WO2022237800A1 - Method for determining average molecular weight of mrna, as well as cap0/1, modified nucleotides and oxides - Google Patents
Method for determining average molecular weight of mrna, as well as cap0/1, modified nucleotides and oxides Download PDFInfo
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- WO2022237800A1 WO2022237800A1 PCT/CN2022/092044 CN2022092044W WO2022237800A1 WO 2022237800 A1 WO2022237800 A1 WO 2022237800A1 CN 2022092044 W CN2022092044 W CN 2022092044W WO 2022237800 A1 WO2022237800 A1 WO 2022237800A1
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- Prior art keywords
- mrna
- amount
- guanosine
- substance
- sample
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- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 150
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 42
- 239000002773 nucleotide Substances 0.000 claims abstract description 49
- 238000005251 capillar electrophoresis Methods 0.000 claims abstract description 22
- 239000000126 substance Substances 0.000 claims description 48
- 229940029575 guanosine Drugs 0.000 claims description 31
- OGHAROSJZRTIOK-KQYNXXCUSA-O 7-methylguanosine Chemical compound C1=2N=C(N)NC(=O)C=2[N+](C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-O 0.000 claims description 27
- DRTQHJPVMGBUCF-XVFCMESISA-N Uridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-XVFCMESISA-N 0.000 claims description 25
- 229930185560 Pseudouridine Natural products 0.000 claims description 20
- PTJWIQPHWPFNBW-UHFFFAOYSA-N Pseudouridine C Natural products OC1C(O)C(CO)OC1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-UHFFFAOYSA-N 0.000 claims description 20
- WGDUUQDYDIIBKT-UHFFFAOYSA-N beta-Pseudouridine Natural products OC1OC(CN2C=CC(=O)NC2=O)C(O)C1O WGDUUQDYDIIBKT-UHFFFAOYSA-N 0.000 claims description 20
- 238000007254 oxidation reaction Methods 0.000 claims description 20
- PTJWIQPHWPFNBW-GBNDHIKLSA-N pseudouridine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1C1=CNC(=O)NC1=O PTJWIQPHWPFNBW-GBNDHIKLSA-N 0.000 claims description 20
- 230000003647 oxidation Effects 0.000 claims description 19
- 239000002777 nucleoside Substances 0.000 claims description 16
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 claims description 15
- DRTQHJPVMGBUCF-PSQAKQOGSA-N beta-L-uridine Natural products O[C@H]1[C@@H](O)[C@H](CO)O[C@@H]1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-PSQAKQOGSA-N 0.000 claims description 13
- DRTQHJPVMGBUCF-UHFFFAOYSA-N uracil arabinoside Natural products OC1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 DRTQHJPVMGBUCF-UHFFFAOYSA-N 0.000 claims description 13
- 229940045145 uridine Drugs 0.000 claims description 13
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- 238000001514 detection method Methods 0.000 claims description 10
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- 238000011534 incubation Methods 0.000 claims description 6
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Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
-
- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
Definitions
- the invention belongs to the field of biomedicine, and specifically relates to a method for measuring the average molecular weight of mRNA in a sample and an application thereof.
- the application particularly relates to a method for measuring the average molecular weight of mRNA, Cap0/1, modified nucleotides and oxides.
- 5'CAP is an important guarantee for the stable existence of mRNA and high-efficiency protein expression.
- the mRNA synthesized by in vitro transcription method is mainly capped by capping analogs and capping enzymes. Whether the final mRNA product is correctly capped and has a qualified capping efficiency becomes the key factor for evaluating mRNA products. important indicators. And for different application scenarios, it is necessary to distinguish the types of mRNA capping, that is, it is necessary to accurately determine the proportion of CAP 0 and CAP 1 of mRNA.
- the existing method for measuring the capping efficiency is to measure the ratio of m7G to A, U, C, and G after hydrolyzing the mRNA to compare the capping efficiency of the mRNA.
- modified nucleotides such as pseudouridine and 5-methylcytosine can effectively inhibit the immunogenicity of mRNA itself, prevent mRNA from being degraded in vivo, prolong the expression time of mRNA in cells, and improve expression efficiency. Therefore, the proportion of modified nucleotides in the entire mRNA is also an important indicator for evaluating mRNA products. Only fully modified mRNA can have better functionality.
- the mRNA transcribed in vitro will inevitably come into contact with oxygen in the air and lead to the oxidation of mRNA.
- the oxidation product is mainly 8-oxoguanosine. 8-oxoguanosine will cause transcription errors and affect the correct expression of proteins. Being able to accurately judge mRNA oxidation products is an important criterion for evaluating mRNA drugs.
- the technical problem to be solved by the present invention is to provide a method for determining the average molecular weight of mRNA and Cap0/1, modified nucleotides in order to overcome the lack of detection methods for capping efficiency in the prior art and the detection of mRNA purity in existing methods. and oxide methods.
- the present invention mainly solves the above-mentioned technical problems through the following technical solutions.
- One of the technical solutions of the present invention is: a method for measuring the average molecular weight of mRNA in a sample, which includes:
- the R is the proportion of the peak area in the interval (a n-1 , a n ).
- n can be any positive integer of 5-100 or 5-50 or 5-20 or 10-100 or 10-50 or 5-20, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, etc.
- the capillary electrophoresis described in step (1) can be carried out according to the routine in this field, preferably using small RNA reagent kit and Capillary electrophoresis was performed with GX Touch TM nuclear acid analyzer to obtain the capillary electrophoresis pattern of the sample.
- the sample also includes a step of removing free nucleotides before capillary electrophoresis; said removing free nucleotides, for example, adopts LiCl precipitation, ion exchange resin chromatography, preparative liquid Chromatography or commercial kits.
- the second technical solution of the present invention is: a method for measuring the amount of mRNA in a sample, which comprises the steps of:
- step 3 According to the quality of mRNA described in step 1) and the average molecular weight of mRNA in the sample obtained in step 2), the amount of mRNA in the sample is obtained.
- the mass concentration in the mRNA can be routinely used in the art, preferably using an ultra-micro spectrophotometer to measure the mass concentration of the mRNA in the sample;
- the ultra-micro spectrophotometer is, for example, Thermo Scientific NanoDrop One or Mettler Toledo UV5 Nano.
- the third technical solution of the present invention is: a method for assaying nucleotide content in mRNA, said assay method comprising:
- step (c) calculating the content of the different nucleotides in the mRNA according to the amount of the different nucleotides in the step (a) and the amount of the mRNA in the step (b);
- steps (a) and (b) have no order requirement, and can be performed separately, simultaneously or successively.
- step (b) it is preferred to measure the content of different nucleotides in the hydrolyzed mRNA by HPLC, and calculate the amount of different nucleotides according to their relative molecular masses. Described hydrolysis comprises the steps:
- step II Add phosphodiesterase I and alkaline phosphatase to the incubation product obtained in step I, and add buffer B, and incubate at 37°C; the incubation time is, for example, 2 hours, and the phosphodiesterase I,
- the volume ratio of alkaline phosphatase and buffer B is, for example, 3.3:3.3:10.6.
- the HPLC measurement described in the present invention can be routine in this field, preferably comprises:
- nucleotides to be determined may be conventional nucleosides constituting mRNA, such as adenosine, uridine, cytidine, and guanosine; they may also be corresponding modified nucleotides Or oxidized nucleotides and other common unconventional nucleosides.
- modified nucleotides described in the present invention preferably include 7-methyl-guanosine, 2'-oxymethyl-guanosine, pseudouridine, 1-methyl-pseudouridine, One or more of 5-methyl-cytidine, 4-acetyl-cytidine and 6-methyl-adenosine.
- CAP0% 7-methylguanosine substance amount/mRNA substance amount ⁇ 100%
- CAP1% 2-oxomethyl-guanosine substance amount/mRNA substance amount ⁇ 100% ⁇ CAP0%;
- Modified nucleotide% the amount of substance to modify nucleotide/(the amount of substance to modify nucleotide+the amount of substance to modify nucleotide) ⁇ 100%;
- Nucleotide % after oxidation the amount of nucleotide substance after oxidation/(the amount of nucleotide substance after oxidation+the amount of nucleotide substance before oxidation) ⁇ 100%.
- the modified nucleotides can be conventional in the art, preferably including: 1-methyl-pseudouridine, 5-methyl-cytidine, 4-acetyl-cytidine and 6- One or more of methyl-adenosine nucleosides.
- the oxidized nucleotide may also be conventional in the art, such as 8-oxo-guanosine.
- the reagents and raw materials used in the present invention are all commercially available.
- the present invention accurately measures the proportion of Cap 0, Cap 1, modified nucleotides and oxidation products in the preparation of mRNA through a new measurement and calculation method. very precise detection method.
- Figure 1 is a capillary electrophoresis map.
- Figure 2 is the profile of hydrolyzed mRNA.
- Figure 3 is the standard curve of cytidine.
- Figure 4 is the standard curve of uridine.
- Figure 5 is a standard curve for guanosine.
- Figure 6 is a standard curve for 7-methyl-guanosine.
- Figure 7 is a standard curve for 2-oxomethyl-guanosine.
- Figure 8 is a standard curve of pseudouridine.
- Figure 9 is a standard curve for 8-oxo-guanine nucleotides.
