA bases.

[0089] The oligonucleotides of this disclosure contain RNA bases and DNA bases in specific ratios, for example, oligonucleotides contain RNA bases and DNA bases in ratios of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 10:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 9:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 8:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 7:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 6:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 5:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 4:1. In some embodiments, oligonucleotides contain RNA bases and DNA bases in a ratio of about 3:1. In some embodiments, the oligonucleotide comprises RNA bases and DNA bases in any ratio suitable for the method of the present invention.

[0090] In some embodiments, a hybrid oligonucleotide complementary to the sequence of the 5' untranslated region of mRNA adjacent to capped or uncapped mRNA is added to an mRNA sample containing capped and uncapped mRNA under conditions that allow the oligonucleotide to anneal to a specific sequence at the 5' end containing the untranslated region. In some embodiments, the oligonucleotide complementary to the sequence of the 5' untranslated region of RNA is, [ka] Therefore, the italicized CTGT represents a DNA base.

[0091] In some embodiments, a hybrid oligonucleotide complementary to the sequence of the 3' untranslated region of mRNA is added to an mRNA sample containing poly(A) tail and / or untailed mRNA under conditions that allow the oligonucleotide to anneal to a specific sequence at the 3' end containing the untranslated region. In some embodiments, the oligonucleotide complementary to the sequence of the 3' untranslated region of RNA is [ka] Therefore, the italicized TTCA represents a DNA base.

[0092] Embodiments of the present disclosure are not limited by the type or size of oligonucleotides. In some embodiments, the oligonucleotides include 10 to 40 nucleotides, for example, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 35 nucleotides, 40 nucleotides, and all individual intermediary amounts.

[0093] The size of the oligonucleotide can be selected to produce a capped fragment (if any) of the desired length. Oligonucleotides can also be designed to hybridize to any region of the 5' untranslated region, depending on where cleavage is desired. That is, they can be positioned within the 5' untranslated region to produce a capped fragment (if any) of any size. In particular, a well-designed oligonucleotide containing small stretches of DNA bases (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 nucleotides) flanked by RNA bases (e.g., 1-15) on both sides (i.e., the "gapmer") can be annealed to an mRNA analyte. By designing such oligonucleotides to bind to complementary bases in the 5' untranslated region of mRNA, selective cleavage via DNA / RNA hybrid recognition by a nuclease, e.g., RNAse H, becomes possible. Similarly, a well-designed oligonucleotide may contain a small stretch of DNA bases (e.g., approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 nucleotides) flanked only at one end (e.g., the 5' or 3' end) by RNA bases (e.g., 1–15).

[0094] In some embodiments, the oligonucleotide is designed to bind directly adjacent to the cap and / or second-to-last base of the mRNA, allowing the resulting cleaved base to consist of the first 1, 2, 3, 4 (or a small number, e.g., up to 15) bases of the mRNA construct. While we do not wish to be bound to any particular theory, the small capped and uncapped fragments are intended to improve the resolution of chromatographic separation of capped and uncapped fragments, thereby improving the accuracy of identification and / or quantification of capped and uncapped species. Generally, the smaller the fragment, the better the separation and quantification. In some embodiments, the first base of the oligonucleotide binds to the second-to-last base of the mRNA adjacent to the cap. In some embodiments, the first base of the oligonucleotide binds adjacent to the second-to-last base of the mRNA. In some embodiments, the first base of the oligonucleotide binds to at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, at least six nucleotides, at least seven nucleotides, at least eight nucleotides, at least nine nucleotides, or at least ten nucleotides from the second-to-last base of the mRNA.

[0095] Annealing of hybrid oligonucleotides to RNA samples The method of the present disclosure includes the step of annealing an RNA sample with a first oligonucleotide probe complementary to the 5'UTR sequence of the RNA and a second oligonucleotide probe complementary to the 3'UTR sequence of the RNA.

[0096] In some embodiments, RNA from exemplary samples and exemplary custom hybrid oligonucleotides is heated to 75°C for 10 minutes to induce denaturation and strand separation, and then the temperature is lowered from 75°C to 23°C and maintained at 23°C for 10 minutes to promote hydrogen bond formation between complementary DNA and RNA sequences. Complementary base pairing between the hybrid oligonucleotide and mRNA in specific regions results in RNA:DNA hybrid formation at the end of the annealing process (Figure 2).

[0097] In some embodiments, the heating temperature is 60°C to 95°C. In some embodiments, the heating temperature is 60°C to 80°C. In some embodiments, the heating temperature is 60°C to 70°C. In some embodiments, the heating temperature is 65°C to 70°C.

[0098] In some embodiments, heating is for 5 minutes, 10 minutes, 15 minutes, or 20 minutes. In some embodiments, RNA is heated at a temperature of 65°C to 70°C for 15 minutes. At higher temperatures, RNA is heated for less than 15 minutes. In some embodiments, RNA is heated at 70°C to 75°C for 10 minutes.

[0099] Digestion of RNA:DNA hybrids In some embodiments, during the digestion step, the RNA:DNA hybrid is incubated with one or more nucleases used to digest or cleave at least one strand of the RNA:DNA hybrid. In some embodiments, multiple nucleases are used in a single reaction to produce, for example, capped fragments. In some embodiments, multiple nucleases are used to produce capped fragments and blunt-end capped fragments. In some embodiments, multiple nucleases are used to produce tail fragments.

[0100] Ribonuclease H is a family of endonuclease enzymes that share a common substrate specificity for the RNA strand of RNA-DNA double helix. RNase H cleaves phosphodiester bonds in the RNA backbone, generating three hydroxyl groups and five phosphate groups. In some embodiments, the appropriate nuclease is RNase H or an enzyme with RNase H-like biochemical activity. RNase H comprises two phylogenetic subtypes, type 1 and type 2. RNase H binds to single-stranded (ss)RNA that hybridizes to complementary single-stranded DNA, and then degrades the RNA portion of the RNA:DNA hybrid. RNase H plays a role in DNA replication, recombination, and repair. In vitro, the enzyme also binds to double-stranded (ds)DNA, ssDNA, ssRNA, and dsRNA, although with lower affinity than when it binds to RNA:DNA hybrids. Several sequences of RNase H are known in the literature, each with slightly different amino acid sequences. This disclosure utilizes any RNase H enzyme, including but not limited to those disclosed in U.S. Patents No. 5,268,289 and No. 5,500,370 (heat-stable RNase H), U.S. Patent No. 6,376,661 (human RNase H), U.S. Patent No. 6,001,652 (human type 2 RNase H), and U.S. Patent No. 6,071,734 (RNase H derived from HBV polymerase). In the methods of this disclosure, nuclease cleavage occurs selectively at specific sites because one or more RNA nucleotides are adjacent to both sides of the DNA base in the hybrid oligonucleotide.

[0101] In some embodiments, the nuclease selectively degrades RNA:DNA hybrids and / or unannealed mRNA, yielding capped and uncapped 5' fragments. In some embodiments, the nuclease selectively degrades RNA:DNA hybrids, yielding tailed and untailed 3' fragments. In some embodiments, at least a portion of the fragment is double-stranded. In some embodiments, the double-stranded portion is at least partially RNA:RNA hybrid. In some embodiments, the double-stranded portion is at least partially RNA:RNA hybrid. The fragments resulting from nuclease treatment may be blunt-ended or shifted. In some embodiments, the fragments consist of 2 to 20 nucleotides (including capped nucleotides, if present), i.e., the fragments resulting from nuclease treatment may be 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 nucleotides.

[0102] In some embodiments, the capped and uncapped fragments contain mRNA of 5 nucleotides or less. In some embodiments, the capped and uncapped fragments contain mRNA of 2 nucleotides or less. In some embodiments, the tail and non-tailed fragments contain mRNA of 5 nucleotides or less. In some embodiments, the tail and non-tailed fragments contain mRNA of 2 nucleotides or less.

[0103] In some embodiments, the suitable nuclease is the S1 nuclease. Further nucleases used alone or in combination include, but are not limited to, Bensonase®, nuclease P1, phosphodiesterase II, RNase A, and RNase T1. In some embodiments, multiple nucleases, such as RNase H and S1 nuclease, are used. Some embodiments further include the addition of a single-stranded DNA nuclease for producing or modifying fragments. In some embodiments, it may be desirable to heat the sample (e.g., to about 60°C to 75°C) or apply the sample to a heated chromatography column to produce capped, uncapped, tailed, or non-tailed fragments.

[0104] In some embodiments, digestion is carried out by incubation with RNAse H and shrimp alkaline phosphatase (rSAP) at 37°C for 40 minutes. In some embodiments, digestion is carried out by incubation with RNAse H and shrimp alkaline phosphatase (rSAP) at 28°C to 45°C. In some embodiments, digestion is carried out by incubation with RNAse H and shrimp alkaline phosphatase (rSAP) for 20 to 60 minutes. The RNAse H enzyme cleaves the phosphodiester bonds between the double-stranded RNA:DNA hybrids produced in the annealing step. Shrimp alkaline phosphatase (rSAP) catalyzes terminal dephosphorylation to prevent self-ligation (Figure 3).

[0105] Chromatographic analysis Some embodiments of this disclosure include chromatographic methods for identifying and quantifying mRNA capping and tailing modifications. Using the chromatographic embodiments of this disclosure, any of the cap structures and cap analogues described herein, as well as the presence or absence of various modifications and / or tails within the cap, can be identified and quantified.

[0106] In some embodiments, capping species and non-tailed species are quantified by single LC-UV, LC-MS, or LC-UV-MS analysis. In some embodiments, capping species and non-tailed species are quantified by single LC-UV analysis. In some embodiments, capping species and non-tailed species are quantified by single LC-MS analysis. In some embodiments, capping species and non-tailed species are quantified by single LC-UV-MS analysis. In some embodiments, capping species and non-tailed species are measured by ultrafast liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS).

[0107] UV detection is particularly valuable in a Good Manufacturing Practice (GMP) environment. In some embodiments, UV detection is at 260 nm. In some embodiments, UV detection is at 280 nm. In some embodiments, UV cells of 0.1 mm to 100 mm are used. In some embodiments, a 10 mm UV cell is used. In some embodiments, a 0.01 mm UV cell is used. In addition to the sample, in some embodiments, standards are injected in parallel for cap 1, cap 0, cap G and uncapped varieties to identify and compare various retention times.

[0108] In some embodiments, the tail species is characterized by single LC-MS or LC-UV-MS analysis. In some embodiments, the tail species is characterized by single LC-MS analysis. In some embodiments, the tail species is characterized by single LC-UV-MS analysis.

[0109] In some embodiments, one or more processes are automated. In some embodiments, one process is automated. In some embodiments, two or more processes are automated. In some embodiments, all processes are automated.

[0110] The nuclease-treated sample is applied to a chromatographic column to separate, for example, capped and uncapped fragments, tails and non-tails. In addition to separating capped fragments from uncapped fragments, chromatography can decompose and quantify methylated caps from unmethylated guanine caps. In some embodiments, the second-to-last methylated base (2'-O-methylated base) can be separated (removed) and quantified from caps that are not methylated at that position. The capped and uncapped fragments are separated by chromatography (i.e., separated from each other) and identified by peaks. The amounts of capped and uncapped fragments can be quantified by standard quantitative chromatography techniques, such as UHPLC peak integration.

[0111] Embodiments of this disclosure utilize chromatography to provide highly resolved (e.g., single-base resolution) capped and uncapped, non-tailed and tailed fragments. The fragments can be efficiently separated by ultra-high-performance liquid chromatography ("UHPLC"). In the context of this disclosure, the term "UHPLC" includes various UHPLC and atmospheric pressure liquid chromatography methods that may be used to perform some embodiments of this disclosure. In some embodiments, the fragments may be separated by reverse-phase ultra-high-performance liquid chromatography (RP-UHPLC). In some embodiments of this disclosure, the main peak in the chromatogram is the capped mRNA fragment. Parameters that may be modified or optimized to increase resolution include gradient conditions, organic modifiers, counterions, temperature, column pore size and particle size, solvent composition and flow rate.

[0112] In some embodiments, the quantitative methods described herein may include one or more steps of ion exchange chromatography-HPLC (e.g., anion exchange HPLC and / or cation exchange HPLC). As is known to those skilled in the art, ion exchangers (e.g., anion exchangers and / or cation exchangers) can be based on a variety of materials with respect to the matrix and the bound charged groups.

[0113] In some embodiments, the fragments are separated by reversed-phase UHPLC. Reverse-phase UHPLC consists of a nonpolar stationary phase and a medium-polar mobile phase. In some embodiments, the stationary phase is, for example, silica treated with RMe2SiCl, where R is C 18 H 37 Or C8H 17 These are linear alkyl groups. Therefore, retention times are inherently longer for more nonpolar molecules, allowing polar molecules to elute more easily. Retention times increase with the addition of polar solvents to the mobile phase and decrease with the addition of more hydrophobic solvents. The properties of a particular RNA molecule as an analyte can play a significant role in its retention properties. Generally, analytes with more nonpolar functional groups (e.g., methyl groups) increase the hydrophobicity of the molecule, resulting in longer retention times. Protocols for high-resolution RNA species using reversed-phase UHPLC that can be adapted for use in embodiments of this disclosure are known in the art (see, for example, U.S. Patent Application Publication No. 2010 / 0048883; Gilar, M., “Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection”, Anal. Biochem. 298:196-206 (2001)).

[0114] Certain embodiments utilize various combinations of chromatographic separations disclosed herein. For example, certain embodiments of this disclosure may utilize reversed-phase ion-pair chromatography, where the separation is based on both hydrophobicity and the number of anions associated with the molecule, and can be used to purify fragments in a single HPLC step. The matrix may be silica-based (see, e.g., Murray et al., Anal. Biochem., 218:177-184 (1994)). In certain embodiments, nonporous inert polymer resins may be used (see, e.g., Huber, CG, “High-resolution liquid chromatography of oligonucleotides on nonporous alkylated styrene-divinylbenzene copolymers”, Anal. Biochem, 212:351-358 (1993)). Other combinations may be equally effective and should be evaluated in relation to the size of the fragment and the modification to be resolved.

[0115] In some embodiments, the capping efficiency profile and / or methylation profile can be determined by strong anion exchange chromatography using an HPLC system. Generally, uncapped mRNA adsorbs to the fixed positive charge of a strong anion exchange column using a mobile phase at a given flow rate, while capped species (with caps having a lower negative charge than uncapped species) elute from the column in proportion to the strength of their ionic interactions with the positively charged column. More negatively charged (more acidic) uncapped species elute more slowly than less negatively charged (less acidic) capped species.

[0116] In certain embodiments, the capped fragment is characterized by the methylation profile associated with that fragment. Typically, the methylation profile reflects and quantifies the efficiency of methylation at the capped guanine base (N-7 position). Further embodiments can also simultaneously quantify the methylation at the 2'-O position of the ribose ring for the second-to-last base (capped 1 structure).

[0117] In some embodiments, the methylation profile can be determined by performing reversed-phase HPLC, either alone or in combination with ion-exchange chromatography. In some embodiments, the “methylation profile” refers to a set of values ​​representing the amount of methylated cap fragments eluting from the column at a given time after the mobile phase has been added to the column. As described above, the retention times of methylated caps and the second-to-last nucleotide, which are inherently more nonpolar, increase and they elute more readily compared to polar molecules. Retention times can be increased by adding a polar solvent to the mobile phase and decreased by adding a more hydrophobic solvent.

