Quality detection method for mRNA capping rate and PolyA distribution

Abstract

The invention provides a quality detection method for mRNA capping rate and PolyA distribution. The quality detection method comprises the following steps: a) mixing mRNA to be detected, a capping rate analysis probe and a PolyA distribution analysis probe to obtain an mRNA-probe structure; b) carrying out enzyme digestion on the mRNA-probe structure by using thermal stable RNase H enzyme to obtain a 5'enzyme digestion fragment and a 3 'enzyme digestion fragment; and c) detecting the 5 '-enzyme digestion fragment and the 3'-enzyme digestion fragment by using liquid chromatography-mass spectrometry, obtaining the capping rate from the 5 '-enzyme digestion fragment, obtaining the PolyA distribution condition from the 3'-enzyme digestion fragment, and realizing simultaneous detection of the 5 '-capping rate and the 3' Poly A. The method can solve the problems of many steps and long time required by detection of the capping rate and the PolyA distribution of mRNA in the prior art, and is suitable for the field of mRNA quality detection.

Description

Technical Field

[0001] The present invention relates to the field of mRNA quality detection, and in particular to a quality detection method for mRNA capping rate and Poly A distribution. Background Art

[0002] Testing mRNA capping rate and Poly A distribution is a common test used in mRNA quality assessment. Since intact mRNA molecules often consist of more than 1000 nucleotides, mass spectrometry cannot directly analyze mRNA molecules of this size. Existing capping rate tests typically use ribonuclease H (RNase H) during sample preparation. RNase H selectively recognizes and cleaves the phosphodiester bonds of RNA within RNA:DNA hybrids, yielding smaller, well-defined 5'-end fragments. Conventional testing of Poly A distribution, on the other hand, uses ribonuclease T1 (RNase T1) during sample preparation. RNase T1 specifically cleaves single-stranded RNA at G residues. It cleaves the phosphodiester bond between the 3'-guanosine residue and the 5'-OH residue of adjacent nucleotides by forming the corresponding intermediate 2',3'-cyclic phosphate.

[0003] Existing LC-MS-based detection of capping rate and Poly A distribution has no correlation in sample preparation, and the corresponding detection is completed through two independent experiments. Summary of the Invention

[0004] The main purpose of the present invention is to provide a method for quality detection of mRNA capping rate and Poly A distribution, so as to solve the problem that the detection of mRNA capping rate and Poly A distribution in the prior art requires many steps and takes a long time.

[0005] To achieve the above-mentioned object, according to a first aspect of the present invention, a method for detecting the quality of mRNA is provided, which comprises: a) mixing an mRNA to be detected, a capping rate analysis probe, and a Poly A distribution analysis probe to obtain an mRNA-probe structure; b) using a thermostable RNase H enzyme to enzymatically digest the mRNA-probe structure to obtain a 5'-enzyme-digested fragment and a 3'-enzyme-digested fragment; c) using liquid chromatography-mass spectrometry to detect the 5'-enzyme-digested fragment and the 3'-enzyme-digested fragment, obtaining the capping rate from the 5'-enzyme-digested fragment, and obtaining the Poly A distribution from the 3'-enzyme-digested fragment, thereby realizing simultaneous detection of the mRNA 5'-capping rate and 3'-Poly A; wherein the capping rate analysis probe can specifically bind to the 5'-end sequence of the mRNA to be detected, and the Poly A distribution analysis probe can specifically bind to the 5'-direction sequence of the 3'-Poly A structure of the mRNA to be detected.

[0006] Furthermore, the molar ratio of the capping rate analysis probe to the mRNA to be detected is 1-10:1, and the molar ratio of the Poly A distribution analysis probe to the mRNA to be detected is 1-10:1.

[0007] Furthermore, the quality detection method includes: mixing 50-200 pmol of the mRNA to be detected, 0.2-1 nmol of the capping rate analysis probe, 0.2-1 nmol of the PolyA distribution analysis probe and 2-10 μL of the thermostable RNaseH enzyme buffer to obtain an mRNA-probe structure; mixing the mRNA-probe structure with 1-10 μL of the thermostable RNaseH enzyme, incubating at 40-60° C. for 25-35 minutes to obtain 5' enzyme-cut fragments and 3' enzyme-cut fragments.

[0008] Furthermore, the quality detection method includes: mixing 100 pmol of the mRNA to be detected, 0.5 nmol of the capping rate analysis probe, 0.5 nmol of the PolyA distribution analysis probe and 6 μL of the thermostable RNase H enzyme buffer to obtain an mRNA-probe structure; mixing the mRNA-probe structure with 5 μL of the thermostable RNase H enzyme, incubating at 50° C. for 25-35 minutes, and obtaining 5' enzyme-cut fragments and 3' enzyme-cut fragments.