- the determination of the chain length distribution of mRNA can be done using instruments such as LabChip GXII Touch HT (PerkinElmer), 2100 Electrophoresis Bioanalyzer Instrument (Agilent), Qsep100 (Bioptic), and all instruments can obtain the same measurement results.
- the distribution of mRNA chain length mentioned in this article is based on the measurement results of LabChip GXII Touch HT as an example.
- mRNA can be purified by LiCl precipitation method, ion exchange resin chromatography, preparative liquid chromatography or commercial kits to completely remove free nucleotides to prevent interference with the determination of various indicators.
- Example 2 mRNA is completely hydrolyzed into single nucleosides and each hydrolyzed component is identified by HPLC
- Endonuclease P1 Endonuclease P1
- phosphodiesterase I Phosphodiesterase I
- alkaline phosphatase Silica Phosphatase
- Zinc chloride, glycerin, sodium acetate, ammonium acetate and magnesium acetate were purchased from Sinopharm Reagent Group. Ultrapure water is taken from Milli-Q pure water machine.
- Uridine, Cytidine and Guanosine were purchased from Merck; Pseudouridine, 7-methylguanosine ), 8-oxo-guanosine (8-oxo-Guanosine) and 2'-oxymethyl-guanosine (2'-O-MethylGuanosine, referred to as 2'-OMe-Guanosine) were purchased from TCI.
- the high-performance liquid chromatography (1260Infinity II bioInert LC) was purchased from Agilent Technologies, and the high-performance liquid chromatography column (XBridge BEH C18 Column, 5 ⁇ m, 4.6mm X 250mm, 1/pk) was purchased from Waters, the benchtop centrifuge (ST8) and constant temperature shaking metal bath (88880028) were purchased from Thermo, the analytical balance (XS205DU) was purchased from Multiparameter, and the micropipette was purchased from RAININ.
- Endonuclease P1 Endonuclease P1 (Endonuclease P1) was prepared as a 1g/L stock solution (20mM potassium acetate, 5mM zinc chloride, 50mM sodium chloride and 50% glycerol) and stored at -20°C.
- Buffer A (141.43mM ammonium acetate, 9.43mM zinc chloride, pH 5.3).
- Buffer B (115.00mM Tris-HCl, 11.50mM magnesium acetate, pH 8.3).
- the nucleotide peak area can be obtained by comparing the retention times of different nucleotides and integrating:
- Pseudouridine (Pseudouridine): 4263.484, Uridine (Uridine): 12.689, 7-methyl Guanosine (7-methyl Guanosine): 18.998, 2'-Oxymethyl-guanosine ( 2'-OMe-Guanosine): 31.655, 8-oxo-Guanosine (8-oxo-Guanosine): 0.
- Uridine, cytidine, guanosine, pseudouridine (Pseudouridine), 7-methylguanosine (7-methyl Guanosine), 8-oxo-guanosine (8 -oxo-Guanosine) and 2'-Oxymethyl-Guanosine (2'-OMe-Guanosine) and other nucleosides were prepared into a gradient concentration aqueous solution and measured by HPLC. The injection volume of each sample was 10 ⁇ L, and the records were different. The retention time of the nucleoside standard substance, and use the normalization method to integrate and calculate the peak area of different nucleosides at different concentrations, and thus draw a nucleoside standard curve based on the gradient concentration of the nucleoside.
- the concentration of different nucleosides in the unit injection volume (10 ⁇ l) can be calculated as follows: Pseudouridine (Pseudouridine): 8.64E-03 ⁇ moL, uridine ( Uridine): 2.97E-05 ⁇ moL, 7-methyl-guanosine (7-methyl Guanosine): 2.96E-05 ⁇ moL, 2'-oxymethyl-guanosine (2'-OMe-Guanosine): 3.02 E-05 ⁇ moL, 8-oxo-guanosine (8-oxo-Guanosine): 0 (not detected).
- Nanodrop can use Thermo Scientific NanoDrop One, Mettler Toledo UV5 Nano and other similar equipment.
- the present invention uses the capillary electrophoresis map combined with an innovative algorithm to accurately calibrate the sample mRNA sequence without knowing the mRNA base sequence and the number of each base to calculate Cap0, Cap1, modified nucleotides and oxidation substance content.
- the molecular weight of mRNA can be accurately calculated as:
- the mRNA molecular weight can be approximated as:
- the average molecular weight of the sample mRNA can then be calculated as follows:
- a is the shortest mRNA base number detectable in capillary electrophoresis
- b is the longest base number detectable in capillary electrophoresis.
- R is the proportion of the peak area in the interval (a n-1 , a n ).
- the average molecular weight of mRNA can be approximated as:
- CAP structure of mRNA is as follows:
- 7-methyl-guanosine (7-methyl Guanosine) is a marker of the CAP0 structure, and each complete mRNA has and only one 7-methyl-guanosine (7-methyl Guanosine):
- the CAP0 ratio of the sample mRNA can be calculated as:
- CAP0% amount of 7-methyl-guanosine (7-methyl Guanosine) substance/amount of mRNA substance ⁇ 100%.
- the complex structure of 7-methyl-guanosine (7-methyl Guanosine) and 2-oxymethyl-guanosine (2-OMe-Guanosine) connected by a triphosphate bond is a marker of the CAP1 structure of mRNA.
- Each complete mRNA has one and only one complex structure of 7-methyl-guanosine (7-methyl Guanosine) and 2'-oxymethyl-guanosine (2'-OMe-Guanosine). Therefore, the proportion of CAP1 can be obtained by measuring the amount of the marker 2'-oxymethyl-guanosine (2'-OMe-Guanosine):
- CAP1% the amount of 2'-oxymethyl-guanosine (2'-OMe-Guanosine) substance/the amount of mRNA substance ⁇ 100% ⁇ CAP0%.
- pseudouridine Pseudouridine
- 1-methyl-pseudouridine 1-Methyl-Pseudouridine
- 5 -Methyl-Cytidine 5-Methyl-Cytidine
- 4-Acetyl-Cytidine 4-Acetyl-Cytidine
- 6-Methyl-Adenesine 6-Methyl-Adenesine
- Pseudouridine (Pseudouridine)% the amount of pseudouridine (Pseudouridine) substance/(uridine (Uridine) substance amount+pseudouridine (Pseudouridine) substance amount) ⁇ 100%.
- 1-Methyl-Pseudouridine (1-Methyl-Pseudouridine)% 1-Methyl-Pseudouridine (1-Methyl-Pseudouridine) amount of substance/(1-Methyl-Pseudouridine nucleus Glycoside (1-Methyl-Pseudouridine) substance amount+uridine nucleoside (Uridine) substance amount) ⁇ 100%.
- 4-Acetyl-Cytidine (4-Acetyl-Cytidine)% Amount of 4-Acetyl-Cytidine/(Amount of 4-Acetyl-Cytidine+Amount of Cytidine) ⁇ 100%.
- the ratio of any other modified nucleotides can be accurately calculated using the same method.
- mRNA will inevitably be oxidized when exposed to air for a long time.
- the oxidation reaction is mainly manifested in the conversion of Guanosine to 8-oxo-Guanosine.
- Oxidation% 8-oxo-guanosine (8-oxo-Guanosine) substance amount/(8-oxo-guanosine nucleoside (8-oxo-Guanosine) substance amount+guanosine nucleoside (Guanosine) substance amount) ⁇ 100%.
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Abstract
A method for determining the average molecular weight of mRNA, as well as Cap0/1, modified nucleotides and oxides. The method comprises: (1) obtaining a capillary electrophoresis diagram of a sample; and (2) calculating the average molecular weight of mRNA according to formula (I). The average molecular weight of mRNA and the proportions of various types of nucleotides obtained after the cleavage of mRNA may be accurately determined by using the present method.
Description
本申请要求申请日为2021/5/12的中国专利申请2021105185771的优先权。本申请引用上述中国专利申请的全文。This application claims the priority of Chinese patent application 2021105185771 with a filing date of 2021/5/12. This application cites the full text of the above-mentioned Chinese patent application.
本发明属于生物医药领域，具体涉及一种测定样品中mRNA平均分子量的方法及其应用，所述应用特别涉及一种测定mRNA平均分子量以及Cap0/1、修饰核苷酸及氧化物的方法。The invention belongs to the field of biomedicine, and specifically relates to a method for measuring the average molecular weight of mRNA in a sample and an application thereof. The application particularly relates to a method for measuring the average molecular weight of mRNA, Cap0/1, modified nucleotides and oxides.
药用mRNA技术的快速发展对mRNA大规模生产的工艺、产率、质量以及成本等提出了很大的挑战，特别是在mRNA疫苗的超大规模生产过程当中，对于mRNA自身的质量控制更是显得尤为重要。The rapid development of medicinal mRNA technology poses great challenges to the process, yield, quality and cost of large-scale production of mRNA. Especially in the process of large-scale production of mRNA vaccines, the quality control of mRNA itself is even more important. especially important.
