CN115346595A - Method for measuring mRNA average molecular weight and Cap0/1, modified nucleotide and oxide - Google Patents
Method for measuring mRNA average molecular weight and Cap0/1, modified nucleotide and oxide Download PDFInfo
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- CN115346595A CN115346595A CN202110518577.1A CN202110518577A CN115346595A CN 115346595 A CN115346595 A CN 115346595A CN 202110518577 A CN202110518577 A CN 202110518577A CN 115346595 A CN115346595 A CN 115346595A
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- mrna
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- guanosine
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for measuring mRNA average molecular weight and Cap0/1, modified nucleotide and oxide. The method comprises the following steps: (1) obtaining a capillary electrophoresis image of the sample; (2) The mRNA average molecular weight was calculated according to the formula (I) of the present invention. The method can be used for accurately measuring the average molecular weight of mRNA and the proportion of various types of nucleotides obtained after mRNA cleavage.
Description
Technical Field
The invention belongs to the field of biological medicine, and particularly relates to a method for measuring the average molecular weight of mRNA in a sample and application thereof, in particular to a method for measuring the average molecular weight of mRNA, cap0/1 and modified nucleotide and oxide.
Background
With the development of nucleic acid drugs, mRNA is considered as a new choice that can be used in drug manufacturing. In 1990, a segment of mRNA was injected into mice and successfully encoded a protein. This mRNA is obtained by a technique known as in vitro transcription. Subsequently, a 1992 study found that injection of the antidiuretic hormone-encoding mRNA successfully induced neuronal activity in the hypothalamus of rats. Although mRNA shows excellent biological activity, mRNA is far from being used in clinical disease therapy due to its instability, strong immunogenicity, and difficulty in vivo delivery.
With the continuous development and research of the technology in the past decades, mRNA becomes a therapeutic means with a wide application prospect, and particularly has outstanding advantages in the aspects of vaccines and protein replacement therapy. In combating viral infectious diseases, mRNA shows some advantages as follows:
mRNA is a platform without infectivity and integration ability, without any potential risk of infection and mutagenic integration. And mRNA is gradually degraded with cellular metabolism, its own half-life can be controlled by various modifications and delivery means. The inherent immunogenicity of the mRNA itself may also be modulated to improve safety of administration.
2. Various modifications may allow mRNA to become more stable and easier to deliver. Efficient mRNA delivery can be achieved by loading the carrier molecule into cytoplasm rapidly to achieve efficient expression. mRNA is a very small genetic information carrier. Clearance of the immune system must be avoided. The mRNA can be designed rapidly as desired.
mRNA can be produced at low cost and high efficiency by a high-efficiency in vitro transcription technique.
The field of mRNA vaccines is now developing very rapidly, and a very large number of preclinical research data have been generated over the past few years.
There are also many items on which clinical trials have been conducted. All this suggests that mRNA vaccines have great potential to meet challenges from a variety of infectious diseases and cancers.
The mRNA serves just as an intermediary in the process of DNA translation and ribosomal protein assembly. The mrnas currently used for vaccine development are largely divided into two categories: non-replicating mrnas derived from viruses and self-amplifying RNAs. A typical non-replicating mRNA vaccine comprises an antigen coding sequence of interest, 5 'and 3' UTR. Self-amplifying RNA not only possesses antigen coding sequence, but also possesses virus replication regulating sequence capable of realizing intracellular self-replication and raising protein expression level.
Clinically used mRNA is obtained by In Vitro Transcription (IVT) techniques. In brief, in vitro transcribed RNA is synthesized using a linearized DNA template with RNA polymerase such as T7, T3 or SP 6. The resulting transcript contained the protein coding sequence, flanked by the non-coding UTR, a 5' cap and a poly A tail. This in vitro synthesized mRNA is completely similar to the mature mRNA molecule in the cytoplasm of eukaryotic cells.
The complexity of mRNA delivery in vivo is also increasingly recognized. Naked mRNA is rapidly degraded by extracellular mRNA enzyme and cannot enter cells to play a role. A number of delivery vehicle agents have been developed for intracellular engulfment of mRNA and to protect mRNA from degradation. Once the mRNA enters the cytoplasm, the cell initiates the translation machinery and proceeds to fold in order to make a functional protein. These properties of mRNA are very advantageous for the development of vaccines and protein replacement therapies. Since both drugs require the correct protein delivery to the interior of the cell for function. All of these in vitro transcribed synthetic mrnas are degraded in normal physiological processes, which also greatly reduces metabolic toxicity.