[0118] In some embodiments, quantitative analysis of the capped fragments can be performed using reversed-phase HPLC column packing with a fully porous C18 adsorbent of 2.5 μm, as an example, as described in Gilar, M., Anal. Biochem. 298:196-206 (2001). Parameters that can be optimized to enhance the mass transfer of oligonucleotides in the stationary phase include high temperature, small adsorbent particle size, and slow mobile phase flow rate. Triethylammonium acetate (TEAA) buffer with UV detection and an optimized TEA-HFIP mobile phase can be used for LC-MS separation and characterization of the capped fragments.

[0119] In some embodiments, ultra-high-performance liquid chromatography ("UHPLC") is used to decompose capped and uncapped fragments, and optionally provide additional quantitative information regarding methylation status. UHPLC generally refers to HPLC techniques using resin particle sizes less than 2.5 μm, which result in a significant increase in efficiency even as flow rate and linear velocity are increased. By using small particles, the rate and peak capacity (number of peaks decomposed per unit time) can be extended. Such techniques utilize the principle of chromatography to perform separation using columns packed with smaller particles and / or higher flow rates, resulting in superior resolution and sensitivity. (See, for example, Swartz, ME, "Ultra Performance Liquid Chromatography (UPLC): an introduction", Separation Science Redefined (2005)).

[0120] In some embodiments, hydrophilic interaction chromatography (HILIC) is used. Typically, HILIC is a variation of reversed-phase chromatography performed using a polar stationary phase that has an affinity for polar analytes. A mixture containing one or more polar analytes to be separated from the mixture is added to the polar stationary phase of a column, and a highly organic mobile phase containing alcohol, acetonitrile and / or aprotic solvent, with a very low percentage of aqueous solvent, buffer or other polar solvent is also added to the column to facilitate the passage of analytes through the stationary phase. Water present in the mobile phase associates with the polar stationary phase, increasing the affinity of polar analytes, such as nucleic acids, to the stationary phase. More polar analytes, such as longer nucleic acids, have a stronger affinity for the stationary phase and are therefore retained in the column longer, thereby enabling the HILIC method to separate nucleic acids by length. HILIC methods using mobile phases suitable for downstream applications such as mass spectrometry allow for the analysis of the mass of purified nucleic acids by mass spectrometry.

[0121] HILIC is carried out by various methods known in the art. Some exemplary, non-limiting volatile salts used in HILIC include ammonium bicarbonate, ammonium acetate, and ammonium formate. Organic solvents include, but are not limited to, methanol, acetonitrile, and isopropanol. Ion pairing agents include, but are not limited to, octylamine, nonafluoro-tert-butyl alcohol, diethylammonium acetate, and dibutylammonium acetate. "Ion pairing agent" or "ion pair" refers to an agent (e.g., a small molecule) that acts as a counterion to a charged (e.g., ionizable or ionizable) functional group on an analyte (e.g., nucleic acid), thereby altering the retention time of the analyte as it moves through the stationary phase of the column. Ion pairing agents are classified as cationic ion pairing agents (interacting with negatively charged functional groups) or anionic ion pairing agents (interacting with positively charged functional groups). Typically, volatile salts and ion-pairing agents are dissolved in an aqueous solution of an organic solvent to prepare the first mobile phase, and then dissolved in a lower concentration aqueous solution of the same organic solvent to prepare the second mobile phase. In each mobile phase, the first and second ion-pairing agents are combined (e.g., in a ratio between 1:10 and 10:1), with a final concentration of 0.1 mM to 100 mM for each ion-pairing agent. A buffer containing volatile salts is added to the mobile phase to achieve a final volatile salt concentration of 1 mM to 100 mM, promoting the ionization of eluted mRNA and reducing the charge state of the mRNA during ionization. The mRNA composition is added to a HILIC column at a temperature of 20°C to 60°C, and the mobile phase is passed through the column. After elution, the purified mRNA is ionized and analyzed by mass spectrometry.

[0122] In some embodiments, liquid chromatography combined with UV detection (LC-UV), liquid chromatography combined with mass spectrometry (LC-MS), or liquid chromatography combined with UV and mass spectrometry (LC-UV-MS) is used for analysis. Current protocols can distinguish between possible RNA-capped species, uncapped species, tailed species, or any combination of non-tailed species. Various embodiments of the chromatography are discussed in more detail below.

[0123] Liquid chromatography-UV (LC-UV) In some embodiments, liquid chromatography uses an ultraviolet (UV) detector, which is an in-line device that provides a continuous signal that can be used to measure the UV absorbance of an LC (e.g., high-performance liquid chromatography, HPLC) eluent and quantify the amount of compound emerging from the LC column. In some embodiments, the UV detector has a fixed wavelength (e.g., 260 nm, 280 nm, etc.). In some embodiments, the UV detector has a tunable wavelength. In some embodiments, the detector is a photodiode array detector. In preferred embodiments, the UV detector is used in a Good Manufacturing Practice (GMP) environment.

[0124] Liquid chromatography-mass spectrometry (LC-MS) LC-MS combines the physical separation capabilities of liquid chromatography (or HPLC) with the mass spectrometry capabilities of mass spectrometry. LC-MS is a highly sensitive and selective technique. Various ionization sources, mass spectrometers, detectors, and statistical methods are used for data analysis.

[0125] An LC-MS method for the separation and identification of oligonucleotides using an aqueous triethylammonium-hexafluoroisopropyl alcohol (TEA HFIP) buffer suitable for MS detection is known in the art (Apffel, A., et al., “New procedure for the use of HPLC-ESI MS for the analysis of nucleotides and oligonucleotides”, J. Chromatogr. A, 777:3-21 (1997)). Alternatively, a triethylammonium bicarbonate mobile phase may be used for oligonucleotide separation with post-column acetonitrile addition to the eluent. The ion-pair buffer can be selected to provide the best MS detection sensitivity.

[0126] In some embodiments, electrospray ionization (ESI) is used as the ionization system for LC-MS. Briefly, the sample is introduced into the LC-MS system via an electrospray probe consisting of a metal capillary tube, and a high voltage is applied to the capillary tube while the sampling orifice is at a low voltage. The heat and voltage applied to the probe produce a fine spray of the sample flow. At low LC flow rates, the potential difference is sufficient to produce the spray. In some embodiments, a nitrogen gas flow is provided for higher LC flow rates. The electric field at the tip of the capillary tube forms positively or negatively charged droplets of the ionized compound, depending on the polarity of the applied voltage. The ESI MS negative mode charges the sample by deprotonation, and the positive ion mode charges the analyte by protonation.

[0127] In some embodiments, the quantification of capped fragments and their methylation status, uncapped fragments, tails, and non-tailed fragments is achieved by automatic integration of the respective peak areas in the HPLC chromatogram. The data may be presented as area percentage values, indicating the percentage of the integrated peak area of ​​a particular species relative to the total integrated peak area of ​​the entire chromatogram.

[0128] In some embodiments, the quantification of capped, uncapped, tailed, and non-tailed fragments can be achieved by other suitable methods, such as liquid chromatography combined with UV and mass spectrometry (MS) based detection (LC-UV-MS).

[0129] In some embodiments of this disclosure, capping species and non-tail species are quantified by single LC-UV, LC-MS, or LC-UV-MS analysis.

[0130] In some embodiments, tail species are characterized by single LC-MS or LC-UV-MS analysis.

[0131] In some embodiments, one or more steps are automated.

[0132] RNA sample Embodiments of this disclosure may be used to quantify capping and tailing modifications of a wide variety of RNA species, including in vitro transcribed mRNA, isolated eukaryotic mRNA, and viral RNA.

[0133] In some embodiments, the methods of the disclosure are used to identify or quantify RNA capping and tailing modifications in linear RNA. In some embodiments, the methods of the disclosure are used to identify or quantify RNA capping and tailing modifications in messenger RNA. In some embodiments, the methods of the disclosure are used to identify or quantify RNA capping and tailing modifications in small RNA.

[0134] In some embodiments, RNA is obtained from the manufacturing process. In some embodiments, RNA is obtained from the final step of manufacturing. In some embodiments, RNA is a deformulated formulation.

[0135] mRNA synthesis mRNA can be synthesized according to any of the various known methods. For example, mRNA can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically carried out using a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitor. The exact conditions vary depending on the specific application.

[0136] In some embodiments, the DNA template is transcribed in vitro for mRNA preparation according to the present disclosure. A suitable DNA template typically has a promoter for in vitro transcription, e.g., a T3, T7, or SP6 promoter, followed by the desired mRNA and a desired nucleotide sequence for terminal signaling.

[0137] mRNA synthesis using T3 RNA polymerase In some embodiments, mRNA is produced using T3 RNA polymerase. T3 RNA polymerase is a DNA-dependent RNA polymerase derived from the T3 bacteriophage that catalyzes RNA formation from DNA in the 5'→3' direction, either single-stranded or double-stranded DNA, and can incorporate modified nucleotides. T3 polymerase is highly promoter-specific and transcribes only DNA downstream of the T3 promoter. T3 binds to the consensus promoter sequence 5'-AATTAACCCTCACTAAAGGGAGA-3' (SEQ ID NO: 3).

[0138] mRNA synthesis using T7 RNA polymerase In some embodiments, mRNA is produced using T7 RNA polymerase. T7 RNA polymerase is a DNA-dependent RNA polymerase derived from the T7 bacteriophage that catalyzes RNA formation from DNA in the 5'→3' direction. T7 polymerase is highly promoter-specific and transcribes only DNA downstream of the T7 promoter. T7 binds to the consensus promoter sequence 5'-TAATACGACTCACTATAGGGAGA-3' (SEQ ID NO: 4). T7 polymerase also requires a double-stranded DNA template and Mg2+ ions as cofactors for RNA synthesis. The error rate is very low.

[0139] mRNA synthesis using SP6 RNA polymerase In some embodiments, mRNA is produced using SP6 RNA polymerase. SP6 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for the SP6 promoter sequence. SP6 polymerase catalyzes the 5'→3' in vitro synthesis of RNA on single-stranded or double-stranded DNA downstream of its promoter, and it incorporates native ribonucleotides and / or modified ribonucleotides and / or labeled ribonucleotides into the polymerized transcript. SP6 binds to the consensus promoter sequence 5'-ATTTAGGTGACACTATAG-3' (SEQ ID NO: 5). Examples of such labeled ribonucleotides include biotin-labeled nucleotides, fluorescein-labeled nucleotides, digoxigenin-labeled nucleotides, aminoallyl-labeled nucleotides, and isotope-labeled nucleotides.

[0140] DNA template Typically, DNA templates are either completely double-stranded or mostly single-stranded with appropriate promoter sequences (e.g., T3, T7, or SP6 promoters).

[0141] Linear plasmid DNA (linear via one or more restriction enzymes), linear genomic DNA fragments (via restriction enzymes and / or physical means), PCR products, and / or synthetic DNA oligonucleotides can be used as templates for in vitro transcription, provided that they contain a double-stranded promoter upstream (and in the correct orientation) of the DNA sequence to be transcribed.

[0142] In some embodiments, the linear DNA template has blunt ends.

[0143] In some embodiments, the transcribed DNA sequence may be optimized to facilitate more efficient transcription and / or translation. For example, the DNA sequence may be optimized with respect to cis-regulatory elements (e.g., TATA boxes, termination signals, and protein-binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements related to transcription; the DNA sequence may be optimized with respect to hidden splice sites, mRNA secondary structure, stable free energy of mRNA, repeat sequences, RNA instability motifs, and / or other elements related to mRNA processing and stability; the DNA sequence may be optimized with respect to codon use bias, codon adaptability, internal chi sites, ribosome-binding sites (e.g., IRES), immature poly-A sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA sequence may be optimized with respect to codon context, codon-anticodon interactions, translation pause sites, and / or other elements related to protein folding. Optimization methods known in the art, such as ThermoFisher's GeneOptimizer and OptimumGene® described in U.S. Patent No. 20110081708, may be used in this disclosure, and their contents are incorporated herein by reference in their entirety.

[0144] In some embodiments, the DNA template includes a 5' and / or 3' untranslated region. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as iron-responsive elements. In some embodiments, the 5' untranslated region may be about 50 to 500 nucleotides long.

[0145] In some embodiments, the 3' untranslated region includes one or more of the following: a polyadenylation signal, a binding site to a protein that affects the stability of the intracellular mRNA position, or one or more binding sites to a miRNA. In some embodiments, the 3' untranslated region may be 50 to 500 nucleotides or longer.

[0146] Exemplary 3' and / or 5'UTR sequences may be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histone, or citrate cycle enzymes) to enhance the stability of the sense mRNA molecule. For example, the 5'UTR sequence may contain a sub-sequence of the CMV earliest 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or extend the half-life of the polynucleotide. Inclusion of sequences or fragments encoding human growth hormone (hGH) at the 3' end or untranslated region of a polynucleotide (e.g., mRNA) to further stabilize the polynucleotide is also contemplated. Generally, these modifications improve the stability and / or pharmacokinetic properties (e.g., half-life) of the polynucleotide compared to their unmodified counterparts, and include modifications made, for example, to improve the resistance of such polynucleotides to nuclease digestion in vivo.

[0147] Large-scale mRNA synthesis In some embodiments, mRNA can be synthesized on a large scale. In some embodiments, mRNA is synthesized in single batches in amounts of at least 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g, 25 g, 500 g, 75 g, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1000 kg, or more. As used herein, the term “batch” refers to the quantity or amount of mRNA synthesized at one time, for example, produced according to a single production setting. A batch may refer to the amount of mRNA synthesized in a single reaction occurring by aliquots of a single enzyme and / or aliquots of a single DNA template for serial synthesis under a series of conditions. mRNA synthesized in a single batch does not include mRNA synthesized at different times that are combined to achieve the desired amount.

[0148] According to this disclosure, RNA polymerase is typically used at a rate of 1 to 100 mg per gram (g) of mRNA produced. In some embodiments, about 1 to 90 mg, 1 to 80 mg, 1 to 60 mg, 1 to 50 mg, 1 to 40 mg, 10 to 100 mg, 10 to 80 mg, 10 to 60 mg, and 10 to 50 mg of RNA polymerase are used per gram of mRNA produced. In some embodiments, about 5 to 20 mg of RNA polymerase is used to produce about 1 gram of mRNA. In some embodiments, about 0.5 to 2 grams of RNA polymerase is used to produce about 100 grams of mRNA. In some embodiments, about 5 to 20 grams of RNA polymerase is used for about 1 kilogram of mRNA. In some embodiments, at least 5 mg of RNA polymerase is used to produce at least 1 gram of mRNA. In some embodiments, at least 500 mg of RNA polymerase is used to produce at least 100 grams of mRNA. In some embodiments, at least 5 grams of RNA polymerase are used to produce at least 1 kilogram of mRNA. In some embodiments, about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg of plasmid DNA is used per gram of mRNA produced. In some embodiments, about 10–30 mg of plasmid DNA is used to produce about 1 gram of mRNA. In some embodiments, about 1–3 grams of plasmid DNA is used to produce about 100 grams of mRNA. In some embodiments, about 10–30 grams of plasmid DNA is used to produce about 1 kilogram of mRNA. In some embodiments, at least 10 mg of plasmid DNA is used to produce at least 1 gram of mRNA. In some embodiments, at least 1 gram of plasmid DNA is used to produce at least 100 grams of mRNA. In some embodiments, at least 10 grams of plasmid DNA is used to produce at least 1 kilogram of mRNA.