5’CAP作为mRNA的重要结构，是mRNA稳定存在并进行蛋白质高效率表达的重要保证。现阶段通过体外转录方法合成的mRNA主要通过帽子类似物和加帽酶的方法对mRNA进行加帽，最终的mRNA产品是否进行了正确的加帽并取得合格的加帽效率则成为了评价mRNA产品的重要指标。并且对于不同的应用场景需要对mRNA加帽的种类进行区分，也就是需要精确测定mRNA的CAP 0和CAP 1的占比。现有加帽效率的测定方法是将mRNA水解后测定m7G与A，U，C，G四种碱基的比例来比较出mRNA的加帽效率，但是由于体外转录的工艺限制，mRNA的纯度无法达到100％，所以基于此类方法不能够准确测定mRNA的加帽效率。同时对于不同种类不同序列的mRNA需要准确知道各碱基的个数才可以进行测量，所以方法并不具有通用性。As an important structure of mRNA, 5'CAP is an important guarantee for the stable existence of mRNA and high-efficiency protein expression. At this stage, the mRNA synthesized by in vitro transcription method is mainly capped by capping analogs and capping enzymes. Whether the final mRNA product is correctly capped and has a qualified capping efficiency becomes the key factor for evaluating mRNA products. important indicators. And for different application scenarios, it is necessary to distinguish the types of mRNA capping, that is, it is necessary to accurately determine the proportion of CAP 0 and CAP 1 of mRNA. The existing method for measuring the capping efficiency is to measure the ratio of m7G to A, U, C, and G after hydrolyzing the mRNA to compare the capping efficiency of the mRNA. However, due to the limitation of the in vitro transcription process, the purity of the mRNA cannot It reaches 100%, so the capping efficiency of mRNA cannot be accurately determined based on this method. At the same time, for different kinds of mRNAs with different sequences, it is necessary to accurately know the number of each base before they can be measured, so the method is not universal.
此外，利用常规ATP、GTP、CTP、UTP并体外转录技术取得的mRNA 会引发机体的自有免疫反应，导致mRNA的降解。修饰核苷酸如假尿苷、5甲基胞嘧啶的加入可以有效的抑制mRNA自身的免疫原性，防止mRNA在体内被降解，并可以延长mRNA在细胞内的表达时间，提高表达效率。所以修饰核苷酸在整个mRNA的占比也是评价mRNA产品的重要指标，经过充分修饰的mRNA才能够拥有较好的功能性。In addition, the mRNA obtained by conventional ATP, GTP, CTP, UTP and in vitro transcription techniques will trigger the body's own immune response, leading to the degradation of mRNA. The addition of modified nucleotides such as pseudouridine and 5-methylcytosine can effectively inhibit the immunogenicity of mRNA itself, prevent mRNA from being degraded in vivo, prolong the expression time of mRNA in cells, and improve expression efficiency. Therefore, the proportion of modified nucleotides in the entire mRNA is also an important indicator for evaluating mRNA products. Only fully modified mRNA can have better functionality.
体外转录的mRNA不可避免的会与空气中的氧气发生接触并导致mRNA的氧化，这当中的氧化产物则以8氧鸟苷为主，8氧鸟苷会导致转录出错，影响蛋白的正确表达。能够准确的判断mRNA氧化产物是评判mRNA药物的一个重要标准。The mRNA transcribed in vitro will inevitably come into contact with oxygen in the air and lead to the oxidation of mRNA. The oxidation product is mainly 8-oxoguanosine. 8-oxoguanosine will cause transcription errors and affect the correct expression of proteins. Being able to accurately judge mRNA oxidation products is an important criterion for evaluating mRNA drugs.
迄今为止，非常多的方法已经被开发用于mRNA产品的检测，比如通过电泳来判断mRNA的长度及纯度，通过PCR及测序技术判断mRNA的序列正确性及Poly A尾巴长度。通过HPLC，GC/MS，LC/MS的方法对加帽效率，修饰核苷酸占比和氧化产物的检测也已经被开发出来。例如专利申请WO2017149139A1中所涉及的方法，然而该方法仅适用于mRNA的纯度为100％时的加帽效率测定。受限于mRNA的生产工艺，mRNA纯度不可能达到100％，所以有大量截短的mRNA仍然会被加帽，这样会导致mRNA的加帽效率测定时会大于100％，所以该方法在原理上是不精确的。可见，现有的所有方法因无法精确测定待测样品中mRNA的物质的量导致难以精确测定加帽效率。So far, many methods have been developed for the detection of mRNA products, such as judging the length and purity of mRNA by electrophoresis, and judging the correctness of mRNA sequence and the length of Poly A tail by PCR and sequencing technology. The detection of capping efficiency, proportion of modified nucleotides and oxidation products by HPLC, GC/MS, LC/MS methods has also been developed. For example, the method involved in the patent application WO2017149139A1, however, this method is only applicable to the determination of capping efficiency when the purity of mRNA is 100%. Limited by the production process of mRNA, the purity of mRNA cannot reach 100%, so a large number of truncated mRNA will still be capped, which will cause the mRNA capping efficiency to be greater than 100%, so the method is in principle is imprecise. It can be seen that all existing methods are difficult to accurately measure the capping efficiency due to the inability to accurately measure the amount of mRNA in the sample to be tested.
发明内容Contents of the invention
本发明所要解决的技术问题是为克服现有技术中缺乏加帽效率的检测方法以及现有方法依赖于mRNA纯度的检测的缺陷，提供一种测定mRNA平均分子量以及Cap0/1、修饰核苷酸及氧化物的方法。The technical problem to be solved by the present invention is to provide a method for determining the average molecular weight of mRNA and Cap0/1, modified nucleotides in order to overcome the lack of detection methods for capping efficiency in the prior art and the detection of mRNA purity in existing methods. and oxide methods.
现有技术中因mRNA纯度检测并不准确，所以基于此类方法测定的加帽效率、修饰核苷酸占比及mRNA氧化的结果并不准确。本方法创造性地 利用毛细管电泳对mRNA的纯度及链长分布进行精确的测定，并将mRNA完全水解后对RNA的例如，7-甲基-鸟苷，2-甲氧基-鸟苷，体外转录时添加的修饰核苷酸，8-氧-鸟苷等特征性化合物进行测定，无需预先测定或知道mRNA的序列及各种不同碱基的含量，通过本发明登载的算法即可精确测得mRNA的CAP0、CAP1，修饰核苷酸及氧化物比例。实现对mRNA质量的精确评价。In the prior art, the detection of mRNA purity is not accurate, so the results of capping efficiency, proportion of modified nucleotides and mRNA oxidation based on this method are not accurate. This method creatively uses capillary electrophoresis to accurately measure the purity and chain length distribution of mRNA, and after the mRNA is completely hydrolyzed, for example, 7-methyl-guanosine, 2-methoxy-guanosine, in vitro transcription When adding modified nucleotides, 8-oxo-guanosine and other characteristic compounds to measure, without pre-determining or knowing the sequence of mRNA and the content of various bases, mRNA can be accurately measured by the algorithm published in the present invention The proportion of CAP0, CAP1, modified nucleotides and oxides. Accurate evaluation of mRNA quality is achieved.
本发明主要通过以下技术方案解决上述技术问题。The present invention mainly solves the above-mentioned technical problems through the following technical solutions.
本发明的技术方案之一为：一种测定样品中mRNA平均分子量的方法，其包括：One of the technical solutions of the present invention is: a method for measuring the average molecular weight of mRNA in a sample, which includes:
(1)获得样品的毛细管电泳图；(1) Obtain the capillary electrophoresis pattern of the sample;
(2)根据公式(I)(2) According to formula (I)
计算得mRNA平均分子量，其中，a为毛细管电泳中所检测到的最短mRNA的碱基个数，b为最长碱基个数，f(N)＝mRNA所占比例，N＝mRNA总碱基个数。The average molecular weight of mRNA is calculated, wherein, a is the number of bases of the shortest mRNA detected in capillary electrophoresis, b is the number of longest bases, f(N)=proportion of mRNA, N=total bases of mRNA number.
在本发明一实施方案中，将毛细管电泳图横坐标分为n等分并命名为a
1、a
2…a
n-1、a
n，其中a
n＝b，所述n≥5，优选地，n≥10；通过如下公式(II)对所述公式(1)进行求解：
In one embodiment of the present invention, the abscissa of the capillary electrophoresis graph is divided into n equal parts and named as a 1 , a 2 ... a n-1 , a n , where a n = b, and n≥5, preferably , n≥10; Solve described formula (1) by following formula (II):
所述R为区间(a
n-1,a
n)内的峰面积占比。
The R is the proportion of the peak area in the interval (a n-1 , a n ).
其中，关于n的数值做如下说明：n无限趋近于正无穷时计算结果无限趋近真实值，因此，在本发明中若取样更多，则结果相对更为精确，但是n取值过大会造成计算十分繁琐，影响效率。本发明中，n可为5～100或5～50或5～20或10～100或10～50或5～20的任何正整数，例如5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、30、40、50等。Among them, the numerical value of n is explained as follows: when n is infinitely close to positive infinity, the calculation result is infinitely close to the true value, therefore, if more samples are taken in the present invention, the result is relatively more accurate, but the value of n is too large The calculation is very complicated and affects the efficiency. In the present invention, n can be any positive integer of 5-100 or 5-50 or 5-20 or 10-100 or 10-50 or 5-20, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, etc.