In recent years, many companies and research institutes have conducted research and development on mRNA, and attempts to reduce the immunogenicity of mRNA itself have been made to promote drug administration. The mRNA can be introduced more easily by optimizing its sequence. The mRNA can also be made to express the antigen of interest in cells with high efficiency using a highly effective non-toxic delivery vehicle. In order to more effectively initiate the immune response mechanism, a number of novel adjuvants have also been developed to improve the effectiveness of mRNA vaccines.
The translation efficiency and stability of mRNA are very important. The 5 'and 3' UTRs flanking the protein coding sequence directly influence the translation efficiency and stability of the mRNA and thus directly determine the effectiveness of the vaccine. This moiety can greatly improve the half-life and expression efficiency of medicinal mrnas by introducing different sequences from viruses to eukaryotes. 5' Cap structure is also an essential part for efficient expression of mRNA. The Cap enzyme can add Cap structure to mRNA by vaccinia virus during in vitro transcription. Of course, this structure can be added using an organic synthetic product or an inverted Cap analog. Poly A tails also play an important role in regulating mRNA function, so an optimized Poly A needs to be added to the mRNA by template or tailing enzyme. The use of different codons may also affect the expression of the protein. tRNA, a commonly used synonymous codon, which changes a rare codon to one that is commonly used, is also a common method of increasing the efficiency of protein translation. There are also studies that increasing the abundance of GC bases can also increase the stability of mRNA in vitro and the efficiency of protein translation in vivo.
Although the translation efficiency of the protein can be improved by changing synonymous codons of the mRNA or using some modified nucleotides, the method also has the possibility of changing the secondary structure of the mRNA, influencing the dynamics and simultaneously influencing the folding of the protein. The secondary structure of different proteins will affect the expression of different reading frames of epitope peptides expressed by effector T lymphocytes. All of these details may influence the immune response elicited by the mRNA vaccine.
The rapid development of medicinal mRNA technology also provides great challenges for the process, yield, quality, cost and the like of mRNA large-scale production, and especially plays an important role in controlling the quality of mRNA in the process of producing mRNA vaccines in a very large scale.
5' CAP is an important structure of mRNA and is an important guarantee that mRNA exists stably and that protein is expressed efficiently. At present, mRNA synthesized by an in vitro transcription method is mainly capped by a capping analogue and a capping enzyme method, and whether a final mRNA product is correctly capped or not and qualified capping efficiency is obtained becomes an important index for evaluating the mRNA product. And for different application scenes, mRNA capping types need to be distinguished, namely the CAP0 and CAP1 ratio of mRNA needs to be accurately measured. The existing method for measuring the capping efficiency is to measure the ratio of m7G to four bases of A, U, C and G after mRNA is hydrolyzed to compare the capping efficiency of the mRNA, but because the process limitation of in vitro transcription is caused, the purity of the mRNA cannot reach 100 percent, so the capping efficiency of the mRNA cannot be accurately measured based on the method. Meanwhile, for different kinds of mRNA with different sequences, the number of each base needs to be accurately known for measurement, so the method is not universal.
In addition, mRNA obtained by conventional ATP, GTP, CTP, UTP in vitro transcription techniques triggers the body's own immune response, leading to degradation of the mRNA. The modified nucleotides such as pseudouridine and 5 methylcytosine can effectively inhibit the immunogenicity of mRNA, prevent the mRNA from being degraded in vivo, prolong the expression time of the mRNA in cells and improve the expression efficiency. Therefore, the proportion of the modified nucleotide in the whole mRNA is also an important index for evaluating mRNA products, and the fully modified mRNA can have better functionality.
The mRNA transcribed in vitro inevitably comes into contact with oxygen in the air and causes the oxidation of mRNA, wherein the oxidation product is 8-oxygen guanosine, and 8-oxygen guanosine causes transcription errors and influences the correct expression of protein. The ability to accurately determine mRNA oxidation products is an important criterion for the evaluation of mRNA drugs.
To date, a great number of methods have been developed for detecting mRNA products, such as determining the length and purity of mRNA by electrophoresis, and determining the sequence correctness of mRNA and the length of Poly A tail by PCR and sequencing techniques. Methods of HPLC, GC/MS, LC/MS for capping efficiency, modified nucleotide ratios and detection of oxidation products have also been developed. For example, the method referred to in patent application WO2017149139A1, which however is only suitable for capping efficiency measurements when the purity of the mRNA is 100%. Because of the limitations of the mRNA production process, mRNA purity cannot reach 100%, so that a large amount of truncated mRNA can still be capped, which can result in mRNA capping efficiency measurements of more than 100%, and the method is not accurate in principle. It can be seen that all of the existing methods are difficult to accurately determine the capping efficiency due to the inability to accurately determine the amount of mRNA species in the sample to be tested.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for measuring the average molecular weight and Cap0/1 of mRNA, and modifying nucleotide and oxide, aiming at overcoming the defects that the prior art lacks a detection method with capping efficiency and the prior art relies on the detection of mRNA purity.