[0149] In some embodiments, the concentration of RNA polymerase in the reaction mixture may be about 1 nM to 100 nM, 1 nM to 90 nM, 1 nM to 80 nM, 1 nM to 70 nM, 1 nM to 60 nM, 1 nM to 50 nM, 1 nM to 40 nM, 1 nM to 30 nM, 1 nM to 20 nM, or about 1 nM to 10 nM. In certain embodiments, the concentration of RNA polymerase is about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. RNA polymerase concentrations of 100 to 10000 units / ml can be used. For example, concentrations of 100 to 9000 units / ml, 100 to 8000 units / ml, 100 to 7000 units / ml, 100 to 6000 units / ml, 100 to 5000 units / ml, 100 to 1000 units / ml, 200 to 2000 units / ml, 500 to 1000 units / ml, 500 to 2000 units / ml, 500 to 3000 units / ml, 500 to 4000 units / ml, 500 to 5000 units / ml, 500 to 6000 units / ml, 1000 to 7500 units / ml, and 2500 to 5000 units / ml can be used.

[0150] The concentrations of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture range from approximately 0.1 mM to approximately 10 mM, for example, approximately 1 mM to approximately 10 mM, approximately 2 mM to approximately 10 mM, approximately 3 mM to approximately 10 mM, approximately 1 mM to approximately 8 mM, approximately 1 mM to approximately 6 mM, approximately 3 mM to approximately 10 mM, approximately 3 mM to approximately 8 mM, approximately 3 mM to approximately 6 mM, and approximately 4 mM to approximately 5 mM. In some embodiments, each ribonucleotide is present in the reaction mixture at approximately 5 mM. In some embodiments, the total concentration of rNTPs used (e.g., ATP, GTP, CTP, and UTP combined) is in the reaction range of 1 mM to 40 mM. In some embodiments, the total concentration of rNTPs used in the reaction (e.g., a combination of ATP, GTP, CTP, and UTP) is in the range of 1 mM to 30 mM, or 1 mM to 28 mM, or 1 mM to 25 mM, or 1 mM to 20 mM. In some embodiments, the total rNTP concentration is less than 30 mM. In some embodiments, the total rNTP concentration is less than 25 mM. In some embodiments, the total rNTP concentration is less than 20 mM. In some embodiments, the total rNTP concentration is less than 15 mM. In some embodiments, the total rNTP concentration is less than 10 mM.

[0151] RNA polymerase reaction buffers typically contain salt / buffers, such as Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, sodium chloride, and magnesium chloride.

[0152] The pH of the reaction mixture may be approximately 6–8.5, 6.5–8.0, or 7.0–7.5, and in some embodiments, the pH is 7.5.

[0153] Combine a linear or linear DNA template (for example, in an amount / concentration sufficient to provide the desired amount of RNA, as described above), RNA polymerase reaction buffer, and RNA polymerase to form a reaction mixture. Incubate the reaction mixture at approximately 37°C to 42°C for 30 minutes to 6 hours, for example, approximately 60 to 90 minutes.

[0154] In some embodiments, approximately 5 mM NTP, approximately 0.05 mg / mL RNA polymerase, and approximately 0.1 mg / mL DNA template are incubated in a suitable RNA polymerase reaction buffer (final reaction mixture pH approximately 7.5) at approximately 37°C to approximately 42°C for 60 to 90 minutes.

[0155] In some embodiments, the reaction mixture comprises a linear double-stranded DNA template having an RNA polymerase-specific promoter, RNA polymerase, an RNase inhibitor, pyrophosphatase, 29 mM NTP, 10 mM DTT, and a reaction buffer (where 10 × 800 mM HEPES, 20 mM spermidine, and 250 mM MgCl2, pH 7.7), and a sufficient amount (QS) of RNase-free water to reach the desired reaction volume. The reaction mixture is then incubated at 37°C for 60 minutes. The polymerase reaction is then quenched by adding DNase I and DNase I buffer (where 10 × 100 mM Tris-HCl, 5 mM MgCl2, and 25 mM CaCl2, pH 7.6) to facilitate the digestion of the double-stranded DNA template in providing the purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[0156] In some embodiments, the reaction mixture comprises NTP at a concentration in the range of 1 to 10 mM, DNA template at a concentration in the range of 0.01 to 0.5 mg / ml, and RNA polymerase at a concentration in the range of 0.01 to 0.1 mg / ml. For example, the reaction mixture comprises NTP at a concentration of 5 mM, DNA template at a concentration of 0.1 mg / ml, and RNA polymerase at a concentration of 0.05 mg / ml.

[0157] In some embodiments, RNA contains one or more modified nucleotides.

[0158] In some embodiments, the RNA is not modified.

[0159] Capped RNA mRNA and other RNAs possess a 5' end "cap" structure that plays a crucial biological role in splicing, translation, and prevention of mRNA degradation, thereby contributing to mRNA stability. The 5' cap plays a role in the processing and maturation of RNA transcripts in the nucleus, such as premRNA splicing, mRNA transport from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA into proteins.

[0160] The 5' cap structure is recognized by eukaryotic translation initiation factors 4E and eIF4E, initiating protein synthesis (see Shatkin, AJ, Cell, 40:223-24 (1985); Furuichi, et al., Nature, 266:235 (1977); Sonenberg, N., Prog. Nuc. Acid Res Mol Biol, 35:173-207 (1988)). There are specific cap-binding proteins that are components of the mechanism necessary for mRNA translation initiation (see, for example, Shatkin, AJ, Cell, 40:223-24 (1985); Sonenberg, N., Prog. Nuc. Acid Res Mol Biol, 35:173-207 (1988)). mRNA caps are recognized by the translation initiation factor eIF4E (Gingras, et al., Ann. Rev. Biochem. 68:913-963 (1999); Rhoads, RE, J. Biol. Chem. 274:30337-3040 (1999)). The 5' cap structure protects mRNA from 5'-exonuclease activity and resulting degradation (Ross, J., Mol. Biol. Med. 5:1-14 (1988); Green, MR et al., Cell, 32:681-694 (1983)). Since the primary transcripts of many eukaryotic and eukaryotic viral genes require splicing to remove intercalating sequences (introns) within the coding region of these transcripts, caps also function in stabilizing pre-mRNA.

[0161] Translation efficiency is increased by capping, and capped RNA has been reported to be translated more efficiently than uncapped transcripts in various in vitro translation systems, such as rabbit reticulocyte lysates or wheat germ translation systems (see, for example, Shimotohno, K., et al., Proc. Natl. Acad. Sci. USA, 74:2734-2738 (1977); Paterson and Rosenberg, Nature, 279:692 (1979)). Increased mRNA stability and resistance to exonucleases are contributing factors.

[0162] The 2'-O methylation at the second-to-last nucleotide of the 5' position of mRNA functions as a molecular signature that identifies host and invading pathogen mRNA, such as viral mRNA, that lacks this feature; therefore, the 5' cap also plays a role in pathogen defense.

[0163] mRNA caps are typically added enzymatically.

[0164] In some embodiments of the methods provided herein, the capping species is cap 1, cap 0, cap G, or uncapped. In some embodiments, the capping species is cap 1. In some embodiments, the capping species is cap 0. In some embodiments, the capping species is cap G. In some embodiments, the capping species is uncapped.

[0165] In some embodiments, the capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA. In some embodiments, the capping species is m7Gpppm7GGACA. In some embodiments, the capping species is m7GpppGGACA. In some embodiments, the capping species is GpppGGACA. In some embodiments, the capping species is GGACA or pppGGACA. In some embodiments, the uncapped species is GGACA or pppGGACA.

[0166] In some embodiments, the relative amount of each capping species is the percentage of the total amount of the capping species in the sample calculated by dividing the peak area under the curve of the capping species of interest by the sum of the peak areas under the curves representing cap1, cap0, capG, and uncapped species and multiplying by 100.

[0167] The methods of the invention described herein are generally suitable for the identification and quantification of any type of standard or non-standard mRNA cap.

[0168] Standard mRNA cap structure In some embodiments, the cap has the structure of formula I.

Chemical formula

[0169] Naturally occurring cap structures include 7-methylguanosine linked via a triphosphate bridge to the 5' end of the first transcribed nucleotide, resulting in a dinucleotide cap of m 7 G(5’)ppp(5’)N (where N is any nucleoside). In some embodiments, the m 7 G cap is m 7 G(5’)ppp(5’)G.

[0170] The standard cap of most eukaryotic and viral mRNA consists of N7-methylguanosine (m7G) linked to the first nucleotide of the RNA by an inverse 5'-5' triphosphate crosslink. Cap 0 is the dominant form in lower eukaryotes, namely m7GpppN. In higher eukaryotes, the first two 5' nucleotides of the 5'UTR can be 2'-O-methylated to produce m7GpppNm (cap 1) and m7GpppNmpNm (cap 2) structures.

[0171] The cap is added to the nucleus and catalyzed by the enzyme guanylyltransferase. The addition of the cap to the 5' end of RNA occurs immediately after transcription initiation. The terminal nucleoside is typically guanosine and is reversed from all other nucleotides, namely G(5')ppp(5')GpNpNp.

[0172] In some embodiments, the cap is a cap 0 structure. The cap 0 structure lacks 2'-O-methyl residues on ribose bound to bases 1 and 2. In some embodiments, the cap is a cap 1 structure. The cap 1 structure has a 2'-O-methyl residue on base 1. In some embodiments, the cap is a cap 2 structure. The cap 2 structure has 2'-O-methyl residues bound to both bases 1 and 2 (Figure 4B).

[0173] Non-standard mRNA cap structure Several non-standard mRNA caps, such as the GpppX variant and the nn-methylated guanosine cap (GpppN), have also been recognized. Some cap variants are further methylated at N6 of Am(m6Am). Multiple methylations also occur on the 5'G of the cap, for example, on the di- and trimethylguanosine cap (m2,2,7GpppN), for example, on nuclear and nucleolar small RNAs, telomerase RNAs, and some viral RNAs. Mammalian U6 and 7SK RNAs contain phosphate methylation of the unprocessed 5' triphosphate (mpppN).

[0174] The typical cap of mRNA produced by in vitro transcription is m 7 The structure is G(5')ppp(5')G, which is used as a dinucleotide cap in in vitro transcription by T7 or SP6 RNA polymerase to obtain RNA with a cap structure at its 5' end. A common method for in vitro synthesis of capped mRNA is to use m as a transcription initiator. 7 G(5')ppp(5')G(「m 7 It uses pre-formed dinucleotides in the form of "GpppG". These are pseudosymmetric dinucleotides. 7 The disadvantage of using G(5')ppp(5')G is that G or m 7 One of the 3'-OH groups in the G portion tends to act as an initiating nucleophile for transcription elongation. In other words, m 7 If 3'-OH groups are present on both the G and G portions, up to half of the mRNA will incorporate the cap in an improper orientation. This means that, depending on the ionic conditions of the transcription reaction, the mRNA will be affected. 7 G(5')pppG(pN) n and G(5')pppm 7 Two isomer RNAs in the G(pN)n form are synthesized in nearly equal proportions. Variation in isomerism can negatively impact in vitro translation and is undesirable for homogeneous therapeutic products.

[0175] To date, the common form of synthetic dinucleotide caps used in in vitro translation experiments is the Anti-Reverse Cap Analog ("ARCA"), which is generally a modified cap analog in which the 2' or 3'OH group is replaced with -OCH3. ARCA and triple-methylated cap analogs are incorporated in the forward direction. 7Chemical modification of G results in the cap being incorporated only in a forward orientation, even when the 2'OH group is not involved in the phosphodiester bond. (Jemielity, J. et al., “Novel 'anti-reverse' cap analogs with superior translational properties”, RNA, 9:1108-1122 (2003)). Selective procedures for methylation of guanosine in N7 and 3'O-methylation and 5' diphosphate synthesis have been established (Kore, A. and Parmar, G. Nucleosides, Nucleotides, and Nucleic Acids, 25:337-340, (2006) and Kore, AR, et al. Nucleosides, Nucleotides, and Nucleic Acids, 25(3):307-14, (2006).

[0176] Cap analogue The cap analogue may be any modified "G" base (e.g., one or more modified guanine nucleotides), or may contain such a base. A suitable cap analogue may include m 7 GpppG, m 7 GpppA, m 7 GpppC; non-methylated cap analogues (e.g., GpppG); dimethylated cap analogues (e.g., m 2,7 GpppG), trimethylated cap analogs (e.g., m 2,2,7 GpppG), dimethylated symmetric cap analogs (e.g., m 7 Gpppm 7 G) or anti-reverse cap analogues (e.g., ARCA; m 7,2’Ome GpppG,m 7,2’d GpppG,m 7,3’Ome GpppG,m 7,3’d This includes, but is not limited to, chemical structures selected from the group consisting of GpppG and their tetraphosphate derivatives.

[0177] various m 7 G-cap analogues are known in the art, and many of them are commercially available. These include the above-mentioned m7 This includes GpppG, as well as the ARCA3'-OCH3 and 2'-OCH3 cap analogs described above (Jemielity, J. et al., RNA, 9:1108-1122 (2003)). Further cap analogs for use in embodiments of this disclosure include N7-benzylated dinucleoside tetraphosphate analogs (described in Grudzien, E. et al., RNA, 10:1479-1487 (2004)), phosphorothioate cap analogs (described in Grudzien-Nogalska, E. et al., RNA, 13:1745-1755 (2007)), and cap analogs (including biotinylated cap analogs) described in U.S. Patent Nos. 8,093,367 and 8,304,529, which are incorporated herein by reference.

[0178] In some embodiments, the mRNA is uncapped. Uncapped mRNA may be present in the sample (i.e., as a result of incomplete capping in the in vitro transcription reaction), and / or may be provided as a standard to be measured in parallel to quantify the level of uncapped species in the sample.

[0179] Production of capped mRNA Capped mRNA suitable for identification and / or quantification by the methods disclosed herein can be produced by any method known in the art.

[0180] In some embodiments, capped mRNA is produced by in vitro transcription, first developed by Krieg and Melton (Methods Enzymol., 1987, 155:397-415) for RNA synthesis using RNA phage polymerase. Typically, these reactions involve a buffer containing at least phage RNA polymerase (T7, T3, or SP6), a DNA template containing a phage polymerase promoter, nucleotides (ATP, CTP, GTP, and UTP), and magnesium salts. RNA synthesis yields can be optimized by increasing nucleotide concentrations, adjusting magnesium concentrations, and including inorganic pyrophosphatases (U.S. Patent No. 5,256,555; Gurevich, et al., Anal. Biochem., 195:207-213 (1991); Sampson, JR and Uhlenbeck, OC, Proc. Natl. Acad. Sci. USA. 85, 1033-1037 (1988); Wyatt, JR, et al., Biotechniques, 11:764-769 (1991)). Some embodiments utilize commercially available kits for large-scale synthesis of in vitro transcripts (e.g., MEGAscript®, Ambion). The RNA synthesized in these reactions typically features a 5' terminal nucleotide with a triphosphate group at the 5' position of ribose. Typically, depending on the combination of RNA polymerase and promoter used, this nucleotide is guanosine, but may also be adenosine (see, for example, Coleman, TM, et al., Nucleic Acids Res., 32:e14 (2004)). In these reactions, all four nucleotides are typically present in equimolar concentrations, and none of them are limiting.

[0181] In some embodiments, all components are combined and incubated at approximately 37°C to promote RNA polymerization in a single-batch reaction until the reaction is complete. Typically, batch reactions are used for convenience and to obtain larger quantities of RNA. In some embodiments, a “fed-batch” system (see, e.g., Jeffrey A. Kern, Batch and Fed-batch strategies for large-scale production of RNA by in vitro transcription (University of Colorado). (1997)) is used to increase the efficiency of the in vitro transcription reaction. All components are combined, but additional amounts of several reagents, such as nucleotides and magnesium, are added over time to maintain constant reaction conditions when the reaction is scaled up in a single batch. In some embodiments, the additional amounts of reagents required are not a linear increase from smaller reactions. Furthermore, in some embodiments, the reaction pH can be maintained at 7.4 by monitoring the reaction pH over time and adding KOH as needed.