[0009] Furthermore, the chromatographic conditions in the liquid chromatography-mass spectrometry were as follows: chromatographic column: C18 chromatographic column; mobile phase: mobile phase A, mobile phase B; flow rate: 0.2-0.4 mL / min; column temperature 40-80° C.; detection wavelength 260 nm; injection volume: 10 μL; mobile phase A was an aqueous solution containing 1 wt% HFmIP, 0.1 wt% DIEA, and 1 μM EDTA; mobile phase B was a 35% (v / v) acetonitrile-water solution containing 0.075 wt% HFmIP, 0.0375 wt% DIEA, and 1 μM EDTA; gradient elution, and the elution conditions are shown in Table 1 below:

[0010] Table 1

[0011] Time (min) Mobile phase A (%) Mobile phase B (%) 0.00 95.0 5.0 15.00 50.0 50.0 16.50 25.0 75.0 17.50 25.0 75.0 18.00 95.0 5.0 20.00 95.0 5.0 .

[0012] Furthermore, the mass spectrometry conditions in the liquid chromatography-mass spectrometry were: full scan, negative ion mode, and a mass range of 400Da-5000Da.

[0013] Furthermore, the length of the capping rate analysis probe is 10-20 bp.

[0014] Furthermore, the length of the PolyA distribution analysis probe is 10-20 bp.

[0015] Furthermore, the capping rate analysis probe includes ribonucleotides and deoxyribonucleotides; preferably, the 5' end and the 3' end of the capping rate analysis probe are both fragments consisting of ribonucleotides, and the middle part is a fragment consisting of deoxyribonucleotides.

[0016] Furthermore, the PolyA distribution analysis probe includes ribonucleotides and deoxyribonucleotides; preferably, the 5' end and the 3' end of the PolyA distribution analysis probe are both fragments composed of ribonucleotides, and the middle part is a fragment composed of deoxyribonucleotides.

[0017] The technical solution of the present invention is applied, and the above-mentioned mRNA quality detection method is used to simultaneously perform enzyme digestion on the capping structure (i.e., the 5' end sequence) and the PolyA structure (i.e., the 3' end sequence) in the mRNA to be detected using a thermostable RNaseH enzyme to obtain the expected 5' digestion fragment and the 3' digestion fragment; further, the 5' digestion fragment and the 3' digestion fragment are detected using liquid chromatography-mass spectrometry (LC-MS) to realize the analysis of the capping structure and the PolyA structure in the mRNA fragment. In the above-mentioned quality detection method, the capping rate and PolyA distribution of mRNA can be simultaneously analyzed in one reaction system, reducing the required experimental steps and time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute part of this application, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0019] Figure 1 The LC-MS result diagram of the one-step combined enzyme digestion experiment in Example 1 of the present invention is shown.

[0020] Figure 2 The figure shows the LC-MS result of detecting the capping rate using RNase H alone according to Example 2 of the present invention.

[0021] Figure 3 The figure shows the LC-MS result of detecting the distribution of Poly A using RNase T1 alone according to Example 2 of the present invention.

[0022] Figure 4 The LC-MS result diagram of the one-step combined enzyme digestion experiment in Example 3 of the present invention is shown.

[0023] Figure 5 The figure shows the LC-MS result of detecting the capping rate using RNase H alone according to Example 4 of the present invention.

[0024] Figure 6The figure shows the LC-MS result of detecting the distribution of Poly A using RNase T1 alone according to Example 4 of the present invention. DETAILED DESCRIPTION

[0025] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0026] As mentioned in the background, the quality attribute detection process of mRNA molecules often requires simultaneous monitoring of capping rate and Poly A distribution. When the two experiments are performed separately, the sample preparation of the two methods is unrelated, which is time-consuming and complicated.

[0027] In this application, the inventors have developed a novel quality detection method for mRNA capping rate and Poly A distribution, completing the detection of capping rate and Poly A distribution in a single pre-treatment and detection step, also known as a "one-step combined enzyme digestion assay." Based on this, a series of protection schemes for this application were proposed.