In the prior art, because mRNA purity detection is not accurate, the capping efficiency, the proportion of modified nucleotides and mRNA oxidation results measured by the method are not accurate. The method creatively utilizes capillary electrophoresis to accurately measure the purity and chain length distribution of mRNA, measures characteristic compounds of RNA such as 7-methyl-guanosine, 2-methoxy-guanosine, modified nucleotide added during in vitro transcription, 8-oxygen-guanosine and the like after the mRNA is completely hydrolyzed, does not need to measure or know the sequence of the mRNA and the content of various different bases in advance, and can accurately measure CAP0, CAP1, the modified nucleotide and the oxide ratio of the mRNA by the algorithm loaded by the method. And accurate evaluation of mRNA quality is realized.
The invention mainly solves the technical problems through the following technical scheme.
One of the technical schemes of the invention is as follows: a method of determining the average molecular weight of mRNA in a sample, comprising:
(1) Obtaining a capillary electrophoresis image of the sample;
(2) According to formula (I)
Calculating the average molecular weight of the mRNA, wherein a is the number of the bases of the shortest mRNA detected in capillary electrophoresis, b is the number of the longest bases, f (N) = the proportion of the mRNA, and N = the total number of the bases of the mRNA.
In one embodiment of the present invention, the horizontal axis of the capillary electrophoresis chart is divided into n equal parts and designated as a 1 、a 2 …a n-1 、a n Wherein a is n = b, n.gtoreq.5, preferably n.gtoreq.10; solving said formula (1) by the following formula (II):
r is the interval (a) n-1 ,a n ) Peak area ratio of inner.
Wherein the numerical value of n is as follows: if n approaches to positive infinity, the calculation result approaches to a true value infinitely, so that in the invention, if more samples are sampled, the result is relatively more accurate, but the calculation is very complicated due to the fact that the value of n is too large, and the efficiency is influenced. In the present invention, n may be any positive integer from 5 to 100, or from 5 to 50, or from 5 to 20, or from 10 to 100, or from 10 to 50, or from 5 to 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 in step (1) can be carried out according to the conventional methods in the art, and is preferably used
Small RNA reagent kit and
GX Touch TM the nucleic acid analyzer was subjected to capillary electrophoresis to obtain a capillary electrophoresis chart of the sample.
In a preferred embodiment of the invention, the sample further comprises a step of removing free nucleotides before performing capillary electrophoresis; the removal of free nucleotides is carried out, for example, by LiCl precipitation, ion exchange resin chromatography, preparative liquid chromatography or commercial kits.
The second technical scheme of the invention is as follows: a method of determining the amount of a substance of mRNA in a sample, comprising the steps of:
1) Measuring the mass concentration of mRNA in the sample, and calculating the mass of mRNA in the sample;
2) Determining the average molecular weight of mRNA in the sample according to the method of one of the claims;
3) The amount of substance of mRNA in the sample is determined from the mass of mRNA in step 1) and the average molecular weight of mRNA in the sample obtained in step 2).
In step 1), the mass concentration of mRNA in the sample can be determined by conventional methods in the art, preferably by using a ultraspectrophotometer; the ultramicro spectrophotometer is for example Thermo Scientific NanoDrop One or Mettler Toledo UV5 Nano.
In step 3), the "amount of substance" mentioned can be calculated by methods conventional in the art, i.e.: the amount of substance of mRNA = mass of mRNA/mRNA average molecular weight.
The third technical scheme of the invention is as follows: a method for determining the nucleotide content of an mRNA, the method comprising:
(a) Obtaining the amount of the substance of the mRNA by the method according to the second embodiment;
(b) Determining the amount of a substance that differs from said nucleotide;
(c) Calculating the amount of the different nucleotides in the mRNA based on the amount of the substance of the different nucleotides in step (a) and the amount of the substance of the mRNA in step (b);
the steps (a) and (b) have no sequence requirement, and can be carried out respectively, simultaneously or sequentially.