[0182] RNA transcription typically begins with a nucleoside triphosphate (usually purine, A, or G). In vitro transcription typically involves a DNA template containing a phage RNA polymerase such as T7, T3, or SP6, a phage polymerase promoter, and a buffer containing nucleotides (ATP, GTP, CTP, and UTP) and magnesium salts. Capped RNA synthesis involves the use of capping analogs in the transcription reaction (e.g., m 7 The incorporation of GpppG is included, and in some embodiments, is incorporated by the addition of recombinant guanylyltransferase. Excess m 7 GpppG to GTP(4:1) increases the chances that each transcript has a 5' cap.

[0183] Kits for capping in vitro transcribed mRNA are commercially available, including the mMESSAGE mMACHINE® kit (Ambion, Inc., Austin, Tex). These kits typically produce 20% uncapped RNA from 80% capped RNA, but the total RNA yield decreases as GTP concentration becomes rate-limiting, as GTP is required for transcript elongation.

[0184] As the ratio of cap analogs to GTP increases in the reaction, the ratio of capped RNA to uncapped RNA increases proportionally. Increasing the ratio of cap analogs to GTP in the transcription reaction reduces the total RNA yield because the GTP concentration is limited when the total concentrations of caps and GTP are kept constant. Therefore, the final RNA yield depends on the GTP concentration required for transcript elongation. Other nucleotides (ATP, CTP, UTP) are present in excess.

[0185] Accordingly, this disclosure provides an improved method for simultaneously identifying and quantifying mRNA capping and tailing modifications in a single sample (e.g., a representative aliquot sample from an in vitro synthesis reaction, RNA from a manufacturing process, or a deformed formulation) without requiring the purification of intermediates that would result in sample loss and / or a decrease in measurement accuracy.

[0186] In some embodiments, mRNA is synthesized by in vitro transcription from a plasmid DNA template encoding a selected gene. In some embodiments, the in vitro transcription includes the addition of a 5' cap structure cap 1 having a 2'-O-methyl residue at the 2'OH group of the ribose ring of base 1 by enzymatic conjugation of GTP via guanylyltransferase. In some embodiments, the in vitro transcription includes the addition of a 5' cap structure cap 0 lacking a 2'-O-methyl residue by enzymatic conjugation of GTP via guanylyltransferase. In some embodiments, the in vitro transcription includes the addition of a 5' cap of any of the cap structures disclosed herein by enzymatic conjugation of GTP via guanylyltransferase.

[0187] RNA tailing The presence of a "tail" at the 3' end helps protect mRNA from exonuclease degradation. The 3' tail can be added before, after, or simultaneously with the 5' cap. Typically, the tail structure contains a poly-A and / or poly-C tail (A, adenosine; C, cytosine).

[0188] In some embodiments, the poly-A tail is added co-transferentially. In some embodiments, the poly-A tail is added post-transferentially. In some embodiments, the poly-C tail is added co-transferentially. In some embodiments, the poly-C tail is added post-transferentially.

[0189] In some embodiments, the poly-A tail is 25 to 5,000 nucleotides long. In some embodiments, the poly-A tail is 25 nucleotides long. In some embodiments, the poly-A tail is 50 nucleotides long. In some embodiments, the poly-A tail is 75 nucleotides long. In some embodiments, the poly-A tail is 100 nucleotides long. In some embodiments, the poly-A tail is 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, 500 to 550, 550 to 600, 600 to 650, 650 to 700, 700 to 750, 750 to 800, 800 to 850, 850 to 900, 900 to 950, and 950 to 1,000 nucleotides long, including all individual intermediary amounts. In some embodiments, the poly-A tail has a nucleotide length of 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, 3500-4000, 4000-4500, or 4500-5000, and includes all individual intermediary amounts.

[0190] In some embodiments, the poly(A) or poly(C) tail at the 3' end of the mRNA contains at least 25-50, 50-100, 100-200, 200-300, 300-500, or 500-800 adenine nucleotides or cytosine nucleotides, including all individual mediating amounts.

[0191] In some embodiments, the tail structure includes combinations of poly-A and poly-C tails having various lengths as described herein. In some embodiments, the poly-A tail structure contains at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, the poly-A tail structure contains at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0192] In some embodiments, the non-tail species is UGCAUC, where U is unmodified uridine. In some embodiments, the non-tail species is U*GCAU*C, where U* is N1-methylpseudolidine.

[0193] In some embodiments, the relative amount of non-tail species is a percentage calculated by dividing the area under the peak of non-tail species by the sum of the areas under the peaks of cap 1, cap 0, cap G, and the uncapped areas, and multiplying by 100.

[0194] As described herein, the addition of a 5' cap and / or 3' tail facilitates the detection of interrupted mRNA transcripts generated during in vitro synthesis, because in the absence of capping and / or tailing, the size of the interrupted mRNA transcripts might be too small to detect. Therefore, in some embodiments, the 5' cap and / or 3' tail are added to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of interrupted transcripts present in the mRNA). In some embodiments, the 5' cap and / or 3' tail are added to the synthesized mRNA before the mRNA is purified. In other embodiments, the 5' cap and / or 3' tail are added to the synthesized mRNA after the mRNA has been purified.

[0195] RNA modification In some embodiments, the linear RNA of this disclosure may include one, two, three or more modifications. In some embodiments, the modified nucleotides are located in the coding region. In some embodiments, the modified nucleotides are located in the untranslated region.

[0196] In some embodiments, the modification stabilizes RNA and increases its resistance to degradation compared to unmodified nucleotides. In some embodiments, the modified nucleotide enhances the biological function of nucleic acid molecules, for example, by increasing binding to RNA-binding proteins or by increasing translation.

[0197] In some embodiments, the modified nucleotide is one or more of N1-methylpseudridine, 5-methoxyuridine, N6-methyladenosine, pseudouridine, or 5-methylcytosine.

[0198] In some embodiments, the modified nucleotide is N1-methylpseudridine. In some embodiments, the modified nucleotide is 5-methoxyuridine. In some embodiments, the modified nucleotide is N6-methyladenosine. In some embodiments, the modified nucleotide is pseudouridine. In some embodiments, the modified nucleotide is 5-methylcytosine.

[0199] In some embodiments, the modified nucleotides are 100%. In some embodiments, the modified nucleotides are less than 50%. In some embodiments, the modified nucleotides are less than 20%. In some embodiments, the modified nucleotides are less than 10%.

[0200] The linear polynucleotides of this disclosure are approximately 0% to approximately 100% modified nucleotides (relating to the overall nucleotide content, or to one or more types of nucleotides, i.e., A, G, T / U, or C) or any percentage of mediating nucleotides (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% It may contain values ​​ranging from % to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, and 95% to 100% (including all values ​​and subranges in between).

[0201] In some embodiments, the polynucleotide is 100% modified. In some embodiments, the polynucleotide is at least 50% modified, for example, at least 50% of the nucleotide is modified. In some embodiments, the polynucleotide is at least 75% modified, for example, at least 75% of the nucleotide is modified. In some embodiments, the polynucleotide is at least 20% modified, for example, at least 20% of the nucleotide is modified. In some embodiments, the polynucleotide is at least 10% modified, for example, at least 10% of the nucleotide is modified. Since each nucleotide (sugar, base, and phosphate portion, e.g., bond) can be modified, it should be understood that any modification to any part of a nucleotide or nucleoside constitutes modification.

[0202] In some embodiments, modifications are structural modifications and / or chemical modifications. In some embodiments, chemical modifications are nucleotide and / or nucleoside modifications, including nucleic acid base modifications and / or sugar modifications, as well as skeletal linkage modifications (i.e., nucleoside-to-nucleoside links, e.g., linked phosphates, phosphodiester bonds, and phosphodiester skeletons). In some embodiments, structural modifications include secondary and / or tertiary structural modifications.

[0203] In some embodiments, modifications include the modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0204] In some embodiments, one, two, or more (depending on the context) nucleoside modifications or nucleotide modifications may be incorporated into the polynucleotides of the Disclosure.

[0205] In some embodiments, a polynucleotide (e.g., RNA) includes at least one modification described herein. In other embodiments, a polynucleotide includes two, three, four, or more (depending on the context) chemical modifications described herein. The modifications may be a combination of nucleic acid base (purine and / or pyrimidine), sugar, and backbone (nucleoside-to-nucleoside) bond modifications. The modifications may be located at one or more nucleotides of the polynucleotide. In some embodiments, all nucleotides of the polynucleotide are chemically modified. In some embodiments, all nucleotides of a biologically functional nucleic acid sequence are chemically modified.

[0206] In some embodiments, the polynucleotide is modified by at least 10% in just one component of the nucleotide, such component being a nucleic acid base, sugar, or bond between nucleosides. For example, the modification may be performed on at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleic acid base, sugar, or bond of the polynucleotides described herein.

[0207] In some embodiments, polynucleotides are designed with a patterned series of sugars, nucleic acid bases, or ligation modifications.

[0208] In some embodiments, the polynucleotides include modifications to maximize stability.

[0209] In other embodiments, the polynucleotide includes modifications that reduce its stability.

[0210] In some embodiments, modified nucleosides and nucleotides include modified nucleic acid bases. Examples of nucleic acid bases in RNA include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). Examples of nucleic acid bases in DNA include, but are not limited to, adenine (A), guanine (G), cytosine (C), and thymine (T).

[0211] In some embodiments, the modified nucleic acid base is modified uracil (U). Examples of nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, and 2-thiouridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine (I 5 U) or 5-bromo-uridine (br 5 U)), 3-methyluridine(m 3 U), 5-methoxyuridine (mo 5 U), Uridine 5-oxyacetic acid (cmo 5 U), Uridine 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudolidine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-uridine (m 5 U, i.e., having the nucleic acid base deoxythymine), 1-methylpseudolidine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), pseudouracil (ψ), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m 3 ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psuduridine, 4-methoxy-2-thio-psuduridine, N1-methylpsuduridine (1-methylpsuduridine (m 1 Also known as ψ), 3-(3-amino-3-carboxypropyl)uridine (acp 3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5 Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine are included.

[0212] In some embodiments, the modified nucleobase is a modified cytosine (C). Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine(s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine(k2C), α-thio-cytidine, 2'-O-methyl-cytidine(Cm), 5,2'-O-dimethyl-cytidine(m 5 Cm), N4-acetyl-2'-O-methyl-cytidine(ac 4 Cm), N4,2'-O-dimethyl-cytidine(m 4 Cm), 5-formyl-2'-O-methyl-cytidine(f 5 Cm), N4,N4,2'-O-trimethyl-cytidine(m 4 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine are included.

[0213] In some embodiments, the modified nucleobase is a modified adenine (A). Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine(m 1 A), 2-methyl-adenosine(m 2 A), N6-methyl-adenosine(m 6A) 2-methylthio-N6-methyladenosine (ms 2 m 6 A) N6-isopentenyl-adenosine (i 6 A) 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A) N6-(cis-hydroxyisopentenyl)adenosine (io 6 A) 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A) N6-Glycinylcarbamoyl-adenosine (g 6 A) N6-Threonylcarbamoyl-adenosine (t 6 A) N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A) 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A) N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A) 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A) N6-acetyl-adenosine (ac 6 A) 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Examples include Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ala-adenosine, 2'-F-adenosine, 2'-OH-ala-adenosine, and N6-(19-amino-pentaaxanonadecyl)-adenosine.

[0214] In some embodiments, the modified nucleic acid base is modified guanine (G). Exemplary nucleic acid bases and nucleosides having modified guanine include inosine (I) and 1-methyl-inosine (m). 1 I) Viosin (imG), Methyl Viosin (mimG), 4-Demethyl Viosin (imG-14), Iso Viosin (imG2), Vibutosin (yW), Peroxy Vibutosin (o2yW), Hydroxy Vibutosin (OHyW), Unmodified Hydroxy Vibutosin (OHyW*), 7-Deaza-Guanosine, Quosin (Q), Epoxy Quosin (oQ), Galactosyl Quosin (galQ), Mannosyl Quosin (manQ), 7-Cyano-7-Deaza-Guanosine (preQ0), 7-Aminomethyl-7-Deaza-Guanosine (preQ1), Archaea (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2, N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1 Examples include Im) and 2'-O-ribosylguanosine (phosphate) (Gr(p)).

[0215] In some embodiments, the nucleic acid bases of the nucleotides are independently selected from purines, pyrimidines, purines, or pyrimidine analogs. In some embodiments, the nucleic acid bases and / or analogs are independently selected from adenine, guanine, uracil, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudoluracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl Selected from naturally occurring and synthetic derivatives of bases, including but not limited to, other 8-substituted adenines and guanines, 5-halos, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinon, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; and 1,3,5-triazine.

[0216] In some embodiments, the polynucleotides include nucleoside modifications. In some embodiments, one or more atoms of the pyrimidine nucleic acid bases are substituted or replaced with, for example, optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or optionally substituted or halo (e.g., chloro or fluoro) atoms or groups.

[0217] In some embodiments, all uracil nucleosides of the polynucleotides of the disclosure are modified. In some embodiments, all guanine nucleosides of the polynucleotides of the disclosure are modified. In some embodiments, all cytosine nucleosides of the polynucleotides of the disclosure are modified. In some embodiments, all thymine nucleosides of the polynucleotides of the disclosure are modified. In some embodiments, all adenine nucleosides of the polynucleotides of the disclosure are modified. In some embodiments, the modifications to each nucleic acid base are the same. In some embodiments, the modifications to each nucleic acid base are different.

[0218] In some embodiments, the modifications of the modified nucleoside and nucleotide are located on the sugar subunit. In some embodiments, the polynucleotides described herein include at least one sugar modification. Generally, RNA includes a sugar subunit: ribose, which is a five-membered ring having oxygen. In some embodiments, the 2'-hydroxyl group (OH) may be modified or substituted with several different substituents. Exemplary substitutions at the 2'-OH- position include, but are not limited to, H, halo, and optionally substituted C. 1~6 Alkyl, and in some cases substituted C 1~6 Alkoxy; C may be substituted. 6~10 Aryloxy; C may be substituted. 3~8 Cycloalkyl; optionally substituted C 3~8 Cycloalkoxy; optionally substituted C 6~10 Aryloxy; C may be substituted. 6~10 Aryl-C 1~6 Alkoxy, and in some cases substituted C 1~12(Heterocyclyl)oxy; sugar (e.g., ribose, pentose, or any as described herein); polyethylene glycol (PEG) O(CH2CH2O)nCH2CH2OR (where R is H or optionally substituted alkyl, and n is an integer from 0 to 20) (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20 (including all values and subranges therebetween)); the 2'-hydroxyl is C 1~6 an alkylene bridge or C 1~6 a "locked" nucleic acid (LNA) connected to the 4'-carbon of the same ribose sugar by an alkylene bridge or a heteroalkylene bridge, where exemplary bridges include a methylene bridge, a propylene bridge, an ether bridge, or an amino bridge; aminoalkyl; aminoalkoxy; amino; and amino acid.

[0219] Other exemplary sugar modifications include substitution of oxygen (O) in ribose (e.g., by S, Se, or an alkylene such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); cyclocondensation of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6-membered or 7-membered ring having additional carbon atoms or heteroatoms, such as in the case of anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino which also have a phosphoramidate backbone); polycyclic forms (e.g., tricyclo); and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA where ribose is replaced by glycol units linked by phosphodiester bonds), threose nucleic acid (TNA, where ribose is replaced by α-L-threofuranosyl- (3'→2')), and peptide nucleic acid (PNA where 2-amino-ethyl-glycine bonds replace the ribose and phosphodiester backbone).

[0220] In some embodiments, the sugar subunit contains one or more carbons having a stereochemical configuration opposite to that of the corresponding carbon in ribose. In some embodiments, the polynucleotides described herein include nucleotides that contain, as the sugar, for example, arabinose.