步骤(1)中所述毛细管电泳可按照本领域常规进行，较佳地使用
small RNA reagent kit以及
GX Touch
TM nucleic acid analyzer进行毛细管电泳，以获得样品的毛细管电泳图。
The capillary electrophoresis described in step (1) can be carried out according to the routine in this field, preferably using small RNA reagent kit and Capillary electrophoresis was performed with GX Touch TM nuclear acid analyzer to obtain the capillary electrophoresis pattern of the sample.
在本发明一较佳实施方案中，所述样品在进行毛细管电泳前还包括除去游离核苷酸的步骤；所述除去游离核苷酸例如采用LiCl沉淀法，离子交换树脂层析，制备级液相色谱或商业用试剂盒进行。In a preferred embodiment of the present invention, the sample also includes a step of removing free nucleotides before capillary electrophoresis; said removing free nucleotides, for example, adopts LiCl precipitation, ion exchange resin chromatography, preparative liquid Chromatography or commercial kits.
本发明的技术方案之二为：一种测定样品中mRNA的物质的量的方法，其包括如下步骤：The second technical solution of the present invention is: a method for measuring the amount of mRNA in a sample, which comprises the steps of:
1)测定样品中mRNA中的质量浓度，计算样品中mRNA的质量；1) measure the mass concentration in the mRNA in the sample, and calculate the quality of the mRNA in the sample;
2)根据如技术方案之一所述的方法，测定样品中mRNA平均分子量；2) According to the method described in one of the technical solutions, measure the average molecular weight of mRNA in the sample;
3)根据步骤1)所述的mRNA的质量和步骤2)中所得样品中mRNA平均分子量得出样品中mRNA的物质的量。3) According to the quality of mRNA described in step 1) and the average molecular weight of mRNA in the sample obtained in step 2), the amount of mRNA in the sample is obtained.
步骤1)中，mRNA中的质量浓度可通过本领域常规，较佳地使用超微量分光光度计测定样品中mRNA的质量浓度；所述超微量分光光度计例如为Thermo Scientific NanoDrop One或者Mettler Toledo UV5 Nano。In step 1), the mass concentration in the mRNA can be routinely used in the art, preferably using an ultra-micro spectrophotometer to measure the mass concentration of the mRNA in the sample; the ultra-micro spectrophotometer is, for example, Thermo Scientific NanoDrop One or Mettler Toledo UV5 Nano.
步骤3)中，所提及的“物质的量”可通过本领域常规方法进行计算，即：mRNA的物质的量＝mRNA的质量/mRNA平均分子量。In step 3), the "substance amount" mentioned can be calculated by conventional methods in the art, ie: mRNA substance amount=mRNA mass/mRNA average molecular weight.
本发明的技术方案之三为：一种mRNA中核苷酸含量的测定方法，所述测定方法包括：The third technical solution of the present invention is: a method for assaying nucleotide content in mRNA, said assay method comprising:
(a)通过如技术方案之二所述的方法获得所述mRNA的物质的量；(a) obtain the amount of the substance of the mRNA by the method as described in the second technical scheme;
(b)测定不同的所述核苷酸的物质的量；(b) determining the amount of a different substance of said nucleotide;
(c)根据步骤(a)中不同的所述核苷酸的物质的量和步骤(b)中所述mRNA的物质的量，计算不同的所述核苷酸在mRNA中的含量；(c) calculating the content of the different nucleotides in the mRNA according to the amount of the different nucleotides in the step (a) and the amount of the mRNA in the step (b);
以上步骤(a)和(b)没有顺序要求，能够分别、同时进行或者先后进行。The above steps (a) and (b) have no order requirement, and can be performed separately, simultaneously or successively.
以上步骤(b)中，优选通过HPLC测定水解的mRNA中不同核苷酸的含量，根据其相对分子质量计算得到不同核苷酸的物质的量。所述水解包括 如下步骤：In the above step (b), it is preferred to measure the content of different nucleotides in the hydrolyzed mRNA by HPLC, and calculate the amount of different nucleotides according to their relative molecular masses. Described hydrolysis comprises the steps:
I.将待水解mRNA与核酸内切酶P1混合，并加入缓冲液A后42℃孵育；所述孵育的时间例如为2小时；所述待水解mRNA、核酸内切酶P1与缓冲液A的体积比例如为100:10:33；1. Mix mRNA to be hydrolyzed with endonuclease P1, and add buffer A to incubate at 42° C.; the incubation time is, for example, 2 hours; the mRNA to be hydrolyzed, endonuclease P1 and buffer A The volume ratio is, for example, 100:10:33;
II.向步骤I所得孵育产物中添加磷酸二酯酶I和碱性磷酸酶，并加入缓冲液B，于37℃孵育；所述孵育的时间例如为2小时，所述磷酸二酯酶I、碱性磷酸酶以及缓冲液B的体积比例如为3.3:3.3:10.6。II. Add phosphodiesterase I and alkaline phosphatase to the incubation product obtained in step I, and add buffer B, and incubate at 37°C; the incubation time is, for example, 2 hours, and the phosphodiesterase I, The volume ratio of alkaline phosphatase and buffer B is, for example, 3.3:3.3:10.6.
本发明中所述的HPLC测定可为本领域常规，较佳地包括：The HPLC measurement described in the present invention can be routine in this field, preferably comprises:
i)使用不同浓度的特定核苷酸的标准品制作浓度与峰面积的标准曲线；i) using different concentrations of specific nucleotide standards to make a standard curve of concentration and peak area;
ii)获得不同的所述核苷酸的峰面积后，对应所述标准曲线计算得所述核苷酸的浓度。ii) After obtaining different peak areas of the nucleotides, calculate the concentration of the nucleotides corresponding to the standard curve.
以上所述HPLC的参数例如为：The parameters of the above-mentioned HPLC are, for example:
柱温25℃，流速0.85mL/min，检测波长254nm，流动相A为5mM醋酸铵水溶液，流动相B为40％乙腈水溶液，梯度100％A—20％B，50min。Column temperature is 25°C, flow rate is 0.85mL/min, detection wavelength is 254nm, mobile phase A is 5mM ammonium acetate aqueous solution, mobile phase B is 40% acetonitrile aqueous solution, gradient 100%A-20%B, 50min.
所测定的核苷酸的种类可为常规的、构成mRNA的核苷，例如腺嘌呤核苷、尿嘧啶核苷、胞嘧啶核苷以及鸟嘌呤核苷；也可为其相应的修饰核苷酸或者氧化后核苷酸以及其他常见的非常规核苷。The types of nucleotides to be determined may be conventional nucleosides constituting mRNA, such as adenosine, uridine, cytidine, and guanosine; they may also be corresponding modified nucleotides Or oxidized nucleotides and other common unconventional nucleosides.
本发明中所述修饰核苷酸优选包括7-甲基-鸟嘌呤核苷、2’-氧甲基-鸟嘌呤核苷、假尿嘧啶核苷、1-甲基-假尿嘧啶核苷、5-甲基-胞嘧啶核苷、4-乙酰基-胞嘧啶核苷以及6-甲基-腺嘌呤核苷中的一种或多种。The modified nucleotides described in the present invention preferably include 7-methyl-guanosine, 2'-oxymethyl-guanosine, pseudouridine, 1-methyl-pseudouridine, One or more of 5-methyl-cytidine, 4-acetyl-cytidine and 6-methyl-adenosine.
在本发明一较佳实施方案中：In a preferred embodiment of the present invention:
a.所述7-甲基鸟嘌呤核苷的占比通过如下公式获得：a. The proportion of the 7-methylguanosine is obtained by the following formula:
CAP0％＝7-甲基鸟嘌呤核苷物质的量/mRNA物质的量×100％；CAP0%=7-methylguanosine substance amount/mRNA substance amount×100%;
b.所述2-氧甲基-鸟嘌呤核苷的占比通过如下公式获得：b. The ratio of the 2-oxymethyl-guanosine is obtained by the following formula:
CAP1％＝2-氧甲基-鸟嘌呤核苷物质的量/mRNA物质的量×100％×CAP0％；CAP1%=2-oxomethyl-guanosine substance amount/mRNA substance amount×100%×CAP0%;
c.所述修饰核苷酸的占比通过如下公式计算：c. The ratio of the modified nucleotides is calculated by the following formula:
修饰核苷酸％＝修饰核苷酸的物质的量/(修饰核苷酸的物质的量+修饰前核苷酸的物质的量)×100％；Modified nucleotide% = the amount of substance to modify nucleotide/(the amount of substance to modify nucleotide+the amount of substance to modify nucleotide) × 100%;
d.所述氧化后核苷酸的占比通过如下公式计算：d. The proportion of the oxidized nucleotide is calculated by the following formula:
氧化后核苷酸％＝氧化后核苷酸的物质的量/(氧化后核苷酸的物质的量+氧化前核苷酸的物质的量)×100％。Nucleotide % after oxidation = the amount of nucleotide substance after oxidation/(the amount of nucleotide substance after oxidation+the amount of nucleotide substance before oxidation)×100%.
所述修饰核苷酸可为本领域常规，较佳地包括：1-甲基-假尿嘧啶核苷、5-甲基-胞嘧啶核苷、4-乙酰基-胞嘧啶核苷以及6-甲基-腺嘌呤核苷中的一种或多种。The modified nucleotides can be conventional in the art, preferably including: 1-methyl-pseudouridine, 5-methyl-cytidine, 4-acetyl-cytidine and 6- One or more of methyl-adenosine nucleosides.