In the above step (b), the content of different nucleotides in the hydrolyzed mRNA is preferably determined by HPLC, and the amount of the substance of different nucleotides is calculated from the relative molecular mass thereof. The hydrolysis comprises the following steps:
I. mixing mRNA to be hydrolyzed with endonuclease P1, adding a buffer solution A, and incubating at 42 ℃; the incubation time is for example 2 hours; the volume ratio of the mRNA to be hydrolyzed, endonuclease P1 and buffer A is, for example, 100;
adding phosphodiesterase I and alkaline phosphatase to the incubation product obtained in the step I, adding a buffer B, and incubating at 37 ℃; the incubation time is, for example, 2 hours, and the volume ratio of phosphodiesterase I, alkaline phosphatase, and buffer B is, for example, 3.3.
The HPLC assay described in the present invention may be conventional in the art, and preferably includes:
i) Preparing a standard curve of concentration and peak area by using standard substances of specific nucleotide with different concentrations;
ii) after obtaining peak areas of different said nucleotides, calculating the concentration of said nucleotides corresponding to said standard curve.
The parameters of the above mentioned HPLC are for example:
column temperature 25 deg.C, flow rate 0.85mL/min, detection wavelength 254nm, mobile phase A5 mM ammonium acetate in water, mobile phase B40% acetonitrile in water, gradient 100% A-20% B,50min.
The kind of nucleotide to be determined may be conventional nucleosides constituting mRNA, such as adenosine, uridine, cytosine and guanosine; and may be their corresponding modified or oxidized nucleotides as well as other commonly unconventional nucleosides.
The modified nucleotide in the present invention preferably includes one or more of 7-methyl-guanosine, 2' -oxymethyl-guanosine, pseudouridine, 1-methyl-pseudouridine, 5-methyl-cytidine, 4-acetyl-cytidine and 6-methyl-adenosine.
In a preferred embodiment of the present invention:
a. the proportion of the 7-methyl guanosine is obtained by the following formula:
CAP0% = amount of 7-methylguanosine substance/amount of mRNA substance × 100%;
b. the ratio of the 2-oxymethyl-guanosine is obtained by the following formula:
CAP1% = amount of 2-oxymethyl-guanosine species/amount of mRNA species × 100% × CAP0%;
c. the proportion of the modified nucleotide is calculated by the following formula:
modified nucleotide% = amount of substance of modified nucleotide/(amount of substance of modified nucleotide + amount of substance of nucleotide before modification) × 100%;
d. the proportion of the oxidized nucleotide is calculated by the following formula:
nucleotide% after oxidation = amount of substance of nucleotide after oxidation/(amount of substance of nucleotide after oxidation + amount of substance of nucleotide before oxidation) × 100%.
Such modified nucleotides may be conventional in the art, and preferably include: one or more of 1-methyl-pseudouridine, 5-methyl-cytosine nucleoside, 4-acetyl-cytosine nucleoside, and 6-methyl-adenine nucleoside.
The oxidized nucleotide may also be conventional in the art, such as 8-oxo-guanosine.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the invention accurately measures the ratios of Cap0, cap1, modified nucleotide and oxidation product in the preparation of mRNA by a new measuring and calculating method, and provides a very accurate detection method for the quality control of the mRNA for medicine and scientific research.
Drawings
FIG. 1 is a capillary electrophoresis pattern.
FIG. 2 is a hydrolyzed mRNA profile.
FIG. 3 is a standard curve for cytosine nucleosides.
FIG. 4 is a standard curve of uridine.
FIG. 5 is a standard curve for guanosine.
FIG. 6 is a standard curve for 7-methyl-guanosine.
FIG. 7 is a standard curve for 2-oxymethyl-guanosine.
FIG. 8 is a standard curve for pseudouridine.
FIG. 9 is a standard curve for 8-oxo-guanine nucleotide.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
EXAMPLE 1 removal of free nucleotides
The measurement of the chain length distribution of mRNA can be carried out using an apparatus such as LabChip GXII Touch HT (Perkinelmer), 2100Electrophoresis Bioanalyzer Instrument (Agilent), qsep100 (Bioptic), etc., and all the measurements can give the same measurement results. The mRNA chain length distributions mentioned herein are exemplified by the results of LabChip GXII Touch HT assay. mRNA can be purified by methods such as LiCl precipitation, ion exchange resin chromatography, preparative liquid chromatography or commercial kits, and free nucleotides are completely removed to prevent interference in measurement of various indexes.
Example 2 complete hydrolysis of mRNA to mononucleosides and HPLC-based identification of each hydrolyzed fraction
1) Experimental reagents and equipment:
endonuclease P1 (Endonuclease P1), phosphodiesterase I (Phosphodiesterase I) and Alkaline Phosphatase (Shump Alkaline Phosphatase) were purchased from Sigma Aldrich.