[0221] Non-limiting examples of sugar modifications can include the modifications provided in Table 1. In some embodiments, the polynucleotides of the present disclosure have one or more nucleotides having the modifications provided in Table 1. In some embodiments, each of the nucleotides of the polynucleotides described herein bears any one of the modifications as shown in Table 1 or bears none of the modifications as shown in Table 1.

[0222] [Table 1]

[0223] [Table 2]

[0224] [Table 3]

[0225] In some embodiments, at least one of the 2'-positions of the sugar (OH in RNA or H in DNA) of the nucleotides of the polynucleotide is substituted with -O-methoxyethyl, called 2'-OMe. In some embodiments, at least one of the 2'-positions of the sugar (OH in RNA or H in DNA) of the nucleotides of the polynucleotide is substituted with -F, called 2'-F. In some embodiments, the sugar modification is one or more locked nucleic acids (LNAs). In some embodiments, the polynucleotide is fully 2'-MOE-sugar modified.

[0226] In some embodiments, one or more modifications are present in the nucleoside bond (linked phosphate or phosphodiester bond or phosphodiester skeleton). In the context of polynucleotide skeletons, the terms "phosphate" and "phosphodiester" are used interchangeably.

[0227] In some embodiments, the skeletal phosphate group is modified by substituting one or more oxygen atoms with different substituents. In some embodiments, the modified nucleosides and nucleotides include substitution of the unmodified phosphate moiety by another nucleoside bond, as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, methylphosphonates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramides, phosphorodiamidates, alkyl or arylphosphonates, and phosphotryesters. In phosphorodithionates, both unbonded oxygen atoms are replaced by sulfur. Phosphate linkers are also modified by substituting the bonded oxygen with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylene-phosphonate).

[0228] The α-thio-substituted phosphate moiety is provided to confer stability to RNA and DNA polynucleotides via non-natural phosphorothioate backbone binding. Phosphorothioate-bound DNA and RNA exhibit increased nuclease resistance, followed by a longer half-life in the cellular environment. Phosphorothioate-bound polynucleotide molecules are also expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0229] In some embodiments, the polynucleotides of the present disclosure include, as modifications, at least one phosphorothioate bond, an internucleotide methylphosphonate bond, a 5'-(E)-vinylphosphonate (5'-E-VP), and a phosphate mimetic.

[0230] In some embodiments, the nucleoside bonds of polynucleotides may be partially or completely modified.

[0231] In some embodiments, the modified nucleotides incorporated into the polynucleotide include, for example, 2'-O-methyl modified or 2'-O-methoxyethyl modified nucleotides (2'-OMe and 2'-MOE modified, respectively), and α-thionucleosides (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thiocytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudridine).

[0232] In some embodiments, different sugar modifications, nucleic acid base modifications, and / or internucleoside bonds (e.g., in the main chain structure) are introduced at various positions in the polynucleotides described herein. Those skilled in the art will understand that nucleotide analogs or other modifications can be placed at any position in the polynucleotide such that the function of the polynucleotide is not substantially impaired.

[0233] In some embodiments, one or more modified nucleotides are 2'-O-methyl or phosphorothioate modified nucleotides. Therefore, in some embodiments, one or more modified nucleotides include 2'-O-methyl modification. In some embodiments, one or more modified nucleotides include phosphorothioate modification.

[0234] In some embodiments, one or more modified nucleotides are selected from 2'-O-methyl-3'-phosphorothioate, 2'-O-methyl, 2'-ribo-3'-phosphorothioate, 2'-fluoro, 2'-O-methoxyethylmorpholino (PMO), locked nucleic acid (LNA), deoxy, or 5'-phosphate modified nucleotides. Therefore, in some embodiments, one or more modified nucleotides are 2'-O-methyl-3'-phosphorothioate. In some embodiments, one or more modified nucleotides are 2'-O-methyl nucleotides. In some embodiments, one or more modified nucleotides are 2'-ribo-3'-phosphorothioate. In some embodiments, one or more modified nucleotides are 2'-fluoro nucleotides. In some embodiments, one or more modified nucleotides are locked nucleic acid (LNA). In some embodiments, one or more modifications include 2'-O-methoxyethylmorpholino (PMO). In some embodiments, one or more modifications include deoxy modifications. In some embodiments, one or more modifications include 5'-phosphate modifications.

[0235] Various modified RNA bases are known in the art, including, for example, 2'-O-methoxyethyl bases (2'-MOE) such as 2-methoxyethoxy A, 2-methoxyethoxy MeC, 2-methoxyethoxy G, and 2-methoxyethoxy T. Other modified bases include, for example, 2'-O-methyl RNA bases and fluoro bases. Various fluoro bases are known, including, for example, Fluoro C, Fluoro U, Fluoro A, and Fluoro G bases. Various 2'OMethyl modifications can also be used in conjunction with the methods described herein. For example, the following RNAs containing one or more of the following 2'-O-methyl modifications can be used in conjunction with the described methods. 2'-OMe-5-methyl-rC, 2'-OMe-rT, 2'-OMe-rI, 2'-OMe-2-amino-rA, aminolinker-C6-rC, aminolinker-C6-rU, 2'-OMe-5-Br-rU, 2'-OMe-5-I-rU, 2'-OMe-7-Deaza-rG.

[0236] In some embodiments, the RNA includes one or more of the following modifications: phosphorothioate, 2'-O-methyl, 2'-fluoro(2'F), DNA. In some embodiments, the RNA includes 2'-OMe modifications at the 3' and 5' ends. In some embodiments, the RNA includes one or more of the following modifications: 2'-O-2-methoxyethyl (MOE), locked nucleic acid, cross-linked nucleic acid, unlocked nucleic acid, peptide nucleic acid, morpholino nucleic acid. In some embodiments, the RNA includes one or more of the following base modifications: 2,6-diaminopurine, 2-aminopurine, pseudouracil, N1-methyl-pseudracil, 5'-methylcytosine, N6-methyladenosine, 2'pyrimidinone (zebralin), thymine. Other modified bases include, for example, 2-aminopurine, 5-bromo-dU, deoxyuridine, 2,6-diaminopurine (2-amino-dA), dideoxy-C, deoxyinosine, hydroxymethyl-dC, inverted-dT, Iso-dG, Iso-dC, inverted-dideoxy-T, 5-methyl-dC, 5-methyl-dC, 5-nitroindole, Super T(registered trademark), 2'-Fr(C,U), 2'-NH2-r(C,U), 2,2'-anhydro-U, 3'-deoxy-r(A,C,G,U), 3'-O-methyl-r(A,C,G,U), rT, rI, 5-methyl-rC, 2-amino-rA, rSpacer (debasic), 7-deaza-rG, 7-deaza-rA, 8-oxo-rG, 5-halogenated-rU, ​​and N-alkylated-rN.

[0237] In some embodiments, other chemically modified RNAs are used herein. For example, the RNA can include modified bases such as, for example, 5’, Int, 3’ azide (NHS ester); 5’ hexynyl; 5’, Int, 3’ 5-octadiynyl dU; 5’, Int biotin (azide); 5’, Int 6-FAM (azide); and 5’, Int 5-TAMRA (azide). Other examples of RNA nucleotide modifications that can be used with the methods described herein include, for example, phosphorylation modifications such as 5’-phosphorylation and 3’-phosphorylation. The RNA can also have one or more of the following modifications. Amino modification, biotinylation, thiol modification, alkyne modification, adenylation, azide (NHS ester), cholesterol-TEG, and digoxigenin (NHS ester).

[0238] RNA Production In some aspects, the disclosure provides a method for producing RNA having a quantified percentage of capped mRNA and non-tailed mRNA, comprising: (a) providing in vitro synthesized RNA; (b) annealing the produced RNA with an oligonucleotide complementary to a sequence within the 5’ UTR of the RNA and an oligonucleotide complementary to a sequence within the 3’ UTR of the RNA; (c) treating the RNA with a nuclease to cleave the RNA into cap fragments and tail fragments; (d) identifying capping species and non-tailed species by liquid chromatography (LC-UV) using UV detection and / or identifying and measuring capping species, non-tailed species, and characterizing the polyA tail by liquid chromatography (LC-MS) combined with mass spectrometry and / or liquid chromatography (LC-UV-MS) combined with mass spectrometry using UV detection; (e) quantifying the relative amount of each capping species, quantifying the relative amount of non-tailed species using LC-UV or LC-MS or LC-UV-MS, and characterizing the polyA tail using LC-MS or LC-UV-MS, simultaneously in a single sample, thereby producing RNA comprising a quantified percentage of capped mRNA and non-tailed mRNA.

[0239] In some embodiments, RNA is produced. In some embodiments, in vitro transcription RNA is provided. In vitro transcription is carried out by synthesizing mRNA using a linear DNA template, nucleotides, and enzymes, for example, in a disposable bioreactor. In various embodiments, synthesis includes designing the DNA template, codon optimization, promoter selection (e.g., T7, SP6), selection of 5'UTR and 3'UTR sequences, and optionally, inclusion of modified nucleotides to enhance protein translation, improve stability, or reduce immunogenicity. Strain optimization and screening are performed to expand suitable templates and select suitable strains to generate a GMP cell bank. In some embodiments, the mRNA product is co-transcribed, or post-synthesis processing is performed, such as enzymatic capping and tailing.

[0240] In some embodiments, a 5' cap is enzymatically added to the RNA from the in vitro synthesis reaction after transcription. In some embodiments, the in vitro synthesized RNA does not contain a 5' cap.

[0241] In some embodiments, the 3' tail is encoded in the plasmid during in vitro synthesis.

[0242] In some embodiments, a 3' tail is enzymatically added to the RNA from step (a) after transcription.

[0243] In some embodiments, the RNA does not contain a 3' tail.

[0244] In some embodiments, RNA is not spliced.

[0245] In some embodiments, RNA is spliced.

[0246] In some embodiments, a 5' cap is enzymatically added to the RNA from step (a) after transcription, and the 3' tail is encoded in the plasmid during in vitro synthesis.

[0247] In some embodiments, a 5' cap is enzymatically added to the RNA from step (a) after transcription, and the RNA does not contain a 3' tail.

[0248] In some embodiments, the RNA does not contain a 5' cap, and the 3' tail is encoded in the plasmid during in vitro synthesis.

[0249] In some embodiments, the RNA does not contain a 5' cap, and the RNA does not contain a 3' tail.

[0250] In various embodiments, nucleotides, reagents, enzymes, and process conditions, such as temperature and incubation time, are selected. The mRNA is then isolated and purified using magnetic bead-based, affinity, or other chromatography and filtration techniques such as ultrafiltration / diafiltration (UF / DF).

[0251] mRNA is tested for various quality attributes, such as sequence verification and UTR identity verification by PCR. In some embodiments, RNA content is quantified, for example, by UV measurement. Potency is tested by activity assays, such as in vitro transcription assays. Purity and integrity are tested by the methods of this disclosure to evaluate the percentage of intact and fragmented mRNA, 5' capping efficiency and 3' poly(A) (%) or length, and to assess the presence of undesirable immunostimulatory RNA byproducts. The prepared RNA drug material is further tested for the presence of residual impurities (e.g., dsRNA, protein or template) and for safety.

[0252] Quality control of manufactured RNA In some embodiments, the present disclosure provides a quality control assay for a manufacturing lot, which includes (a) providing a manufactured RNA sample containing a 5' cap and / or 3' tail, (b) quantifying RNA capping efficiency and tailing efficiency by a method provided herein, and (c) comparing the capping and tailing efficiency in step (b) with the capping and tailing efficiency in a reference sample, wherein a decrease in the amount of capping species compared to the reference sample indicates 5' degradation, and an increase in the amount of non-tailed species and / or different poly-A tail lengths or polydispersity indicates 3' degradation, thereby simultaneously monitoring 5' and 3' degradation of the RNA product and determining the product quality of the manufactured lot.

[0253] In some embodiments, the reference sample is a stable RNA sample with greater than 75% integrity. In some embodiments, the reference sample is a stable RNA sample with integrity of 75–80%, 80–85%, 85–90%, 90–95%, or 95–100% (including all values ​​and partial ranges in between). In some embodiments, the reference sample is a sample from a manufacturing lot that has been previously tested and found to be suitable for release. In some embodiments, the reference sample is from one or more previous batches to ensure batch-to-batch consistency.

[0254] Purified mRNA therapeutics or vaccines (i.e., mRNA preparations) are formulated by combining mRNA with a delivery vehicle such as LNPs or other lipids or carbohydrates. Once formulated, the mRNA-containing preparation is processed into the final vaccine or therapeutic, sterilized, aseptically filled, and packaged. Each lot of the filled packages is evaluated for quality control and batch-to-batch consistency before the release of the manufactured lot. Lots that pass the quality control assay are stored in ultra-low temperature freezers for storage, distribution, and delivery.

[0255] In some embodiments, the RNA product is therapeutic mRNA.

[0256] In some embodiments, the RNA product is an mRNA vaccine.

[0257] kit This disclosure further provides kits comprising various reagents and materials useful for carrying out the methods of the present invention as disclosed herein. The procedures described herein may be carried out in a diagnostic laboratory, laboratory, or commercial laboratory. This disclosure provides kits that can be used in these different settings.

[0258] For example, materials and reagents for quantifying mRNA capping and tailing efficiency in mRNA samples by enzymatic manipulation and chromatographic separation (LC-UV, LC-MS, or LC-UV-MS) combined with UV and / or mass spectrometry can be assembled together in a kit. In certain embodiments, the kit includes a chromatographic column, as well as agents for separating capped mRNA species, uncapped mRNA species, and untailed mRNA species on the column, and instructions for using the kit according to the methods of this disclosure.

[0259] In some embodiments, each kit includes customized reagents to make the treatment target-specific. Thus, to detect / quantify the mRNA capping and tailing efficiency of a specific target, the kit includes a hybrid oligonucleotide reagent of a design sequence that specifically anneals adjacent to the 5' and 3' ends of the target. In some embodiments, the kit includes a nuclease for constructing the capped fragment, e.g., RNase H and / or S1 nuclease. In some embodiments, the kit further includes in vitro transcription and capping reagents, enzymes, and instructions for their use.

[0260] The kits or other products described herein include one or more containers for holding various reagents. Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), and ampoules. The containers may be made from a variety of materials, such as glass or plastic.

[0261] In some embodiments, the kits of the present disclosure include appropriate standards or control levels or standards or control samples for determining the control levels described herein. In some embodiments, the kits of the present disclosure include instructions for using the kit according to one or more methods of the present disclosure. In some embodiments, the kits further include instructions for in vitro transfer and capping.

[0262] A first set of representative embodiments of the present disclosure 1. A single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications, (a) To provide a sample comprising RNA and optionally having a 5' cap and / or a 3' poly-A tail, wherein the sample further comprises a first oligonucleotide probe complementary to the sequence in the 5' untranslated region (UTR) of the RNA and a second oligonucleotide probe complementary to the sequence in the 3' untranslated region (UTR) of the RNA. (b) Annealing the RNA sample with a first oligonucleotide probe complementary to the sequence in the 5'UTR of the RNA and a second oligonucleotide probe complementary to the sequence in the 3'UTR of the RNA, (c) The RNA sample from step (b) is treated with a nuclease to cleave the RNA into cap and tail fragments, (d) i. Identifying capping species in capping fragments by measuring the retention time of peaks in chromatograms generated from liquid chromatography using ultraviolet detection (LC-UV) and / or mass spectra generated by LC (LC-MS) or LC-UV-MS combined with mass spectrometry. ii. Identifying non-tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS, and iii. Identifying tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS. Therefore, using the sample from step (c), perform LC-UV, LC-MS, or LC-UV-MS. This allows for the simultaneous identification of RNA capping modifications and tailing modifications in a single sample, A single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications, including [specific modifications].