所述氧化后核苷酸也可为本领域常规，例如8-氧-鸟嘌呤核苷。The oxidized nucleotide may also be conventional in the art, such as 8-oxo-guanosine.
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.
本发明所用试剂和原料均市售可得。The reagents and raw materials used in the present invention are all commercially available.
本发明的积极进步效果在于：The positive progress effect of the present invention is:
本发明通过一种新的测定及计算方法对mRNA制备中的Cap 0、Cap 1、修饰核苷酸及氧化产物占比进行精确的测定，本发明对于药用及科研用的mRNA的质量控制提供了非常精确的检测方法。The present invention accurately measures the proportion of Cap 0, Cap 1, modified nucleotides and oxidation products in the preparation of mRNA through a new measurement and calculation method. very precise detection method.
图1为毛细管电泳图谱。Figure 1 is a capillary electrophoresis map.
图2为水解mRNA图谱。Figure 2 is the profile of hydrolyzed mRNA.
图3为胞嘧啶核苷的标准曲线。Figure 3 is the standard curve of cytidine.
图4为尿嘧啶核苷的标准曲线。Figure 4 is the standard curve of uridine.
图5为鸟嘌呤核苷的标准曲线。Figure 5 is a standard curve for guanosine.
图6为7-甲基-鸟嘌呤核苷的标准曲线。Figure 6 is a standard curve for 7-methyl-guanosine.
图7为2-氧甲基-鸟嘌呤核苷的标准曲线。Figure 7 is a standard curve for 2-oxomethyl-guanosine.
图8为假尿嘧啶核苷的标准曲线。Figure 8 is a standard curve of pseudouridine.
图9为8-氧-鸟嘌呤核苷酸的标准曲线。Figure 9 is a standard curve for 8-oxo-guanine nucleotides.
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。The present invention is further illustrated below by means of examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions.
实施例1 游离核苷酸的去除Example 1 The removal of free nucleotides
mRNA的链长分布测定可以使用LabChip GXII Touch HT(PerkinElmer)，2100Electrophoresis Bioanalyzer Instrument(Agilent)，Qsep100(Bioptic)等仪器完成，所有仪器测定都可以得到相同的测定结果。本文中提到的mRNA链长分布都以LabChip GXII Touch HT的测定结果为例。mRNA可采用LiCl沉淀法，离子交换树脂层析，制备级液相色谱或商业用试剂盒等方法进行纯化，完全除去游离核苷酸以防止对各项指标测定产生干扰。The determination of the chain length distribution of mRNA can be done using instruments such as LabChip GXII Touch HT (PerkinElmer), 2100 Electrophoresis Bioanalyzer Instrument (Agilent), Qsep100 (Bioptic), and all instruments can obtain the same measurement results. The distribution of mRNA chain length mentioned in this article is based on the measurement results of LabChip GXII Touch HT as an example. mRNA can be purified by LiCl precipitation method, ion exchange resin chromatography, preparative liquid chromatography or commercial kits to completely remove free nucleotides to prevent interference with the determination of various indicators.
实施例2 mRNA完全水解为单核苷并利用HPLC对各水解后的组分进行鉴定Example 2 mRNA is completely hydrolyzed into single nucleosides and each hydrolyzed component is identified by HPLC
1)实验试剂与设备：1) Experimental reagents and equipment:
核酸内切酶P1(Endonuclease P1)、磷酸二酯酶I(Phosphodiesterase I)以及碱性磷酸酶(Shrimp Alkaline Phosphatase)购于Sigma Aldrich。Endonuclease P1 (Endonuclease P1), phosphodiesterase I (Phosphodiesterase I) and alkaline phosphatase (Shrimp Alkaline Phosphatase) were purchased from Sigma Aldrich.
氯化锌、甘油、醋酸钠、醋酸铵以及醋酸镁均采购自国药试剂集团。超纯水则都取自Milli-Q纯水机。Zinc chloride, glycerin, sodium acetate, ammonium acetate and magnesium acetate were purchased from Sinopharm Reagent Group. Ultrapure water is taken from Milli-Q pure water machine.
尿嘧啶核苷(Uridine)、胞嘧啶核苷(Cytidine)以及鸟嘌呤核苷(Guanosine)均购于Merck；假尿嘧啶核苷(Pseudouridine)、7-甲基鸟嘌呤核苷(7-methyl Guanosine)、8-氧-鸟嘌呤核苷(8-oxo-Guanosine)以及2’-氧甲基-鸟嘌呤核苷(2’-O-MethylGuanosine，简称2’-OMe-Guanosine)购于TCI。Uridine, Cytidine and Guanosine were purchased from Merck; Pseudouridine, 7-methylguanosine ), 8-oxo-guanosine (8-oxo-Guanosine) and 2'-oxymethyl-guanosine (2'-O-MethylGuanosine, referred to as 2'-OMe-Guanosine) were purchased from TCI.
高效液相色谱仪(1260Infinity II bioInert LC)购置于安捷伦科技，高效液相色谱柱(XBridge BEH C18 Column,
5μm,4.6mm X 250mm,1/pk)购置于Waters，台式离心机(ST8)与恒温振荡金属浴(88880028)购置于Thermo，分析天平(XS205DU)购置于Multiparameter，微量移液器购置于RAININ。
The high-performance liquid chromatography (1260Infinity II bioInert LC) was purchased from Agilent Technologies, and the high-performance liquid chromatography column (XBridge BEH C18 Column, 5μm, 4.6mm X 250mm, 1/pk) was purchased from Waters, the benchtop centrifuge (ST8) and constant temperature shaking metal bath (88880028) were purchased from Thermo, the analytical balance (XS205DU) was purchased from Multiparameter, and the micropipette was purchased from RAININ.
2)实验方法与操作：2) Experimental method and operation:
在贮存液(110mM Tris-HCl缓冲液、100mM氯化钠、15mM氯化镁、50％甘油，pH 8.9)中加入7g/L磷酸二酯酶I(Phosphodiesterase I)，配制为磷酸二酯酶I(Phosphodiesterase I)贮存液，储存于-20℃。Add 7g/L phosphodiesterase I (Phosphodiesterase I) to the storage solution (110mM Tris-HCl buffer solution, 100mM sodium chloride, 15mM magnesium chloride, 50% glycerol, pH 8.9) to prepare phosphodiesterase I (Phosphodiesterase I) I) Stock solution, stored at -20°C.
核酸内切酶P1(Endonuclease P1)配制为1g/L的贮存液(20mM醋酸钾、5mM氯化锌、50mM氯化钠以及50％甘油)储存于-20℃。Endonuclease P1 (Endonuclease P1) was prepared as a 1g/L stock solution (20mM potassium acetate, 5mM zinc chloride, 50mM sodium chloride and 50% glycerol) and stored at -20°C.
缓冲液A(141.43mM醋酸铵、9.43mM氯化锌，pH 5.3)。Buffer A (141.43mM ammonium acetate, 9.43mM zinc chloride, pH 5.3).
缓冲液B(115.00mM Tris-HCl、11.50mM醋酸镁，pH 8.3)。Buffer B (115.00mM Tris-HCl, 11.50mM magnesium acetate, pH 8.3).
将100μL待测mRNA(3.018mg/mL)样品加入2μL稀释10倍的核酸内切酶P1(Endonuclease P1)，并加入33μL缓冲液A后42℃孵育2小时，加入3.3μL磷酸二酯酶I(Phosphodiesterase I)贮存液和碱性磷酸酶(Shrimp Alkaline Phosphatase)(0.1U/μL)补加缓冲液B10.6μL并继续37℃孵育2小时。mRNA降解完成后17000g离心10min，取出上清液(体积153.3μL，mRNA降解物1.969mg/mL)检测备用。将HPLC调节至如下参数：柱温25℃，流速0.85mL/min，检测波长254nm，流动相A为5mM醋酸铵水溶液，流动相B为40％乙腈水溶液，梯度100％A—20％B，50min。可取得水解mRNA图谱如图2所示。Add 100 μL of the mRNA (3.018 mg/mL) sample to be tested to 2 μL of 10-fold diluted endonuclease P1 (Endonuclease P1), add 33 μL of buffer A, incubate at 42°C for 2 hours, add 3.3 μL of phosphodiesterase I ( Phosphodiesterase I) stock solution and alkaline phosphatase (Shrimp Alkaline Phosphatase) (0.1U/μL) were supplemented with 10.6 μL of buffer B and continued to incubate at 37°C for 2 hours. After mRNA degradation was completed, centrifuge at 17,000 g for 10 min, and remove the supernatant (volume 153.3 μL, mRNA degradation product 1.969 mg/mL) for detection and use. Adjust the HPLC to the following parameters: column temperature 25°C, flow rate 0.85mL/min, detection wavelength 254nm, mobile phase A is 5mM ammonium acetate aqueous solution, mobile phase B is 40% acetonitrile aqueous solution, gradient 100%A—20%B, 50min . The obtained hydrolyzed mRNA profile is shown in Figure 2.