Zinc chloride, glycerin, sodium acetate, ammonium acetate and magnesium acetate were purchased from the national pharmaceutical products group. The ultrapure water is taken from a Milli-Q water purifier.
Uridine (Uridine), cytidine (Cytidine), and Guanosine (Guanosine) were purchased from Merck; pseudouridine (Pseudouridine), 7-MethylGuanosine (7-methyl Guanosine), 8-oxo-Guanosine (8-oxo-Guanosine) and 2' -oxymethyl-Guanosine (2 ' -O-methyl Guanosine, 2' -OMe-Guanosine for short) were purchased from TCI.
High performance liquid chromatography (1260 Infinity II bioInert LC) was purchased from agilent technologies, high performance liquid chromatography Column (XBridge BEH C18 Column,
5 μm,4.6mm X250mm, 1/pk) was purchased from Waters, a bench centrifuge (ST 8) and a thermostated metal bath (88880028) was purchased from Thermo, an analytical balance (XS 205 DU) was purchased from Multiparameter, and a micropipette was purchased from RAININ.
2) The experimental method and operation are as follows:
7g/L of Phosphodiesterase I (Phosphodiesterase I) was added to the stock solution (110 mM Tris-HCl buffer, 100mM sodium chloride, 15mM magnesium chloride, 50% glycerol, pH 8.9) to prepare a Phosphodiesterase I stock solution, which was stored at-20 ℃.
Endonuclease P1 (Endonuclease P1) was prepared as a 1g/L stock solution (20 mM potassium acetate, 5mM zinc chloride, 50mM sodium chloride, and 50% glycerol) and stored at-20 ℃.
Buffer A (141.43 mM ammonium acetate, 9.43mM zinc chloride, pH 5.3).
Buffer B (115.00 mM Tris-HCl, 11.50mM magnesium acetate, pH 8.3).
mu.L of a sample of the mRNA to be tested (3.018 mg/mL) was added to 2. Mu.L of 10-fold diluted Endonuclease P1 (Endonuclease P1) and incubated at 42 ℃ for 2 hours after addition of 33. Mu.L of buffer A, 3.3. Mu.L of Phosphodiesterase I stock solution and Alkaline Phosphatase (Shump alkali Phosphatase) (0.1U/. Mu.L) supplemented with buffer B10.6. Mu.L and incubation continued at 37 ℃ for 2 hours. After mRNA degradation was complete, 17000g were centrifuged for 10min, and the supernatant (153.3. Mu.L in volume, 1.969mg/mL of mRNA degradation) was removed and assayed for future use. HPLC was adjusted to the following parameters: column temperature 25 ℃, flow rate 0.85mL/min, detection wavelength 254nm, mobile phase A5 mM ammonium acetate in water, mobile phase B40% acetonitrile in water, gradient 100% A-20% B,50min. The hydrolyzed mRNA profile was obtained as shown in FIG. 2.
Nucleotide peak areas can be obtained by aligning the retention times of different nucleotides and integrating:
pseudouridine (pseudoouridine): 4263.484, uridine (Uridine): 12.689, 7-methylguanosine (7-methyl Guanosine): 18.998,2 '-oxymethyl-Guanosine (2' -OMe-Guanosine): 31.655, 8-oxo-Guanosine (8-oxo-Guanosine): 0.
several nucleosides such as uridine, cytidine, guanosine, pseudouridine (Pseudouridine), 7-methylguanosine (7-methyl Guanosine), 8-oxo-Guanosine (8-oxo-Guanosine), and 2 '-oxymethyl-Guanosine (2' -OMe-Guanosine) were formulated into gradient concentration aqueous solution and measured by HPLC, with each sample having an intake of 10 μ L, the retention times of different nucleoside standards were recorded, and the peak areas of different nucleosides at different concentrations were integrated by normalization method, and a standard curve of nucleosides was drawn from the gradient concentrations of nucleosides.
Each nucleoside standard curve shown in FIGS. 3 to 9 was prepared according to the following tables 1 to 7.
TABLE 1
TABLE 2
TABLE 3
TABLE 4
TABLE 5
TABLE 6
TABLE 7
The concentration of different nucleosides per sample size (10 μ l) can be calculated from the standard curve and peak area of the different nucleosides as follows: pseudouridine (pseudoouridine): 8.64E-03. Mu. MoL, uridine (Uridine): 2.97E-05. Mu. MoL, 7-methyl-Guanosine (7-methyl Guanosine): 2.96E-05. Mu. MoL,2 '-oxymethyl-Guanosine (2' -OMe-Guanosine): 3.02E-05. Mu. MoL, 8-oxo-Guanosine (8-oxo-Guanosine): 0 (not detected).