[0263] 2. A single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency, (a) To provide a sample containing RNA and optionally having a 5' cap and / or a 3' poly-A tail, (b) Annealing the RNA sample with a first oligonucleotide probe complementary to the sequence of the 5' untranslated region (UTR) of the RNA and a second oligonucleotide probe complementary to the sequence of the 3' untranslated region (UTR) of the RNA, (c) The RNA sample from step (b) is treated with a nuclease to cleave the RNA into cap and tail fragments, (d) Performing LC (LC-MS) or LC-UV-MS in combination with ultraviolet detection liquid chromatography (LC-UV) or mass spectrometry, (e) Measure the peak area of ​​each capping species in the capped fragment, and each non-tailed and tailed species in the tailed fragment, (f) Quantify the relative amounts of each capping species and the relative amounts of non-tailed species, characterize the poly(A) tails in the sample from step (d), thereby simultaneously quantifying the RNA capping efficiency and tailing efficiency in the sample. A single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency, including the above.

[0264] 3. The method according to the numbered embodiment 2, wherein characterizing the poly-A tail includes (i) measuring the mass of the poly-A tail species, deconvolution of the mass spectrometry signal, thereby providing the mass distribution of the tail species, and (ii) determining the minimum length, maximum length, average length and polydispersity of the poly-A tail based on the corresponding mass distribution.

[0265] 4. The method according to numbered embodiment 1 or 2, wherein the RNA is in vitro transcribed mRNA.

[0266] 5. The method according to numbered embodiment 1 or 2, wherein RNA is obtained from the manufacturing process.

[0267] 6. The method according to numbered embodiment 1 or 2, wherein RNA is obtained from the active pharmaceutical ingredient (DS) in the final step of the manufacturing process.

[0268] 7. The method according to numbered embodiment 1 or 2, wherein RNA is obtained from a deformulated drug product (DP).

[0269] 8. The method according to any one of the numbered embodiments 1 to 7, wherein the RNA is not modified.

[0270] 9. The method according to any one of the numbered embodiments 1 to 8, wherein the RNA is modified.

[0271] 10. The method according to numbered embodiment 1 or 2, wherein the nuclease is RNAse H.

[0272] 11. The method according to numbered embodiment 1 or 2, wherein the masses of the cap fragment and tail fragment are measured by LC-MS.

[0273] 12. The method according to numbered embodiment 1 or 2, wherein capping species, non-tail species, and tail species are identified from the UV signal by comparing the retention time of the obtained peak with the retention time of a reference standard.

[0274] 13. The method according to any one of the numbered embodiments 1 to 12, wherein the capping type is cap 1, cap 0, cap G, or uncapped.

[0275] 14. The method according to any one of the numbered embodiments 1 to 13, wherein the capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, GGACA, or pppGGACA.

[0276] 15. The method according to numbered embodiment 13 or 14, wherein the uncapped species is GGACA or pppGGACA.

[0277] 16. The method according to any one of the numbered embodiments 1 to 15, wherein the non-tail species is UGCAUC and U is unmodified uridine.

[0278] 17. The method according to any one of the numbered embodiments 1 to 16, wherein the non-tail species is U*GCAU*C and U* is N1-methylpseudolidine.

[0279] 18. The method according to any one of the numbered embodiments 1 to 17, wherein the relative amount of each capping species is a percentage of the total amount of capping species in the sample, calculated by dividing the area under the peak of the capping species of interest by the sum of the total areas under the peaks representing cap 1, cap 0, cap G, and uncapped species, and multiplying by 100.

[0280] 19. The method according to any one of the numbered embodiments 1 to 18, wherein the relative amount of the non-tail species is a percentage calculated by dividing the area under the peak of the non-tail species by the sum of the areas under the peak of cap 1, cap 0, cap G and the uncapped species, and multiplying by 100.

[0281] 20. The method according to any one of the numbered embodiments 1 to 19, wherein the oligonucleotide is approximately 10 to 40 nucleotides in length.

[0282] 21. The method according to any one of the numbered embodiments 1 to 20, wherein the oligonucleotide comprises RNA bases and DNA bases.

[0283] 22. The method according to any one of the numbered embodiments 1 to 21, wherein the oligonucleotide comprises RNA and DNA bases in a ratio of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1.

[0284] 23. The method according to any one of the numbered embodiments 1 to 22, wherein the oligonucleotide is 10 to 40 nucleotides long and contains 4 DNA bases.

[0285] 24. The method according to the numbered embodiment 23, wherein the oligonucleotide complementary to the sequence of the 5' untranslated region of RNA is 3'-CCTGTCUAGCGGACCU-5' (SEQ ID NO: 1), and the italicized CTGT is a DNA base.

[0286] 25. The method according to the numbered embodiment 23, wherein the oligonucleotide complementary to the sequence of the 3' untranslated region of RNA is 3'-GGUCGGAACAGGAUUAUUUUAATTCAA-5' (SEQ ID NO: 2), and the italicized TTCA is a DNA base.

[0287] 26. The method according to any one of the numbered embodiments 1 to 25, wherein capping species and non-tail species are quantified by single LC-UV, LC-MS, or LC-UV-MS analysis.

[0288] 27. The method according to numbered embodiment 1, wherein the tail species is characterized by single LC-MS or LC-UV-MS analysis.

[0289] 28. The method according to any one of the numbered embodiments 1 to 27, wherein one or more steps are automated.

[0290] 29. Oligonucleotides complementary to the 5'UTR or 3'UTR sequence of RNA, wherein the oligonucleotides are approximately 10 to 40 nucleotides long and contain RNA bases and DNA bases, and are complementary to the 5'UTR or 3'UTR sequence of RNA.

[0291] 30. The oligonucleotide according to numbered embodiment 29, wherein the oligonucleotide comprises RNA and DNA bases in a ratio of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1.

[0292] 31. The oligonucleotide according to any one of the numbered embodiments 1 to 30, wherein the oligonucleotide is approximately 10 to 40 nucleotides long and contains 4 DNA bases.

[0293] 32. The oligonucleotide according to any one of the numbered embodiments 1 to 31, wherein the oligonucleotide complementary to the sequence in the 5'UTR of RNA is 3'-CCTGTCUAGCGGACCU-5' (SEQ ID NO: 1), and the italicized CTGT is a DNA base.

[0294] 33. The oligonucleotide according to any one of the numbered embodiments 1 to 32, wherein the oligonucleotide complementary to the sequence in the 3'UTR of RNA is 3'-GGUCGGAACAGGAUUAUUUUAATTCAA-5' (SEQ ID NO: 2), and the italicized TTCA is a DNA base.

[0295] 34. A method for producing RNA having a quantified percentage of capped mRNA and untailed mRNA, (a) To provide in vitro synthetic RNA, (b) Annealing the produced RNA with an oligonucleotide complementary to the sequence in the 5'UTR of the RNA and an oligonucleotide complementary to the sequence in the 3'UTR of the RNA, (c) Treating RNA with a nuclease to cleave the RNA into cap and tail fragments, (d) Identify capping species and non-tail species by liquid chromatography with UV detection (LC-UV), and / or identify and measure the masses of capping species and non-tail species by liquid chromatography combined with mass spectrometry (LC-MS) and / or liquid chromatography combined with mass spectrometry using UV detection (LC-UV-MS), thereby characterizing the poly-A tail. (e) Quantify the relative amount of each capping species, quantify the relative amount of non-tail species using LC-UV, LC-MS, or LC-UV-MS, characterize the poly-A tail using LC-MS or LC-UV-MS, and simultaneously in a single sample, This allows for the production of RNA containing quantified percentages of capped mRNA and untailed mRNA, Methods that include...

[0296] 35. The method according to the numbered embodiment 34, wherein a 5' cap is enzymatically added to the RNA from step (a) after transcription.

[0297] 36. The method according to the numbered embodiment 34, wherein the RNA does not contain a 5' cap.

[0298] The method according to numbered embodiment 34, wherein the 37.3' tail is encoded in the plasmid during in vitro synthesis.

[0299] 38. The method according to the numbered embodiment 34, wherein a 3' tail is enzymatically added to the RNA from step (a) after transcription.

[0300] 39. The method according to the numbered embodiment 34, wherein the RNA does not contain a 3' tail.

[0301] 40. The method according to the numbered embodiment 34, wherein the RNA is not spliced.

[0302] 41. The method according to the numbered embodiment 34, wherein RNA is spliced.

[0303] 42. A quality control assay for a manufacturing lot, wherein the assay is (a) To provide a manufactured RNA sample including a 5' cap and / or a 3' tail, (b) To quantify the RNA capping efficiency and tailing efficiency in a sample by the method described in claim 2, (c) Compare the capping efficiency and tailing efficiency in step (b) with the capping efficiency and tailing efficiency in the reference sample, Includes, The amount of capping species reduced compared to the reference sample indicates 5' decomposition. Increased amount of non-tail species and / or different poly-A tail lengths or polydispersity exhibits 3' decomposition. This includes simultaneously monitoring 5' and 3' degradation of RNA products and determining the quality of the product from the manufactured lot. Quality control assay for manufacturing lots.

[0304] 43. The quality control assay according to numbered embodiment 42, wherein the reference sample is a stable RNA sample having greater than 75% integrity.

[0305] 44. The quality control assay according to numbered embodiment 42, wherein the RNA product is therapeutic mRNA.

[0306] 45. A quality control assay for a numbered embodiment 43 in which the RNA product is an mRNA vaccine.

[0307] A second set of representative embodiments of the present disclosure 1. A single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications, (a) To provide a sample comprising RNA and optionally having a 5' cap and / or a 3' poly-A tail, wherein the sample further comprises a first oligonucleotide probe complementary to the sequence in the 5' untranslated region (UTR) of the RNA and a second oligonucleotide probe complementary to the sequence in the 3' untranslated region (UTR) of the RNA. (b) Annealing the RNA sample with a first oligonucleotide probe complementary to the sequence in the 5'UTR of the RNA and a second oligonucleotide probe complementary to the sequence in the 3'UTR of the RNA, (c) The RNA sample from step (b) is treated with a nuclease to cleave the RNA into cap and tail fragments, (d) i. Identifying capping species in capping fragments by measuring the retention time of peaks in chromatograms generated from liquid chromatography using ultraviolet detection (LC-UV) and / or mass spectra generated by LC (LC-MS) or LC-UV-MS combined with mass spectrometry. ii. Identifying non-tail and tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS. iii. Identifying non-tail and tail species in tail fragments by measuring the retention time of peaks in chromatograms generated from LC-UV and / or mass spectra generated by LC-MS or LC-UV-MS. Therefore, using the sample from step (c), perform LC-UV, LC-MS, or LC-UV-MS. This allows for the simultaneous identification of RNA capping modifications and tailing modifications in a single sample, A single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications, including [specific modifications].

[0308] 2. A single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency, (a) To provide a sample containing RNA and optionally having a 5' cap and / or a 3' poly-A tail, (b) Annealing the RNA sample with a first oligonucleotide probe complementary to the sequence of the 5' untranslated region (UTR) of the RNA and a second oligonucleotide probe complementary to the sequence of the 3' untranslated region (UTR) of the RNA, (c) The RNA sample from step (b) is treated with a nuclease to cleave the RNA into cap and tail fragments, (d) Performing LC (LC-MS) or LC-UV-MS in combination with ultraviolet detection liquid chromatography (LC-UV) or mass spectrometry, (e) Measure the peak area of ​​each capping species in the capped fragment, and each non-tailed and tailed species in the tailed fragment, (f) Quantify the relative amounts of each capping species and the relative amounts of non-tailed species, characterize the poly(A) tails in the sample from step (d), thereby simultaneously quantifying the RNA capping efficiency and tailing efficiency in the sample. A single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency, including the above.

[0309] 3. The method according to the numbered embodiment 2, wherein characterizing the poly-A tail includes (i) measuring the mass of the poly-A tail species, deconvolution of the mass spectrometry signal, thereby providing the mass distribution of the tail species, and (ii) determining the minimum length, maximum length, average length and polydispersity of the poly-A tail based on the corresponding mass distribution.

[0310] 4. The following conditions: (a) RNA is in vitro transcribed mRNA. (b) RNA is obtained from the manufacturing process, (c) RNA is obtained from the active pharmaceutical ingredient (DS) in the final stage of manufacturing. (d) RNA is obtained from deformed drug products (DP), The method according to any one of the numbered embodiments 1 to 3, wherein one or more of the following conditions are met.

[0311] 5. The method according to any one of the numbered embodiments 1 to 4, wherein the RNA is either unmodified or modified.

[0312] 6. The following conditions: (a) The nuclease is RNAse H, (b) The masses of the cap and tail fragments are measured by LC-MS. (c) By comparing the retention time of the obtained peaks with the retention time of a reference standard, the capping species, non-tailed species, and tailed species are identified from the UV signal. (d) The capping type is cap 1, cap 0, cap G, or uncapped. (e) The capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA. (f) The capped species are Cap 1, Cap 0, Cap G, or uncapped, and the uncapped species are GGACA. (g) The capped varieties are cap 1, cap 0, cap G, or uncapped, and the uncapped varieties are pppGGACA. (h) The capping species are m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA, and the non-capping species is GGACA. (i) The capping species are m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA, and the non-capping species is pppGGACA. (j) The non-tail species is UGCAUC, where U is unmodified uridine. (k) The non-tail species is U*GCAU*C, where U* is N1-methylpseudolidine. (l) The capped species are cap 1, cap 0, cap G, or uncapped, and the non-tail species is UGCAUC, where U is unmodified uridine. (m) The capped species is Cap 1, Cap 0, Cap G, or uncapped, and the non-tail species is U*GCAU*C, where U* is N1-methylpseudolidine. (n) The capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA, and the non-tail species is UGCAUC, where U is unmodified uridine. (o) The capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA or pppGACA, and the non-tail species is U*GCAU*C, where U* is N1-methylpseudolidine. (p) The capping species is cap 1, cap 0, cap G, or uncapped, the uncapped species is GGACA or pppGGACA, the non-tail species is UGCAUC, and U is unmodified uridine. (q) The capped species are Cap 1, Cap 0, Cap G, or uncapped; the uncapped species is GGACA or pppGGACA; the non-tailed species is U*GCAU*C, where U* is N1-methylpseudolidine. (r) The capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA; the uncapped species is GGACA or pppGGACA; the untailed species is UGCAUC, where U is unmodified uridine. (s) The capping species is m7Gpppm7GGACA, m7GpppGGACA, GpppGGACA, or GGACA; the uncapped species is GGACA or pppGGACA; the untailed species is U*GCAU*C, where U* is N1-methylpseudolidine. The method according to any one of the numbered embodiments 1 to 5, wherein one or more of the following conditions are met.

[0313] 7. The method according to any one of the numbered embodiments 1 to 6, wherein the relative amount of each capping species is a percentage of the total amount of capping species in the sample, calculated by dividing the area under the peak of the capping species of interest by the sum of the total areas under the peak of cap 1, cap 0, cap G and uncapped species, and multiplying by 100, and / or the relative amount of uncapped species is a percentage calculated by dividing the area under the peak of the uncapped species by the sum of the areas under the peak of cap 1, cap 0, cap G and uncapped species, and multiplying by 100.