通过比对不同核苷酸的保留时间并积分可得到核苷酸峰面积：The nucleotide peak area can be obtained by comparing the retention times of different nucleotides and integrating:
假尿嘧啶核苷(Pseudouridine)：4263.484，尿嘧啶核苷(Uridine)：12.689，7-甲基鸟嘌呤核苷(7-methyl Guanosine)：18.998，2’-氧甲基-鸟嘌呤核苷(2’-OMe-Guanosine)：31.655，8-氧代-鸟嘌呤核苷(8-oxo-Guanosine)：0。Pseudouridine (Pseudouridine): 4263.484, Uridine (Uridine): 12.689, 7-methyl Guanosine (7-methyl Guanosine): 18.998, 2'-Oxymethyl-guanosine ( 2'-OMe-Guanosine): 31.655, 8-oxo-Guanosine (8-oxo-Guanosine): 0.
将尿嘧啶核苷、胞嘧啶核苷、鸟嘌呤核苷、假尿嘧啶核苷(Pseudouridine)、7-甲基鸟嘌呤核苷(7-methyl Guanosine)、8-氧-鸟嘌呤核苷(8-oxo-Guanosine)以及2’-氧甲基-鸟嘌呤核苷(2’-OMe-Guanosine)等多种核苷配制成梯度浓度水溶液利用HPLC进行测量，每个样品进样量10μL，记录不同核苷标准品的保留时间，并利用归一法积分计算不同核苷在不同浓度下的峰面积，并由此依据核苷的梯度浓度绘制核苷的标准曲线。Uridine, cytidine, guanosine, pseudouridine (Pseudouridine), 7-methylguanosine (7-methyl Guanosine), 8-oxo-guanosine (8 -oxo-Guanosine) and 2'-Oxymethyl-Guanosine (2'-OMe-Guanosine) and other nucleosides were prepared into a gradient concentration aqueous solution and measured by HPLC. The injection volume of each sample was 10 μL, and the records were different. The retention time of the nucleoside standard substance, and use the normalization method to integrate and calculate the peak area of different nucleosides at different concentrations, and thus draw a nucleoside standard curve based on the gradient concentration of the nucleoside.
根据如下表格1～7绘制得如图3～图9所示的各核苷标准曲线。The standard curves for each nucleoside as shown in Figures 3 to 9 were drawn according to the following Tables 1 to 7.
表1Table 1
表2Table 2
表3table 3
表4Table 4
表5table 5
表6Table 6
表7Table 7
根据不同核苷的标准曲线和峰面积可以计算得出单位进样量(10μl)中不同核苷的浓度如下所示：假尿嘧啶核苷(Pseudouridine)：8.64E-03μmoL，尿嘧啶核苷(Uridine)：2.97E-05μmoL，7-甲基-鸟嘌呤核苷(7-methyl Guanosine)：2.96E-05μmoL，2’-氧甲基-鸟嘌呤核苷(2’-OMe-Guanosine)：3.02E-05μmoL，8-氧-鸟嘌呤核苷(8-oxo-Guanosine)：0(未检出)。According to the standard curve and peak area of different nucleosides, the concentration of different nucleosides in the unit injection volume (10 μl) can be calculated as follows: Pseudouridine (Pseudouridine): 8.64E-03 μmoL, uridine ( Uridine): 2.97E-05μmoL, 7-methyl-guanosine (7-methyl Guanosine): 2.96E-05μmoL, 2'-oxymethyl-guanosine (2'-OMe-Guanosine): 3.02 E-05 μmoL, 8-oxo-guanosine (8-oxo-Guanosine): 0 (not detected).
3、利用本发明的算法计算mRNA的各项指标。3. Using the algorithm of the present invention to calculate various indexes of mRNA.
1)精确标定样品中mRNA的质量浓度。1) Accurately calibrate the mass concentration of mRNA in the sample.
首先利用Nanodrop精确测定样品中mRNA的质量浓度，Nanodrop可以选用Thermo Scientific NanoDrop One，Mettler Toledo UV5 Nano等类似设备。First, use Nanodrop to accurately measure the mass concentration of mRNA in the sample. Nanodrop can use Thermo Scientific NanoDrop One, Mettler Toledo UV5 Nano and other similar equipment.
2)精确测定样品mRNA的平均分子质量。2) Accurately measure the average molecular mass of the sample mRNA.
考虑到依据现有的体外转录法进行mRNA大规模合成的成品mRNA很难达到100％纯度，所以仅仅使用经验公式：Considering that it is difficult to achieve 100% purity of the finished mRNA for large-scale synthesis of mRNA based on the existing in vitro transcription method, so only the empirical formula is used:
MW(RNA)＝(A×329.2)+(U×306.2)+(C×305.2)+(G×345.2)+159MW(RNA)＝(A×329.2)+(U×306.2)+(C×305.2)+(G×345.2)+159
无法进行准确的mRNA的分子量进行计算。Unable to perform accurate mRNA molecular weight calculations.
本发明使用利用毛细管电泳图谱与创新性算法相结合，无需知晓mRNA碱基序列及各碱基个数的前提下就可以对样品mRNA序列进行精确标定从而计算Cap0、Cap1、修饰核苷酸及氧化物的含量。The present invention uses the capillary electrophoresis map combined with an innovative algorithm to accurately calibrate the sample mRNA sequence without knowing the mRNA base sequence and the number of each base to calculate Cap0, Cap1, modified nucleotides and oxidation substance content.
当mRNA的纯度为100％时，mRNA的分子量的计算方法如下：When the purity of mRNA is 100%, the calculation method of the molecular weight of mRNA is as follows:
mRNA分子量可以精确计算为：The molecular weight of mRNA can be accurately calculated as:
MW＝(A×329.2)+(U×306.2)+(C×305.2)+(G×345.2)+159MW＝(A×329.2)+(U×306.2)+(C×305.2)+(G×345.2)+159
当mRNA长度大于1000nt后mRNA分子量可以近似为：When the mRNA length is greater than 1000nt, the mRNA molecular weight can be approximated as:
MW＝N×320.5+159(N＝mRNA总碱基个数)MW=N×320.5+159 (N=number of total bases in mRNA)
但是基于现有mRNA制备工艺，100％纯度是不能达到的。如果mRNA的纯度无法达到100％，则需要利用mRNA的毛细管电泳图谱进行测定。However, based on the existing mRNA preparation process, 100% purity cannot be achieved. If the purity of mRNA cannot reach 100%, it needs to be determined by capillary electrophoresis pattern of mRNA.
将毛细管电泳图谱曲线定义为函数：Define the CE spectrum curve as a function:
R＝f(N)，N＝mRNA总碱基个数，R＝mRNA所占比例。R=f(N), N=number of total bases in mRNA, R=proportion of mRNA.
样品mRNA的平均分子量则可以由此计算得出：The average molecular weight of the sample mRNA can then be calculated as follows:
a为毛细管电泳图中可检测到的最短mRNA碱基个数，b为毛细管电泳图中可检测到的最长碱基个数。a is the shortest mRNA base number detectable in capillary electrophoresis, b is the longest base number detectable in capillary electrophoresis.
函数f(N)的求解为“求平面坐标系中任意曲线的函数表达式”，求解此函数可以借用高等数学的“多项式拟合方程”，或者借用Python及MatLab进行求解。The solution of the function f(N) is "finding the function expression of any curve in the plane coordinate system". To solve this function, you can use the "polynomial fitting equation" of advanced mathematics, or use Python and MatLab to solve it.
此方程可以借助wolframalpha进行求解(https://www.wolframalpha.com/)，或者采用本发明提供的近似算法：This equation can be solved with the help of wolframalpha (https://www.wolframalpha.com/), or using the approximate algorithm provided by the present invention:
将毛细管电泳图横坐标分为n(n≥10，本实施例中n＝11)等分即a
1,a
2…a
n-1,a
n，其中a
n＝b(本实施例中b＝2946)。其中R为区间(a
n-1,a
n)内的峰面积占比。
The abscissa of the capillary electrophoresis graph is divided into n (n ≥ 10, n=11 in this embodiment) equal parts, namely a 1 , a 2 ... a n-1 , a n , where a n = b (b in this embodiment =2946). Where R is the proportion of the peak area in the interval (a n-1 , a n ).
mRNA平均分子量可以近似计算为：The average molecular weight of mRNA can be approximated as:
表8Table 8
如表8和图1所示，将待测mRNA毛细管电泳图谱进行分割后，得出不同长度区间的mRNA的分配比例。然后代入以上公式可以得出mRNA的平均分子量及单位进样量(10μl)中mRNA的摩尔数，结果见下表9。As shown in Table 8 and FIG. 1, after the capillary electrophoresis pattern of the mRNA to be tested is segmented, the distribution ratio of mRNAs in different length intervals can be obtained. Then by substituting the above formula, the average molecular weight of mRNA and the number of moles of mRNA in unit injection volume (10 μl) can be obtained, and the results are shown in Table 9 below.