3. Various indexes of mRNA are calculated by utilizing the algorithm of the invention.
1) Accurately calibrating the mass concentration of mRNA in the sample.
Firstly, the mass concentration of mRNA in a sample is accurately determined by using a Nanodrop, which can be a similar device such as Thermo Scientific NanoDrop One, mettler Toledo UV5 Nano and the like.
2) The average molecular mass of the sample mRNA is accurately determined.
Considering that the final mRNA synthesized on a large scale according to the existing in vitro transcription method is difficult to reach 100% purity, only empirical formula is used:
MW(RNA)＝(A×329.2)+(U×306.2)+(C×305.2)+(G×345.2)+159
accurate mRNA molecular weight calculations cannot be made.
The invention combines capillary electrophoresis pattern with innovative algorithm, and can accurately calibrate the mRNA sequence of the sample under the premise of not knowing the base sequence of mRNA and the number of each base, thereby calculating the contents of Cap0, cap1, modified nucleotide and oxide.
When the purity of mRNA is 100%, the molecular weight of mRNA is calculated as follows:
the mRNA molecular weight can be accurately calculated as:
MW＝(A×329.2)+(U×306.2)+(C×305.2)+(G×345.2)+159
the molecular weight of mRNA after mRNA length is greater than 1000nt can be approximated as:
MW = N × 320.5+159 (N = total base number of mRNA)
However, 100% purity is not achievable based on current mRNA preparation processes. If the purity of mRNA does not reach 100%, it is necessary to perform measurement by using capillary electrophoresis of mRNA.
Capillary electropherogram curves were defined as a function:
r = f (N), N = total number of bases of mRNA, and R = proportion of mRNA.
The average molecular weight of the sample mRNA can then be calculated from:
a is the number of the shortest base of mRNA detectable in the capillary electrophoresis chart, and b is the number of the longest base detectable in the capillary electrophoresis chart.
The function f (N) is solved by solving a function expression of any curve in a plane coordinate system, and the function can be solved by using a polynomial fitting equation of higher mathematics or by using Python and MatLab.
This equation can be solved by wolframalpha (https:// www. Wolframalpha. Com /), or by the approximation algorithm provided by the present invention:
the abscissa of the capillary electrophoresis chart is divided into n (n ≧ 10, n =11 in this example) halves, i.e., a 1 ,a 2 …a n-1 ,a n Wherein a is n = b (b =2946 in this embodiment). Wherein R is the interval (a) n-1 ,a n ) Peak area ratio of inner.
The mRNA average molecular weight can be calculated approximately as:
TABLE 8
As shown in Table 8 and FIG. 1, the distribution ratios of mRNAs in different length intervals were obtained by dividing the capillary electropherogram of the mRNA to be measured. The average molecular weight of the mRNA and the number of moles of the mRNA per sample amount (10. Mu.l) were obtained by substituting the above formula, and the results are shown in Table 9 below.
TABLE 9
3) CAP0 and CAP1 ratio calculation
The CAP structure of mRNA is shown below:
where 7-methyl-Guanosine (7-methyl Guanosine) is a marker of the CAP0 structure, there is one and only one 7-methyl-Guanosine (7-methyl Guanosine) per intact mRNA:
therefore, by accurately measuring the content of 7-methyl-Guanosine (7-methyl Guanosine), the CAP0 ratio of the mRNA of the sample can be calculated as:
CAP0% = amount of 7-methyl Guanosine (7-methyl Guanosine) substance/amount of mRNA substance × 100%.
According to the experimental result, CAP0% =2.96E-05 μmoL/3.32E-05 μmoL =89%.
The complex structure of 7-methyl-Guanosine (7-methyl Guanosine) and 2-oxymethyl-Guanosine (2-OMe-Guanosine) linked by triphosphate bonds is a marker of CAP1 structure of mRNA, and each complete mRNA has only one complex structure of 7-methyl-Guanosine (7-methyl Guanosine) and 2 '-oxymethyl-Guanosine (2' -OMe-Guanosine). Therefore, the ratio of CAP1 can be obtained by measuring the amount of substance of the marker 2 '-oxymethyl-Guanosine (2' -OMe-Guanosine):
CAP1% = amount of 2 '-oxymethyl-Guanosine (2' -OMe-Guanosine) substance/amount of mRNA substance × 100% × CAP0%.
According to the experimental result, CAP1% =3.02E-05 μmoL/3.32E-05 μmoL × 89% =81%.