[0028] In a first typical embodiment of the present application, a method for quality detection of mRNA capping rate and Poly A distribution is provided, which quality detection method comprises: a) mixing the mRNA to be detected, a capping rate analysis probe and a Poly A distribution analysis probe to obtain an mRNA-probe structure; b) using thermostable RNaseH enzyme (Thermostable RNaseH) to enzymatically digest the mRNA-probe structure to obtain a 5' digested fragment and a 3' digested fragment; c) using liquid chromatography-mass spectrometry to detect the 5' digested fragment (i.e., the 5' end sequence) and the 3' digested fragment (i.e., the 3' end sequence), and simultaneously obtaining the capping rate and Poly A distribution, thereby realizing quality detection of the mRNA; wherein, the capping rate analysis probe can specifically bind to the 5' end sequence of the mRNA to be detected, and the Poly A distribution analysis probe can specifically bind to the sequence in the 5' direction of the 3' Poly A structure of the mRNA to be detected.

[0029] In the existing technology, capping rate analysis is based on the enrichment of probes with enrichment tags after RNaseH digestion, chromatographic separation and mass spectrometry identification; the PolyA analysis process generally uses enzyme digestion methods such as RNase T1 to hydrolyze the mRNA, and the remaining PolyA part is then enriched, chromatographically separated, and identified by mass spectrometry. However, this method does not have the ability of site-specific enzymatic digestion. For mRNA carrying multiple PolyA segments, the fragments will be cut into multiple parts, resulting in the loss of the integrity of the mRNA PolyA distribution.

[0030] The present invention innovates and optimizes the existing technology. The sample preparation of the original two tests is digested with Thermostable RNase H. Thermostable RNase H is used to digest the specific site where the mRNA binds to the probe. The 5' end sequence in the mRNA is captured by the capping rate analysis probe, and the 3' poly (A) structure in the mRNA is captured by the Poly A distribution analysis probe.

[0031] The quality detection method of the present application is to realize the analysis of capping rate and PolyA distribution simultaneously in an experiment for the first time, while reducing the complexity and operation time in the analytical process, significantly improving flux, avoiding the process of enrichment in the prior art. In the present application, capping rate analysis probe and PolyA distribution analysis probe can be combined with the sequence of 5' end sequence and 3'PolyA 5' direction respectively positioned at mRNA to be detected, thereby forming the first and second cleavage sites, and then utilizing thermostable RNaseH enzyme to carry out enzyme digestion to the two cleavage sites, realizing the cutting of 5' end sequence and PolyA sequence. Preferably, the first cleavage site is generally located at the position of 5' end 5-40nt, and the second cleavage site is located at the position of 5' upstream 1-20nt of the first A in the PolyA structure, and those skilled in the art can, according to the sequence of mRNA to be detected, flexibly design the sequence of capping rate analysis probe and PolyA distribution analysis probe. Furthermore, the inventors improved the LC-MS detection sensitivity for target molecules by optimizing the mobile phase and gradient, achieving the goal of completing both capping rate and Poly A distribution analysis in a single experiment, with lower costs and higher efficiency, shortening the mRNA analysis and detection process and time, and achieving the simultaneous completion of sample preparation and on-machine analysis of mRNA capping rate and Poly A distribution within one hour.

[0032] Ribonuclease H (RNase H) was first discovered in calf thymus tissue, and its encoding gene has been cloned into Escherichia coli. It specifically degrades the RNA strand in a DNA:RNA hybrid duplex, producing oligonucleotides and mononucleotides with 3'-OH and 5'-phosphate termini; it cannot degrade single-stranded or double-stranded DNA or RNA.

[0033] The thermostable RNaseH enzyme used in this application is a commonly used tool enzyme that can specifically recognize and cut the phosphodiester bond of the RNA sequence in the RNA:DNA hybrid while keeping the DNA sequence intact. It still has high enzymatic activity at temperatures above 65°C, with a half-life of up to several hours at 70°C and up to about 30 minutes at 95°C. The high temperature resistance of the enzyme makes the heteroduplex RNA:DNA hybrid molecules have higher specificity and more specifically cut the RNA in the hybrid duplex at higher temperatures. The thermostable RNaseH has similar enzymatic properties to common RNaseH, but RNase H is inactivated at above 55°C.

[0034] Preferably, the thermostable RNaseH enzyme is a protein having the amino acid sequence shown in SEQ ID NO: 1, or a protein having a homology of 70% or more with SEQ ID NO: 1 and having the thermostable and high temperature resistance capabilities.

[0035] SEQ ID NO: 1:

[0036] MNPSPRKRVALFTDGACLGNPGPGGWAALLRFHAHEKLLSGGEACTTNNRMELKAAI EGLKALKEPCEVDLYTDSHYLKKAFTEGWLEGWRKRGWRTAEGKPVKNRDLWEALLLAM APHRVRFHFVKGHTGHPENERVDREARRQAQSQAKTPCPPRAPPTLFHEEA.