表9Table 9
3)CAP0及CAP1占比计算3) Calculate the proportion of CAP0 and CAP1
mRNA的CAP结构如下所示：The CAP structure of mRNA is as follows:
其中7-甲基-鸟嘌呤核苷(7-methyl Guanosine)是CAP0结构的标志物，每个完整的mRNA有且仅有一个7-甲基-鸟嘌呤核苷(7-methyl Guanosine)：Among them, 7-methyl-guanosine (7-methyl Guanosine) is a marker of the CAP0 structure, and each complete mRNA has and only one 7-methyl-guanosine (7-methyl Guanosine):
因此可以通过精确测定7-甲基-鸟嘌呤核苷(7-methyl Guanosine)的含量，样品mRNA的CAP0占比可以计算为：Therefore, by accurately measuring the content of 7-methyl-guanosine (7-methyl Guanosine), the CAP0 ratio of the sample mRNA can be calculated as:
CAP0％＝7-甲基-鸟嘌呤核苷(7-methyl Guanosine)物质的量/mRNA物 质的量×100％。CAP0% = amount of 7-methyl-guanosine (7-methyl Guanosine) substance/amount of mRNA substance × 100%.
根据实验测得结果CAP0％＝2.96E-05μmoL/3.32E-05μmoL＝89％。According to the experimental results, CAP0%=2.96E-05 μmoL/3.32E-05 μmoL=89%.
7-甲基-鸟嘌呤核苷(7-methyl Guanosine)与2-氧甲基-鸟嘌呤核苷(2-OMe-Guanosine)通过三磷酸键相连的复合结构是mRNA的CAP1结构的标志物，每个完整的mRNA有且仅有一个7-甲基-鸟嘌呤核苷(7-methyl Guanosine)与2’-氧甲基-鸟嘌呤核苷(2’-OMe-Guanosine)的复合结构。因此通过测定标志物2’-氧甲基-鸟嘌呤核苷(2’-OMe-Guanosine)的物质的量即可得到CAP1的占比：The complex structure of 7-methyl-guanosine (7-methyl Guanosine) and 2-oxymethyl-guanosine (2-OMe-Guanosine) connected by a triphosphate bond is a marker of the CAP1 structure of mRNA. Each complete mRNA has one and only one complex structure of 7-methyl-guanosine (7-methyl Guanosine) and 2'-oxymethyl-guanosine (2'-OMe-Guanosine). Therefore, the proportion of CAP1 can be obtained by measuring the amount of the marker 2'-oxymethyl-guanosine (2'-OMe-Guanosine):
CAP1％＝2’-氧甲基-鸟嘌呤核苷(2’-OMe-Guanosine)物质的量/mRNA物质的量×100％×CAP0％。CAP1%=the amount of 2'-oxymethyl-guanosine (2'-OMe-Guanosine) substance/the amount of mRNA substance×100%×CAP0%.
根据实验结果测得CAP1％＝3.02E-05μmoL/3.32E-05μmoL×89％＝81％。According to the experimental results, CAP1%=3.02E-05 μmoL/3.32E-05 μmoL×89%=81%.
4)修饰核苷酸占比测定4) Determination of the proportion of modified nucleotides
为提高药用mRNA的表达效率并延长表达时间，在体外转录的过程中往往需要加入假尿嘧啶核苷(Pseudouridine)、1-甲基-假尿嘧啶核苷(1-Methyl-Pseudouridine)、5-甲基-胞嘧啶核苷(5-Methyl-Cytidine)、4-乙酰-胞嘧啶核苷(4-Acetyl-Cytidine)以及6-甲基-腺嘌呤核苷(6-Methyl-Adenesine)等修饰核苷酸。In order to improve the expression efficiency of medicinal mRNA and prolong the expression time, it is often necessary to add pseudouridine (Pseudouridine), 1-methyl-pseudouridine (1-Methyl-Pseudouridine), 5 -Methyl-Cytidine (5-Methyl-Cytidine), 4-Acetyl-Cytidine (4-Acetyl-Cytidine) and 6-Methyl-Adenesine (6-Methyl-Adenesine) and other modifications Nucleotides.
为测定相应修饰核苷酸在mRNA样品中的占比可以利用如下公式进行计算。In order to determine the proportion of the corresponding modified nucleotide in the mRNA sample, the following formula can be used for calculation.
假尿嘧啶核苷(Pseudouridine)％＝假尿嘧啶核苷(Pseudouridine)物质的量/(尿嘧啶核苷(Uridine)物质的量+假尿嘧啶核苷(Pseudouridine)物质的量)×100％。Pseudouridine (Pseudouridine)%=the amount of pseudouridine (Pseudouridine) substance/(uridine (Uridine) substance amount+pseudouridine (Pseudouridine) substance amount)×100%.
本实验可以精确测得：假尿嘧啶核苷(Pseudouridine)％＝8.64nmoL/(8.64nmoL+2.97E-02nmoL)×100％＝99.7％。This experiment can accurately measure: Pseudouridine %=8.64nmoL/(8.64nmoL+2.97E-02nmoL)×100%=99.7%.
1-甲基-假尿嘧啶核苷(1-Methyl-Pseudouridine)％＝1-甲基-假尿嘧啶核苷(1-Methyl-Pseudouridine)物质的量/(1-甲基-假尿嘧啶核苷(1-Methyl-Pseudouridine)物质的量+尿嘧啶核苷(Uridine)物质的量)×100％。1-Methyl-Pseudouridine (1-Methyl-Pseudouridine)%=1-Methyl-Pseudouridine (1-Methyl-Pseudouridine) amount of substance/(1-Methyl-Pseudouridine nucleus Glycoside (1-Methyl-Pseudouridine) substance amount+uridine nucleoside (Uridine) substance amount)×100%.
5-甲基-胞嘧啶核苷(5-Methyl-Cytidine)％＝5-甲基-胞嘧啶核苷(5-Methyl-Cytidine)物质的量/(5-甲基-胞嘧啶核苷(5-Methyl-Cytidine)物质的量+胞嘧啶核苷(Cytidine)物质的量)×100％。5-Methyl-Cytidine (5-Methyl-Cytidine) %=5-Methyl-Cytidine (5-Methyl-Cytidine) amount of substance/(5-Methyl-Cytidine (5 - the amount of Methyl-Cytidine) substance+the amount of Cytidine substance)×100%.
4-乙酰基胞嘧啶核苷(4-Acetyl-Cytidine)％＝4-Acetyl-Cytidine物质的量/(4-Acetyl-Cytidine物质的量+Cytidine物质的量)×100％。4-Acetyl-Cytidine (4-Acetyl-Cytidine)%=Amount of 4-Acetyl-Cytidine/(Amount of 4-Acetyl-Cytidine+Amount of Cytidine)×100%.
6-甲基-腺嘌呤核苷(6-Methyl-Adenesine)％＝6-甲基-腺嘌呤核苷(6-Methyl-Adenesine)物质的量/(6-甲基-腺嘌呤核苷(6-Methyl-Adenesine)物质的量+腺嘌呤核苷(Adenesine)物质的量)×100％。6-Methyl-Adenesine (6-Methyl-Adenesine)%=6-Methyl-Adenesine (6-Methyl-Adenesine) amount of substance/(6-Methyl-Adenesine (6 - the amount of Methyl-Adenesine) substance+the amount of adenosine (Adenesine) substance)×100%.
其它任意修饰核苷酸占比都可采用同样的方法进行精确计算。The ratio of any other modified nucleotides can be accurately calculated using the same method.
5)氧化产物占比测定5) Determination of the proportion of oxidation products
mRNA长期与空气接触会不可避免的发生氧化。其氧化反应主要表现在鸟嘌呤核苷(Guanosine)转化为8-氧-鸟嘌呤核苷(8-oxo-Guanosine)。mRNA will inevitably be oxidized when exposed to air for a long time. The oxidation reaction is mainly manifested in the conversion of Guanosine to 8-oxo-Guanosine.
对8-氧-鸟嘌呤核苷(8-oxo-Guanosine)的浓度进行精确测定可以直接得出mRNA的氧化产物含量。Accurate determination of the concentration of 8-oxo-guanosine (8-oxo-Guanosine) can directly obtain the content of oxidation products of mRNA.
Oxidation％＝8-氧-鸟嘌呤核苷(8-oxo-Guanosine)物质的量/(8-氧-鸟嘌呤核苷(8-oxo-Guanosine)物质的量+鸟嘌呤核苷(Guanosine)物质的量)×100％。Oxidation%=8-oxo-guanosine (8-oxo-Guanosine) substance amount/(8-oxo-guanosine nucleoside (8-oxo-Guanosine) substance amount+guanosine nucleoside (Guanosine) substance amount) × 100%.
本实验中mRNA氧化产物未检出。mRNA oxidation products were not detected in this experiment.