4) Modified nucleotide ratio assay
In order to improve the expression efficiency of medicinal mRNA and prolong the expression time, modified nucleotides such as Pseudouridine (Pseudouridine), 1-Methyl-Pseudouridine (1-Methyl-Pseudouridine), 5-Methyl-cytosine nucleoside (5-Methyl-cytosine), 4-Acetyl-cytosine nucleoside (4-Acetyl-cytosine) and 6-Methyl-adenine nucleoside (6-Methyl-adenine) are often required to be added in the in vitro transcription process.
The ratio of the corresponding modified nucleotides in the mRNA sample can be calculated by the following formula.
Pseudouridine (Pseudouridine)% = amount of Pseudouridine (Pseudouridine) substance/(amount of Uridine (Uridine) substance + amount of Pseudouridine (Pseudouridine) substance) × 100%.
This experiment can accurately determine: pseudouridine (pseudoouridine)% =8.64 nmoL/(8.64nmol +2.97E-02 nmoL) × 100% =99.7%.
The amount of 1-Methyl-Pseudouridine (1-Methyl-Pseudouridine)% = 1-Methyl-Pseudouridine (1-Methyl-Pseudouridine) substance/(the amount of 1-Methyl-Pseudouridine (1-Methyl-Pseudouridine) substance + the amount of Uridine (Uridine) substance) × 100%.
5-Methyl-cytosine nucleoside (5-Methyl-Cytidine)% = amount of 5-Methyl-cytosine nucleoside (5-Methyl-Cytidine) substance/(amount of 5-Methyl-cytosine nucleoside (5-Methyl-Cytidine) substance + amount of cytosine nucleoside (Cytidine) substance) × 100%.
4-acetylcytosine nucleoside (4-Acetyl-Cytidine)% = amount of 4-Acetyl-Cytidine substance/(amount of 4-Acetyl-Cytidine substance + amount of Cytidine substance) × 100%.
6-Methyl-adenosine (6-Methyl-adenosines)% = the amount of 6-Methyl-adenosine (6-Methyl-adenosines) substance/(the amount of 6-Methyl-adenosine (6-Methyl-adenosines) substance + the amount of adenosine (adenosines) substance) × 100%.
The ratio of any other modified nucleotide can be accurately calculated by the same method.
5) Determination of oxidation product ratio
The oxidation of mRNA inevitably occurs in long-term contact with air. The oxidation reaction is mainly shown in the conversion of Guanosine (Guanosine) into 8-oxo-Guanosine (8-oxo-Guanosine).
The content of the oxidation product of mRNA can be directly obtained by accurately measuring the concentration of 8-oxo-Guanosine (8-oxo-Guanosine).
Oxidation% = amount of 8-oxo-Guanosine (8-oxo-Guanosine) substance/(amount of 8-oxo-Guanosine (8-oxo-Guanosine) substance + amount of Guanosine (Guanosine) substance) × 100%.
mRNA oxidation products were not detected in this experiment.
Claims (10)
1. A method for determining the average molecular weight of mRNA in a sample, comprising:
(1) Obtaining a capillary electrophoresis image of the sample;
(2) According to formula (I)
∫ b a (N*320.5+159)*f(N)d(N)
Calculating the average molecular weight of the mRNA, wherein a is the number of the bases of the shortest mRNA detected in capillary electrophoresis, b is the number of the longest bases, f (N) = the proportion of the mRNA, and N = the total number of the bases of the mRNA.
2. The method of claim 1, wherein the capillary electropherogram is divided on the abscissa into n equal parts and designated as a 1 、a 2 …a n-1 、a n Wherein a is n = b, n.gtoreq.5, preferably n.gtoreq.10; solving said formula (I) by the following formula (II):
r is an interval (a) n-1 ,a n ) Peak area ratio of inner.
3. The method of claim 1 or 2, wherein in step (1), the method of the present invention is used
Small RNA reagent kit and
GX Touch TM performing capillary electrophoresis on a nucleic acid analyzer to obtain a capillary electrophoresis image of the sample; preferably, the sample further comprises a step of removing free nucleotides before performing capillary electrophoresis; the removal of free nucleotides is carried out, for example, by LiCl precipitationIon exchange resin chromatography, preparative liquid chromatography or commercial kits.
4. A method for determining the amount of a substance of mRNA in a sample, comprising the steps of:
1) Measuring the mass concentration of mRNA in the sample, and calculating the mass of mRNA in the sample;
2) Determining the average molecular weight of mRNA in a sample according to the method of any one of claims 1-3;
3) The amount of substance of mRNA in the sample is determined from the mass of mRNA in step 1) and the average molecular weight of mRNA in the sample obtained in step 2).