[0314] 8. The following conditions: (a) Oligonucleotide probes are approximately 10-40 nucleotides in length. (b) Oligonucleotide probes include RNA bases and DNA bases. (c) Oligonucleotide probes contain RNA bases and DNA bases in ratios of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. (d) Oligonucleotide probes are 10 to 40 nucleotides long and contain 4 DNA bases. (e) The oligonucleotide probe contains RNA and DNA bases, and the oligonucleotide probe complementary to the sequence in the 5' untranslated region of RNA is 3'-CCTGTCUAGCGGACCU-5' (SEQ ID NO: 1), where the italicized CTGT is a DNA base, and (f) The oligonucleotide probe contains RNA and DNA bases, and the oligonucleotide is complementary to the sequence of the 3' untranslated region of the RNA. [ka] And the italicized TTCA are DNA bases. A method according to any one of the numbered embodiments 1 to 7, wherein one or more of the following conditions are met.

[0315] 9. The following conditions: (a) Quantify capping species and non-tail species by single LC-UV, LC-MS, or LC-UV-MS analysis. (b) Tail species are characterized by single LC-MS or LC-UV-MS analysis, and (c) One or more processes are automated, The method according to any one of the numbered embodiments 1 to 8, wherein one or more of the following conditions are met.

[0316] 10. Oligonucleotides complementary to the 5'UTR or 3'UTR sequence of RNA, wherein the oligonucleotide probe is approximately 10-40 nucleotides long and contains RNA bases and DNA bases.

[0317] 11. The following conditions: (a) Oligonucleotide probes contain RNA bases and DNA bases in ratios of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. (b) Oligonucleotide probes are 10-40 nucleotides long and contain 4 DNA bases. (c) Oligonucleotide probes contain RNA bases and DNA bases in ratios of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1, and each oligonucleotide probe is approximately 10-40 nucleotides long and contains 4 DNA bases. (d) The oligonucleotide probe complementary to the sequence in the 5'UTR of RNA is 3'-CCTGTCUAGCGGACCU-5' (SEQ ID NO: 1), where the italicized CTGT is a DNA base, and (e) Oligonucleotide probes complementary to the sequence within the 3'UTR of RNA are [ka] And the italicized TTCA are DNA bases. The method according to the numbered embodiment 11, wherein one or more of the following conditions are met.

[0318] 12. A method for producing RNA having a quantified percentage of capped mRNA and untailed mRNA, (a) A step of providing in vitro synthesized RNA, (b) A step of annealing in vitro synthesized RNA with a first oligonucleotide complementary to the sequence in the 5'UTR of the RNA and a second oligonucleotide complementary to the sequence in the 3'UTR of the RNA, (c) A step of treating RNA with a nuclease to cleave the RNA into cap fragments and tail fragments, (d) A step of characterizing poly-A tails by identifying capping species and non-tail species by liquid chromatography with UV detection (LC-UV), and / or identifying and measuring the masses of capping species and non-tail species by liquid chromatography combined with mass spectrometry (LC-MS) and / or liquid chromatography combined with mass spectrometry using UV detection (LC-UV-MS), (e) Quantify the relative amount of each capping species, quantify the relative amount of non-tail species using LC-UV, LC-MS, or LC-UV-MS, characterize the poly-A tail using LC-MS or LC-UV-MS, and simultaneously in a single sample, This process involves producing RNA containing quantified percentages of capped mRNA and untailed mRNA, Methods that include...

[0319] 13. The following conditions: (a) After transcription, a 5' cap is enzymatically added to the RNA from step (a). (b) RNA does not contain a 5' cap. (c) The 3' tail is encoded in the plasmid during in vitro synthesis. (d) RNA does not contain a 3' tail. (e)RNA is not spliced. (f) RNA is spliced. (g) After transcription, a 5' cap is enzymatically added to the RNA from step (a), and the 3' tail is encoded in the plasmid during in vitro synthesis. (h) After transcription, a 5' cap is enzymatically added to the RNA from step (a), and the RNA does not contain a 3' tail. (i) RNA does not contain a 5' cap, and the 3' tail is encoded in the plasmid during in vitro synthesis. (j) RNA does not contain a 5' cap, RNA does not contain a 3' tail. The method according to the numbered embodiment 12, wherein one or more of the following conditions are met.

[0320] 14. A quality control assay for a manufacturing lot, wherein the assay is (a) To provide a manufactured RNA sample including a 5' cap and / or a 3' tail, (b) To quantify the RNA capping efficiency and tailing efficiency in a sample by the method described in claim 2, (c) Compare the capping efficiency and tailing efficiency in step (b) with the capping efficiency and tailing efficiency in the reference sample, Includes, The amount of capping species reduced compared to the reference sample indicates 5' decomposition. Increased amount of non-tail species and / or different poly-A tail lengths or polydispersity exhibits 3' decomposition. This includes simultaneously monitoring 5' and 3' degradation of RNA products and determining the quality of the product from the manufactured lot. Quality control assay for manufacturing lots.

[0321] 15. The quality control assay according to numbered embodiment 14, wherein the reference sample is a stable RNA sample having more than 75% integrity, and / or the RNA product is therapeutic mRNA, in particular an mRNA vaccine. [Examples]

[0322] Example 1. RNA generation by in vitro synthesis, capping, and tailing. The identification or quantification of mRNA capping and tailing described herein is performed on RNA samples obtained from various sources. For example, in various non-limiting embodiments, the RNA is in vitro transcribed mRNA, the RNA is obtained from a manufacturing process, for example, the RNA is obtained from the final step of manufacturing, or the RNA is obtained from a deformed formulation. In some embodiments, the RNA is unmodified. In some embodiments, the RNA is modified.

[0323] This example illustrates RNA synthesis by in vitro transcription, followed by capping and tailing.

[0324] In short, RNA was synthesized by in vitro transcription from a plasmid DNA template containing a DNA sequence encoding mRNA. The in vitro transcription involved the enzymatic conjugation of GTP via guanylyltransferase to add an exemplary 5' cap structure, e.g., cap 1, having a 2'-O-methyl residue at the 2'OH group of the ribose ring of base 1. Exemplary mRNA capped and uncapped structures present in various embodiments of this disclosure, as well as means for enzymatically inducing them, are shown in Figure 4B. Uncapped mRNA (e.g., in vitro transcription mRNA, pppG-mRNA), capped G (GpppG-mRNA), capped 0 (m7GpppG-mRNA), and capped 1 (m7GpppmG-mRNA).

[0325] The 3' poly(A) tail was incorporated by adding ATP combined with poly(A) polymerase (see detailed reaction conditions below). The in vitro transcript contained the 5' and 3' untranslated regions.

[0326] RNA synthesis was performed under RNAse-free conditions. All tubes, vials, pipette tips, pipettes, and buffers were nuclease-free. RNA was synthesized from a linear DNA template. To prepare the desired RNA precursor (IVT) construct, a mixture of approximately 100 μg of linear DNA, rNTP (3.33 mM), DTT (10 mM), T7 RNA polymerase, RNAse inhibitor, pyrophosphatase, and reaction buffer (10×, 800 mM Hepes (pH 8.0), 20 mM Spermidine, 250 mM MgCl2, pH 7.7) was prepared to a final volume of 2.24 ml using RNase-free water. The reaction mixture was incubated at 37°C for 20–120 minutes. After completion, the mixture was further treated with DNase I for 15 minutes and quenched accordingly.

[0327] The purified RNA product from the aforementioned IVT process was denatured at 65°C for 10 minutes. Separately, a portion of GTP (20 mM), S-adenosylmethionine, RNAse inhibitor, 2'-O-methyltransferase, and guanylyltransferase were mixed with reaction buffer (10x, 500 mM Tris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl2) to a final concentration of 8.3 ml. After denaturation, the RNA was cooled on ice and then added to the reaction mixture. The combined solution was incubated at 37°C for 20–90 minutes. Upon completion, aliquots of ATP (20 mM), poly(A) polymerase, and Tailing reaction buffer (10x, 500 mM Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl2) were added, and the entire reaction mixture was further incubated at 37°C for approximately 20–45 minutes. Once completed, the final reaction mixture was quenched and purified accordingly.

[0328] Overall, this embodiment exemplifies the synthesis of RNA including a 5' cap and a 3' tail by in vitro synthesis as an exemplary method for generating RNA for identification and / or quantification of capping and tailing efficiency according to the present disclosure.

[0329] Example 2: Identification and / or quantification of RNA capping and tailing efficiency in a single-sample method This example demonstrates a single-sample method for the simultaneous identification and / or quantification of RNA capping and tailing efficiency.

[0330] Custom Hybrid Oligonucleotide Design: Initially, custom hybrid oligonucleotides were designed to bind to RNA near the 5' and 3' ends. The hybrid oligonucleotides contained both RNA and DNA bases. In some embodiments, the oligonucleotides contained RNA and DNA bases in ratios of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:1. In some embodiments, the oligonucleotide contained four DNA bases. For example, HO2.16 is a custom-designed hybrid oligonucleotide with 16 nts complementary to the sequence in the 3' UTR of RNA. [ka] The italicized CTGT near the 3' end represents a DNA base, and HO2.27 is an exemplary 27nt custom-designed hybrid oligonucleotide complementary to the RNA 5'UTR sequence. [ka] The italicized TTCA near the 5' end represents four DNA bases. Specifically, oligonucleotides, 10–30 nucleotides long, for example 15–30 nucleotides long, were designed and synthesized to bind to the 5'UTR of the capped RNA, for example, within a few nucleotides (e.g., one or two, or up to 15) of the 5' cap. The binding of the oligonucleotide to the 5'UTR adjacent to the cap established a well-defined region of the DNA:RNA hybrid that is susceptible to RNAse H-mediated cleavage. In some embodiments, the oligonucleotide had 1–15 RNA nucleotides flanking both ends. In some embodiments, the oligonucleotide had 1–15 RNA nucleotides flanking the 3' end.

[0331] RNA Annealing to Custom Hybrid Oligonucleotides for RNA:DNA Hybrid Formation: In the annealing step, mRNA samples having a 5' cap and / or 3' poly(A) tail, synthesized by in vitro transcription as described in Example 1, for example, were annealed with a first oligonucleotide probe complementary to the sequence of the RNA's 5' untranslated region (UTR) and a second oligonucleotide probe complementary to the sequence of the RNA's 3' untranslated region (UTR) (Figure 2). Briefly, RNA from exemplary samples and exemplary custom hybrid oligonucleotides was heated at 75°C for 10 minutes to induce denaturation and strand separation, and then the temperature was lowered from 75°C to 23°C and maintained at 23°C for 10 minutes to promote hydrogen bond formation between the complementary DNA sequence and the RNA sequence. Complementary base pairing between the hybrid oligonucleotide and mRNA in specific regions resulted in RNA:DNA hybrid formation at the end of the annealing step (Figure 2).

[0332] Digestion of RNA:DNA hybrids: In the digestion step, RNA:DNA hybrids were incubated with RNAse H and alkaline phosphatase (rSAP) at 37°C for 40 minutes. The RNAse H enzyme cleaves the phosphodiester bonds between the double-stranded RNA:DNA hybrids generated in the annealing step. Alkaline phosphatase (rSAP) catalyzes terminal dephosphorylation to prevent self-ligation (Figure 3).

[0333] As described above, the hybrid oligonucleotides were designed so that a 4-base DNA molecule is flanked on both sides by one or more RNA nucleotides, providing specificity to the cleavage site by the nuclease. This allowed for selective cleavage of the RNA:DNA hybrid by a nuclease such as RNAse H, generating 5' capped or uncapped fragments of several base lengths (e.g., 2–10 nucleotides including capped nucleotides) without generating significant internal fragments of the mRNA. Larger mRNA fragments were separated from the shorter 5' capped or uncapped fragments during chromatography due to their dramatically different physicochemical properties. Larger mRNA fragments distal to the 5' cap were readily removed, allowing the chromatographic resolution to focus on the shorter 5' capped and uncapped fragments produced by enzymatic digestion. In certain embodiments, this cleavage was achieved using an S1 nuclease or other nuclease, creating blunt-ended fragments marked at the 5' end by a selected cap analyte. Overall, this process provided smaller molecules with improved resolution for detecting both the presence and modification of caps.

[0334] Identification of capped, uncapped, and non-tailed species: In various embodiments, liquid chromatography (LC-UV) or LC-MS or LC-UV-MS combined with UV detection was used to identify capped species in capped fragments by analyzing the peaks of the chromatogram generated from LC-UV and / or the mass spectra generated by LC-MS or LC-UV-MS. In embodiments using LC-UV, a standard was required for comparison. In other embodiments using LC-MS, the mass measurements under each peak were used to identify capped species. Simultaneously, non-tailed and tailed species in tailed fragments in the same sample were identified by analyzing the peaks of the chromatogram generated from LC-UV and / or the mass spectra generated by LC-MS or LC-UV-MS.

[0335] Liquid Chromatography: In some embodiments, the analysis of digested RNA:DNA hybrids was performed, for example, by liquid chromatography. Oligonucleotides produced by digestion were separated, for example, by reverse-phase separation with separation based on the degree of hydrophobicity. For example, oligonucleotides were separated based on ion-pair reverse-phase chromatography using Acquity PREMIER Oligonucleotide BEH C18 130Å, 1, 7μm, 2.1*100mm.

[0336] UV Detection: In some embodiments, UV detection was performed. UV detection was particularly valuable in a Good Manufacturing Practice (GMP) environment. In some embodiments, UV detection was performed at 260 nm. In some embodiments, UV detection was performed at 280 nm. In some embodiments, UV cells ranging from 0.1 mm to 100 mm were used. In some embodiments, a 10 mm UV cell was used. In some embodiments, a 0.01 mm UV cell was used. In addition to the samples, in some embodiments, standards were injected in parallel for cap 1, cap 0, cap G, and uncapped varieties to identify and compare various retention times.

[0337] LC-UV or LC-MS or LC-UV-MS: In some embodiments, UV detection (LC-UV) and / or LC were combined with mass spectrometry (LC-MS) or LC-UV-MS to identify capping species in capped fragments and non-tailed and tailed species in tailed fragments by analyzing the peaks of the chromatogram generated from LC-UV and / or the mass spectrum generated by LC-MS or LC-UV-MS.

[0338] Quantification of capped, uncapped, and untailed species: In some embodiments, the relative amounts of each capped species in the capped fragment, and each untailed and tailed species in the tailed fragment, were quantified by mass spectrometry, UV spectrometry, or chromatographic peak retention time.

[0339] Mass Spectrometry: In some embodiments, the analysis of digested RNA:DNA hybrids was quantitatively performed by mass spectrometry to identify oligonucleotides, which were then quantified using mass-to-charge ratio (m / z) measurements. The mass spectrometer converts oligonucleotide molecules into a charged ionized state. These ionized molecules and fragments were then detected based on their mass-to-charge ratio and retention time. Various ionization and ion analysis systems are known in the art, including, for example, electrospray-source ionization (ESI) four-times-to-flight (Q-TOF) and negative modes including the formation of negative ions to determine the mass-to-charge ratio of sample molecules.

[0340] For example, an exemplary extracted ion chromatogram (EIC) shows the peaks generated for capped species (cap 0, cap 1, cap G), uncapped species, and non-tailed species (Figure 4A). The percentage of any particular capped species (cap 0, cap 1, cap G, or uncapped) was calculated by multiplying the area under the curve of the capped species by 100, which is the sum of the areas under the curves of the capped peaks (cap 0, cap 1, cap G) and uncapped peaks. Similarly, the percentage of non-tailed species was calculated by measuring the area under the curve of non-tailed species relative to the total area under the curve of the capped peaks (cap 0, cap 1, cap G) and uncapped peaks multiplied by 100. In the exemplary EIC shown in Figure 4A, the quantitative results demonstrated the presence of 6.8% non-tailed, 4.2% uncapped, 0.4% cap G, 0.0% cap 0, and 95.4% cap 1 species. In some embodiments, the EIC is generated by, for example, liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-ultraviolet spectroscopy / mass spectrometry (LC-UV / MS), ultrafast liquid chromatography-electrospray ionization multiple reaction monitoring tandem mass spectrometry (UHPLC-ESI-MS), or other mass spectrometry methods. The mass spectrometry signal intensity was plotted against m / z (mass-to-charge ratio).