Claims (10)
- 一种测定样品中mRNA平均分子量的方法，其特征在于，其包括：A method for measuring the average molecular weight of mRNA in a sample, characterized in that it comprises:(1)获得样品的毛细管电泳图；(1) Obtain the capillary electrophoresis pattern of the sample;(2)根据公式(I)(2) According to the formula (I)
计算得mRNA平均分子量，其中，a为毛细管电泳中所检测到的最短mRNA的碱基个数，b为最长碱基个数，f(N)＝mRNA所占比例，N＝mRNA总碱基个数。The average molecular weight of mRNA is calculated, wherein, a is the number of bases of the shortest mRNA detected in capillary electrophoresis, b is the number of longest bases, f(N)=proportion of mRNA, N=total bases of mRNA number. - 如权利要求1所述的方法，其特征在于，将毛细管电泳图横坐标分为n等分并命名为a 1、a 2…a n-1、a n，其中a n＝b，所述n≥5，优选地，n≥10；通过如下公式(II)对所述公式(I)进行求解： The method according to claim 1, characterized in that the abscissa of the capillary electrophoresis graph is divided into n equal parts and named as a 1 , a 2 ... a n-1 , a n , where a n = b, and the n ≥5, preferably, n≥10; the formula (I) is solved by the following formula (II):
所述R为区间(a n-1,a n)内的峰面积占比。 The R is the proportion of the peak area in the interval (a n-1 , a n ). - 如权利要求1或2所述的方法，其特征在于，步骤(1)中，使用small RNA reagent kit以及
GX Touch TMnucleic acid analyzer进行毛细管电泳，以获得样品的毛细管电泳图；优选地，所述样品在进行毛细管电泳前还包括除去游离核苷酸的步骤；所述除去游离核苷酸例如采用LiCl沉淀法，离子交换树脂层析，制备级液相色谱或商业用试剂盒进行。 The method according to claim 1 or 2, characterized in that, in step (1), using
small RNA reagent kit andGX Touch TM nucleic acid analyzer performs capillary electrophoresis to obtain the capillary electrophoresis pattern of the sample; preferably, the sample also includes a step of removing free nucleotides before capillary electrophoresis; the removal of free nucleotides is, for example, carried out by LiCl precipitation method, ion exchange resin chromatography, preparative liquid chromatography or commercial kits. - 一种测定样品中mRNA的物质的量的方法，其特征在于，其包括如下步骤：A method for measuring the amount of mRNA in a sample, characterized in that it comprises the steps of:1)测定样品中mRNA中的质量浓度，计算样品中mRNA的质量；1) measure the mass concentration in the mRNA in the sample, and calculate the quality of the mRNA in the sample;2)根据如权利要求1-3中任一项所述的方法，测定样品中mRNA平均分子量；2) according to the method as described in any one in claim 1-3, measure the average molecular weight of mRNA in the sample;3)根据步骤1)所述的mRNA的质量和步骤2)中所得样品中mRNA 平均分子量得出样品中mRNA的物质的量。3) According to the quality of mRNA described in step 1) and the average molecular weight of mRNA in the sample obtained in step 2), the amount of mRNA in the sample is obtained.
- 如权利要求4所述的方法，其特征在于，步骤1)中使用超微量分光光度计测定样品中mRNA的质量浓度；所述超微量分光光度计例如Thermo Scientific NanoDrop One或者Mettler Toledo UV5 Nano。The method according to claim 4, wherein, step 1) uses an ultra-micro spectrophotometer to measure the mass concentration of mRNA in the sample; said ultra-micro spectrophotometer is such as Thermo Scientific NanoDrop One or Mettler Toledo UV5 Nano.
- 一种mRNA中核苷酸含量的测定方法，其特征在于，所述测定方法包括：A kind of assay method of nucleotide content in mRNA, it is characterized in that, described assay method comprises:(a)通过如权利要求4或5所述的方法获得所述mRNA的物质的量；(a) obtain the amount of the substance of described mRNA by the method as claimed in claim 4 or 5;(b)测定不同的所述核苷酸的物质的量；(b) determining the amount of a different substance of said nucleotide;(c)根据步骤(a)中不同的所述核苷酸的物质的量和步骤(b)中所述mRNA的物质的量，计算不同的所述核苷酸在mRNA中的含量；(c) calculating the content of the different nucleotides in the mRNA according to the amount of the different nucleotides in the step (a) and the amount of the mRNA in the step (b);以上步骤(a)和(b)没有顺序要求，能够分别、同时进行或者先后进行。The above steps (a) and (b) have no order requirement, and can be performed separately, simultaneously or sequentially.
- 如权利要求6所述的测定方法，其特征在于，步骤(b)中，通过HPLC测定水解的mRNA中不同核苷酸的含量，根据其相对分子质量计算得到不同核苷酸的物质的量；The assay method according to claim 6, characterized in that, in step (b), the content of different nucleotides in the mRNA of hydrolysis is measured by HPLC, and the amount of substances of different nucleotides is calculated according to its relative molecular mass;优选地，所述水解包括如下步骤：Preferably, the hydrolysis comprises the steps of:I.将待水解mRNA与核酸内切酶P1混合，并加入缓冲液A后42℃孵育；所述孵育的时间例如为2小时；所述待水解mRNA、核酸内切酶P1与缓冲液A的体积比例如为100:10:33；1. Mix mRNA to be hydrolyzed with endonuclease P1, and add buffer A to incubate at 42° C.; the incubation time is, for example, 2 hours; the mRNA to be hydrolyzed, endonuclease P1 and buffer A The volume ratio is, for example, 100:10:33;II.向步骤I所得孵育产物中添加磷酸二酯酶I和碱性磷酸酶，并加入缓冲液B，于37℃孵育；所述孵育的时间例如为2小时，所述磷酸二酯酶I、碱性磷酸酶以及缓冲液B的体积比例如为3.3:3.3:10.6；II. Add phosphodiesterase I and alkaline phosphatase to the incubation product obtained in step I, and add buffer B, and incubate at 37°C; the incubation time is, for example, 2 hours, and the phosphodiesterase I, The volume ratio of alkaline phosphatase and buffer B is, for example, 3.3:3.3:10.6;和/或，步骤(b)中的所述HPLC测定包括：And/or, the HPLC determination in step (b) comprises:i)使用不同浓度的特定核苷酸的标准品制作浓度与峰面积的标准曲线；i) using different concentrations of specific nucleotide standards to make a standard curve of concentration and peak area;ii)获得不同的所述核苷酸的峰面积后，对应所述标准曲线计算得所述核苷酸的浓度；ii) after obtaining different peak areas of the nucleotides, calculate the concentration of the nucleotides corresponding to the standard curve;其中，所述HPLC的参数例如为：Wherein, the parameter of described HPLC is for example:柱温25℃，流速0.85mL/min，检测波长254nm，流动相A为5mM醋酸铵水溶液，流动相B为40％乙腈水溶液，梯度100％A—20％B，50min。Column temperature is 25°C, flow rate is 0.85mL/min, detection wavelength is 254nm, mobile phase A is 5mM ammonium acetate aqueous solution, mobile phase B is 40% acetonitrile aqueous solution, gradient 100%A-20%B, 50min.
- 如权利要求6或7所述的测定方法，其特征在于，所述核苷酸包括尿嘧啶核苷、胞嘧啶核苷、鸟嘌呤核苷、及其相应的修饰核苷酸以及氧化后核苷酸中的一种或多种；优选地，所述修饰核苷酸包括：7-甲基-鸟嘌呤核苷、2’-氧甲基-鸟嘌呤核苷、假尿嘧啶核苷、1-甲基-假尿嘧啶核苷、5-甲基-胞嘧啶核苷、4-乙酰基-胞嘧啶核苷以及6-甲基-腺嘌呤核苷中的一种或多种。The assay method according to claim 6 or 7, wherein the nucleotides include uridine, cytidine, guanosine, and their corresponding modified nucleotides and oxidized nucleosides One or more of acids; preferably, the modified nucleotides include: 7-methyl-guanosine, 2'-oxymethyl-guanosine, pseudouridine, 1- One or more of methyl-pseudouridine, 5-methyl-cytidine, 4-acetyl-cytidine and 6-methyl-adenosine.
- 如权利要求8所述的测定方法，其特征在于，Assay method as claimed in claim 8, is characterized in that,a.所述7-甲基-鸟嘌呤核苷的占比通过如下公式获得：a. The proportion of the 7-methyl-guanosine is obtained by the following formula:CAP0％＝7-甲基-鸟嘌呤核苷物质的量/mRNA物质的量×100％；CAP0%=7-methyl-guanosine substance amount/mRNA substance amount×100%;b.所述2’-氧甲基-鸟嘌呤核苷的占比通过如下公式获得：b. The ratio of the 2'-oxymethyl-guanosine is obtained by the following formula:CAP1％＝2’-氧甲基-鸟嘌呤核苷物质的量/mRNA物质的量×100％×CAP0％；CAP1%=2'-oxymethyl-guanosine substance amount/mRNA substance amount×100%×CAP0%;c.所述修饰核苷酸的占比通过如下公式计算：c. The ratio of the modified nucleotides is calculated by the following formula:修饰核苷酸％＝修饰核苷酸的物质的量/(修饰核苷酸的物质的量+修饰前核苷酸的物质的量)×100％；Modified nucleotide% = the amount of substance to modify nucleotide/(the amount of substance to modify nucleotide+the amount of substance to modify nucleotide) × 100%;d.所述氧化后核苷酸的占比通过如下公式计算：d. The ratio of the oxidized nucleotides is calculated by the following formula:氧化后核苷酸％＝氧化后核苷酸的物质的量/(氧化后核苷酸的物质的量+氧化前核苷酸的物质的量)×100％。Nucleotide % after oxidation = the amount of nucleotide substance after oxidation/(the amount of nucleotide substance after oxidation+the amount of nucleotide substance before oxidation)×100%.
- 如权利要求8或9所述的测定方法，其特征在于，所述氧化后核苷酸包括8-氧-鸟嘌呤核苷。The assay method according to claim 8 or 9, wherein the oxidized nucleotide comprises 8-oxo-guanosine.
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