5. The method according to claim 4, wherein the mass concentration of mRNA in the sample is measured in step 1) using a ultraspectrophotometer; such as Thermo Scientific NanoDrop One or Mettler Toledo UV5 Nano.
6. A method for determining the nucleotide content of mRNA, comprising:
(a) Obtaining the amount of the substance of the mRNA by the method of claim 4 or 5;
(b) Determining the amount of a substance that differs from said nucleotide;
(c) Calculating the amount of the different nucleotides in the mRNA based on the amount of the substance of the different nucleotides in step (a) and the amount of the substance of the mRNA in step (b);
the steps (a) and (b) have no sequence requirement, and can be carried out respectively, simultaneously or sequentially.
7. The assay method according to claim 6, wherein in the step (b), the content of different nucleotides in the hydrolyzed mRNA is measured by HPLC, and the amount of the substance having different nucleotides is calculated from the relative molecular mass thereof;
preferably, the hydrolysis comprises the steps of:
I. mixing mRNA to be hydrolyzed with endonuclease P1, adding a buffer solution A, and incubating at 42 ℃; the incubation time is for example 2 hours; the volume ratio of the mRNA to be hydrolyzed, endonuclease P1 and buffer A is, for example, 100;
adding phosphodiesterase I and alkaline phosphatase to the incubation product obtained in the step I, adding a buffer B, and incubating at 37 ℃; the incubation time is, for example, 2 hours, and the volume ratio of phosphodiesterase I, alkaline phosphatase, and buffer B is, for example, 3.3;
and/or, the HPLC assay in step (b) comprises:
i) Preparing a standard curve of concentration and peak area by using standard substances of specific nucleotide with different concentrations;
ii) after obtaining peak areas of different said nucleotides, calculating the concentration of said nucleotides corresponding to said standard curve;
wherein, the parameters of the HPLC are as follows:
column temperature 25 deg.C, flow rate 0.85mL/min, detection wavelength 254nm, mobile phase A5 mM ammonium acetate in water, mobile phase B40% acetonitrile in water, gradient 100% A-20% B,50min.
8. The assay of claim 6 or 7, wherein the nucleotides comprise one or more of uridine, cytidine, guanosine, and their corresponding modified nucleotides and oxidized nucleotides; preferably, the modified nucleotides include: one or more of 7-methyl-guanosine, 2' -oxymethyl-guanosine, pseudouridine, 1-methyl-pseudouridine, 5-methyl-cytidine, 4-acetyl-cytidine, and 6-methyl-adenosine.
9. The assay method according to claim 8,
a. the proportion of the 7-methyl-guanosine is obtained by the following formula:
CAP0% = amount of 7-methyl-guanosine substance/amount of mRNA substance × 100%;
b. the ratio of the 2' -oxymethyl-guanosine is obtained by the following formula:
CAP1% = amount of 2' -oxymethyl-guanosine species/amount of mRNA species × 100% × CAP0%;
c. the proportion of the modified nucleotide is calculated by the following formula:
modified nucleotide% = amount of substance of modified nucleotide/(amount of substance of modified nucleotide + amount of substance of nucleotide before modification) × 100%;
d. the proportion of the oxidized nucleotide is calculated by the following formula:
nucleotide% after oxidation = amount of substance of nucleotide after oxidation/(amount of substance of nucleotide after oxidation + amount of substance of nucleotide before oxidation) × 100%.
10. The assay of claim 8 or 9, wherein the oxidized nucleotides comprise 8-oxo-guanosine.
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| CN117778523B (en) * | 2024-02-26 | 2024-05-28 | 苏州近岸蛋白质科技股份有限公司 | Poly (A) polymerase activity determination method |
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| CN105911127A (en) * | 2016-04-14 | 2016-08-31 | 浙江工商大学 | Rapid detection method of fish parvalbumin by capillary electrophoresis |
| CN107389774A (en) * | 2017-07-19 | 2017-11-24 | 山西晋城无烟煤矿业集团有限责任公司 | A kind of capillary electrophoresis detection method of metal cation in liquid |
| CN110954587B (en) * | 2019-12-11 | 2020-11-20 | 中国科学院化学研究所 | High-reproducibility capillary electrophoresis method based on migration charge density |
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| CN117778523B (en) * | 2024-02-26 | 2024-05-28 | 苏州近岸蛋白质科技股份有限公司 | Poly (A) polymerase activity determination method |
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