[0341] To determine poly(A) tailing, the mass of the poly(A) tail species was measured, followed by deconvolution of the mass spectrometry signal to obtain the mass distribution of the tail species. Figure 5A shows the mean deconvolution mass spectrum of an exemplary mRNA used to study poly(A) tailing in an exemplary drug substance. In the graph, the mass spectrometry signal intensity is plotted against m / z (mass-to-charge ratio).

[0342] Based on the corresponding mass distribution, the minimum, maximum, average length, and polydispersity of the poly-A tail were determined. For example, a plot of the percentage of mass spectrometry signal intensity against the poly-A tail length reveals the polydispersity of poly-A for an exemplary mRNA (Figure 5B).

[0343] In some embodiments, the method was automated.

[0344] It is understood that the methods described in this embodiment are applicable to any RNA, including mRNA, therapeutic mRNA from any source, and mRNA vaccines targeting any disease, including viral diseases. Some exemplary mRNA vaccines include, among others, vaccines against coronaviruses (e.g., SARS, SARS-CoV-2, MERS), influenza, respiratory syncytial virus, and various types of cancer, including pancreatic cancer, colorectal cancer, and melanoma.

[0345] Overall, the method of this disclosure provides a powerful combination of simultaneous qualitative and / or quantitative assessment of capped, uncapped, or untailed mRNA species for determining the quality stability of mRNA, applicable to a wide variety of uses, particularly therapeutic applications, such as mRNA vaccines.

[0346] Equivalents and scope Those skilled in the art will be able to recognize or confirm, by mere conventional experimentation, many equivalents to the specific embodiments described herein. The scope of this disclosure is not limited to the foregoing and is as set forth in the appended claims.

[0347] In the claims, articles such as “a,” “an,” and “it” can mean one or more unless there is a contradictory statement or the context makes another meaning obvious. Thus, for example, a reference to “antibody” includes multiple such antibodies, and a reference to “cell” includes a reference to one or more cells known to those skilled in the art. A claim or statement containing “or” between one or more members of a group is deemed satisfied if one, two or more, or all of the members of that group are present in, used in, or otherwise related to a given product or process, unless the opposite meaning is stated or the context makes another interpretation obvious. The disclosure includes embodiments in which exactly one member of a group is present in, used in, or otherwise related to a given product or process. The disclosure includes embodiments in which more than one, or all, members of a group are presented in, used in, or otherwise related to a given product or process. Furthermore, it should be understood that this disclosure includes all variations, combinations, and substitutions in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the enumerated claims are introduced into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Furthermore, if a composition is described in a claim, it should be understood that unless otherwise indicated, or unless it is obvious to a person skilled in the art that this would result in a contradiction or inconsistency, this includes a method of using the composition for any of the purposes disclosed herein and a method of producing the composition according to any of the manufacturing methods disclosed herein or other methods known in the art.

[0348] If elements are presented as a list, for example in Markush group format, it should be understood that each subgroup of the elements is also disclosed, and any element can be removed from a group. Generally, if this disclosure or an aspect thereof is referred to as including certain elements, features, etc., it should be understood that certain embodiments of this disclosure or an aspect thereof consist of, or are essentially, such elements, features, etc. For simplicity, these embodiments are not specifically described herein. Note that the term “comprising” is intended to be open and allow for the inclusion of additional elements or processes.

[0349] Where a range is indicated, it includes the endpoints. Furthermore, unless otherwise clearly indicated by the context, and unless it is understood by those skilled in the art, values ​​expressed as a range may be assumed to be any specific value or subrange within the state range in different embodiments of this disclosure, up to one-tenth of the lower limit of the range, unless the context clearly indicates otherwise.

[0350] Furthermore, it should be understood that any particular embodiment of the present disclosure contained in the prior art may be expressly excluded from one or more of the claims. Such embodiments may be excluded even if the exclusion is not expressly stated herein, as they are considered to be known to those skilled in the art. Any particular embodiment of the compositions of the present disclosure may be excluded from any one or more embodiments for any reason, whether or not it relates to the existence of the prior art.

[0351] The publications discussed above and throughout this text are provided solely for the purpose of their disclosure prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment by the inventors that they have no prior rights to such disclosures.

[0352] Other Embodiments Those skilled in the art will readily understand that the foregoing represents only specific preferred embodiments of the present disclosure. Various changes and modifications to the above procedures and compositions can be made without departing from the spirit or scope of the present disclosure, as described in the following claims.

Claims

1. A single-sample method for identifying ribonucleic acid (RNA) capping modifications and tailing modifications, (a) To provide a sample comprising RNA and optionally having a 5' cap and / or a 3' poly-A tail, wherein the sample further comprises a first oligonucleotide probe complementary to the sequence in the 5' untranslated region (5'UTR) of the RNA, and a second oligonucleotide probe complementary to the sequence in the 3' untranslated region (3'UTR) of the RNA. (b) Annealing the RNA sample with a first oligonucleotide probe complementary to the sequence in the 5' UTR of the RNA and a second oligonucleotide probe complementary to the sequence in the 3' UTR of the RNA, (c) The RNA sample from step (b) is treated with a nuclease to cleave the RNA into cap fragments and tail fragments, (d) i. Identifying the capping species in the capping fragment by measuring the retention time of the peaks in the chromatogram generated from liquid chromatography (LC-UV) using ultraviolet detection, and / or LC (LC-MS) or LC-UV-MS combined with mass spectrometry. ii. Identifying non-tail species in the tail fragment by measuring the retention time of peaks in the chromatogram generated from LC-UV and / or the mass spectrum generated by LC-MS or LC-UV-MS, and iii. Identifying the tail species in the tail fragment by measuring the retention time of the peaks in the chromatogram generated from LC-UV and / or the mass spectrum generated by LC-MS or LC-UV-MS. Therefore, using the sample from step (c), perform LC-UV, LC-MS, or LC-UV-MS. This allows for the simultaneous identification of RNA capping modifications and tailing modifications in the single sample, A single-sample method for identifying ribonucleic acid (RNA) capping and tailing modifications, including [specific modifications].

2. The single sample method according to claim 1, wherein the first base of the first oligonucleotide probe is adjacent to the second-to-last base of the RNA or to the second-to-last base of the RNA, for example, by binding to at least 2 to 10 nucleotides from the second-to-last base of the RNA.

3. A single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency, (a) To provide a sample containing RNA and optionally having a 5' cap and / or a 3' poly-A tail, (b) Annealing the RNA sample with a first oligonucleotide probe complementary to the sequence of the 5' untranslated region (5'UTR) of the RNA and a second oligonucleotide probe complementary to the sequence of the 3' untranslated region (3'UTR) of the RNA, (c) The RNA sample from step (b) is treated with a nuclease to cleave the RNA into cap fragments and tail fragments, (d) Performing LC (LC-MS) or LC-UV-MS in combination with liquid chromatography-UV or mass spectrometry using ultraviolet detection, (e) Measuring the peak area of ​​each capping species in the capped fragment, and each non-tail and tail species in the tailed fragment, (f) Quantify the relative amounts of each capping species and the relative amounts of non-tail species, characterize the poly(A) tails in the sample from step (d), thereby simultaneously quantifying the RNA capping efficiency and tailing efficiency in the sample. A single-sample method for simultaneously quantifying RNA capping efficiency and tailing efficiency, including the above.

4. The single sample method according to claim 3, wherein the first base of the first oligonucleotide probe is adjacent to the second-to-last base of the RNA or, for example, binds to at least 2 to 10 nucleotides from the second-to-last base of the RNA.

5. The method according to claim 3 or 4, wherein characterizing the poly-A tail comprises (i) measuring the mass of the poly-A tail species, deconvolution of the mass spectrometry signal, thereby providing a mass distribution of the tail species, and (ii) determining the minimum length, maximum length, average length and polydispersity of the poly-A tail based on the corresponding mass distribution.

6. The method according to any one of claims 1 to 5, wherein the RNA is in vitro transcribed mRNA.

7. The method according to any one of claims 1 to 6, wherein the RNA is obtained from the manufacturing process.

8. The method according to claim 7, wherein the RNA is obtained from the active pharmaceutical ingredient (DS) in the final step of the manufacturing process.

9. The method according to any one of claims 1 to 6, wherein the RNA is obtained from a deformulated drug product (DP).

10. The method according to any one of claims 1 to 9, wherein the RNA is not modified.

11. The method according to any one of claims 1 to 9, wherein the RNA is modified.

12. The method according to any one of claims 1 to 11, wherein the nuclease is RNAse H.

13. The method according to any one of claims 1 to 12, wherein the masses of the cap fragment and the tail fragment are measured by LC-MS.

14. The method according to any one of claims 1 to 13, wherein the capping species, non-tail species, and tail species are identified from the UV signal by comparing the retention time of the obtained peak with the retention time of a reference standard.

15. The method according to any one of claims 1 to 14, wherein the capping type is cap 1, cap 0, cap G, or not capped.

16. The method according to any one of claims 1 to 15, wherein the capping species is m7Gpppmm7GGACA, m7GpppGGACA, GpppGGACA, GGACA, or pppGGACA.

17. The method according to claim 15 or 16, wherein the uncapped species is GGACA or pppGGACA.

18. The method according to any one of claims 1 to 17, wherein the non-tail species is UGCAUC, and U is unmodified uridine.

19. The method according to any one of claims 1 to 17, wherein the non-tail species is U*GCAU*C, and U* is N1-methylpsuduridine.

20. The method according to any one of claims 1 to 19, wherein the relative amount of each of the capping species is a percentage of the total amount of capping species in the sample, calculated by dividing the area under the peak of the capping species of interest by the sum of the total areas under the peak representing cap 1, cap 0, cap G, and uncapped species, and multiplying by 100.

21. The method according to any one of claims 1 to 20, wherein the relative amount of the non-tail species is a percentage calculated by dividing the area under the peak of the non-tail species by the sum of the areas under the peak of cap 1, cap 0, cap G, and the uncapped species, and multiplying by 100.

22. The method according to any one of claims 1 to 21, wherein the oligonucleotide probe is about 10 to 40 nucleotides long.

23. The method according to any one of claims 1 to 22, wherein the oligonucleotide probe comprises RNA and DNA bases.

24. The method according to any one of claims 1 to 23, wherein the oligonucleotide probe comprises RNA bases and DNA bases in a ratio of about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, or about 3:

1.

25. The method according to any one of claims 1 to 24, wherein the oligonucleotide probe is 10 to 40 nucleotides long and contains four DNA bases.

26. The method according to claim 25, wherein the oligonucleotide probe complementary to the sequence in the 5' untranslated region of the RNA is 3'-CCTGTCUAGCGACCU-5' (SEQ ID NO: 1), and the italicized CTGT is a DNA base.

27. The method according to claim 25, wherein the oligonucleotide probe complementary to the sequence in the 3' untranslated region of the RNA is 3'-GGUCGGAACAGGAAUUAUUUAATTCAA-5' (SEQ ID NO: 2), and the italicized TTCA is a DNA base.

28. The method according to any one of claims 1 to 27, wherein capping species and non-tail species are quantified by single LC-UV, LC-MS, or LC-UV-MS analysis.

29. The method according to claim 1 or 2, wherein the tail species is characterized by single LC-MS or LC-UV-MS analysis.

30. The method according to any one of claims 1 to 29, wherein one or more steps are automated.

31. An oligonucleotide complementary to the sequence of the 5' untranslated region (5'UTR) or the 3' untranslated region (3'UTR) of the RNA, wherein the oligonucleotide is approximately 10 to 40 nucleotides long and contains RNA bases and DNA bases.

32. The oligonucleotide according to claim 31, wherein the oligonucleotide comprises RNA and DNA bases in a ratio of approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, or 3:

1.

33. The oligonucleotide according to claim 31 or 32, wherein the oligonucleotide is about 10 to 40 nucleotides long and contains four DNA bases.

34. The oligonucleotide according to any one of claims 31 to 33, wherein the oligonucleotide complementary to the sequence in the 5' UTR of the RNA is 3'-CCTGTCUAGCGGACCU-5' (SEQ ID NO: 1), and the italicized CTGT is a DNA base.

35. The oligonucleotide according to any one of claims 31 to 33, wherein the oligonucleotide complementary to the sequence in the 3'UTR of the RNA is 3'-GGUCGGAACAGGAAUUAUUUAATTCAA-5' (SEQ ID NO: 2), and the italicized TTCA is a DNA base.

36. A method for producing RNA having a quantified percentage of capped mRNA and untailed mRNA, (a) A step of providing in vitro synthetic RNA, (b) Annealing the in vitro synthesized RNA with a first oligonucleotide complementary to the sequence in the 5' untranslated region (5'UTR) of the RNA and a second oligonucleotide complementary to the sequence in the 3' untranslated region (3'UTR) of the RNA, (c) A step of treating the RNA with a nuclease to cut the RNA into a cap fragment and a tail fragment, (d) A step of characterizing the poly-A tail by identifying the capping species and non-tail species by liquid chromatography with UV detection (LC-UV), and / or by liquid chromatography combined with mass spectrometry (LC-MS) and / or by liquid chromatography combined with mass spectrometry using UV detection (LC-UV-MS), (e) Quantify the relative amount of each capping species, quantify the relative amount of non-tail species using LC-UV, LC-MS, or LC-UV-MS, and simultaneously characterize the poly-A tail in a single sample using LC-MS or LC-UV-MS. This process involves producing RNA containing quantified percentages of capped mRNA and untailed mRNA, Methods that include...

37. The method according to claim 36, wherein the first base of the first oligonucleotide is attached to the second-to-last base of the RNA or adjacent to the second-to-last base of the RNA, for example, by binding to at least 2 to 10 nucleotides from the second-to-last base of the RNA.

38. The method according to claim 36 or 37, wherein a 5' cap is enzymatically added to the RNA from step (a) after transcription.

39. The method according to claim 36 or 37, wherein the RNA does not contain a 5' cap.

40. The method according to any one of claims 36 to 39, wherein the 3' tail is encoded in the plasmid during in vitro synthesis.

41. The method according to any one of claims 36 to 39, wherein a 3' tail is enzymatically added to the RNA from step (a) after transcription.

42. The method according to any one of claims 36 to 39, wherein the RNA does not contain a 3' tail.

43. The method according to any one of claims 36 to 42, wherein the RNA is not spliced.

44. The method according to any one of claims 36 to 42, wherein the RNA is spliced.

45. A quality control assay for a manufacturing lot, wherein the assay is (a) To provide a manufactured RNA sample containing a 5' cap and / or a 3' tail, (b) Quantifying the RNA capping efficiency and tailing efficiency in the sample by the method described in claim 3 or 4, (c) The capping efficiency and tailing efficiency in step (b) are compared with the capping efficiency and tailing efficiency in the reference sample, Includes, The amount of capping species reduced compared to the reference sample indicates 5' decomposition. An increased amount of non-tail species and / or different poly-A tail lengths or polydispersity exhibits 3' decomposition. This includes simultaneously monitoring the 5' and 3' degradation of RNA products and determining the quality of the product from the manufactured lot. Quality control assay for manufacturing lots.

46. The quality control assay according to claim 45, wherein the reference sample is a stable RNA sample having more than 75% integrity.

47. The quality control assay according to claim 45 or 46, wherein the RNA product is therapeutic mRNA.

48. The quality control assay according to claim 45 or 46, wherein the RNA product is an mRNA vaccine.

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