[0037] In a preferred embodiment, the molar ratio of the capping rate analysis probe to the mRNA to be detected is 1-10:1 (including but not limited to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1), and the molar ratio of the Poly A distribution analysis probe to the mRNA to be detected is 1-10:1 (including but not limited to 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1).

[0038] In a preferred embodiment, the quality detection method comprises: 50-200 pmol (including but not limited to 50, 75, 100, 125, 150, 175, 200 pmol) of the mRNA to be detected, 0.2-1 nmol (including but not limited to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 nmol) of the capping rate analysis probe, 0.2-1 nmol (including but not limited to 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1 nmol) of the Poly A distribution analysis probe and 2-10 μL (including but not limited to 2, 3 The mRNA-probe structure is mixed with 1-10 μL (including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μL) of thermostable RNase H enzyme buffer to obtain an mRNA-probe structure; the mRNA-probe structure is mixed with 1-10 μL (including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μL) of thermostable RNase H enzyme, and incubated at 40-60° C. (including but not limited to 40, 42, 45, 47, 50, 52, 55, 57 or 60° C.) for 25-35 min (including but not limited to 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 min) to obtain a 5' digestion fragment and a 3' digestion fragment.

[0039] In a preferred embodiment, the quality detection method includes: mixing 100 pmol of the mRNA to be detected, 0.5 nmol of the capping rate analysis probe, 0.5 nmol of the PolyA distribution analysis probe, and 6 μL of the thermostable RNase H enzyme buffer to obtain an mRNA-probe structure; mixing the mRNA-probe structure with 5 μL of the thermostable RNase H enzyme, incubating at 50° C. for 25-35 minutes, and obtaining the expected 5' enzyme-cut fragment (i.e., 5' end sequence) and 3' enzyme-cut fragment (i.e., 3' end sequence).

[0040] In a preferred embodiment, the chromatographic conditions in the liquid chromatography-mass spectrometry are as follows: chromatographic column: BEH C18 chromatographic column; mobile phase: mobile phase A-mobile phase B; flow rate: 0.2-0.4 mL / min (including but not limited to 0.3 mL / min); column temperature 40-80° C. (including but not limited to 75° C.); detection wavelength 260 nm; injection volume: 10 μL; mobile phase A is an aqueous solution containing 1 wt% HFmIP (hexafluoromethyl isopropanol), 0.1 wt% DIEA (N,N-diisopropylethylamine) and 1 μM EDTA; mobile phase B is a 35% (v / v) acetonitrile-water solution containing 0.075 wt% HFmIP, 0.0375 wt% DIEA and 1 μM EDTA; gradient elution, the elution conditions are shown in Table 1:

[0041] The above-mentioned chromatographic conditions can be used to separate mRNA fragments obtained by enzyme digestion with Thermostable RNase H, and the fragments to be detected can be separated to achieve subsequent mass spectrometry detection.

[0042] In a preferred embodiment, the mass spectrometry conditions in liquid chromatography-mass spectrometry are: full scan, negative ion mode, and a mass range of 400Da-5000Da.

[0043] In a preferred embodiment, the mass spectrometry conditions in liquid chromatography-mass spectrometry are shown in Table 2 below:

[0044] Table 2

[0045]

[0046]

[0047] In a preferred embodiment, the length of the capping rate analysis probe is 10-20 bp, including but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bp.

[0048] In a preferred embodiment, the length of the Poly A distribution analysis probe is 10-20 bp, including but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bp.

[0049] In a preferred embodiment, the capping rate analysis probe includes ribonucleotides and deoxyribonucleotides; preferably, the 5' end and the 3' end of the capping rate analysis probe are both fragments consisting of ribonucleotides, and the middle part is a fragment consisting of deoxyribonucleotides.

[0050] In a preferred embodiment, the PolyA distribution analysis probe includes ribonucleotides and deoxyribonucleotides; preferably, the 5' end and the 3' end of the PolyA distribution analysis probe are both fragments composed of ribonucleotides, and the middle part is a fragment composed of deoxyribonucleotides.

[0051] The capping rate analysis probe and Poly A distribution analysis probe used in the above-mentioned method of the present application are each divided into three parts. The first part starting from the 5' portion is 2-8 nt ribonucleotides, the second part is 2-4 nt deoxyribonucleotides, and the third part is 2-8 nt ribonucleotides. The first, second, and third parts are sequentially connected along the 5'-3' direction via phosphodiester bonds to form a single-stranded probe. The total number of nucleotides in the probe is within 20, and the short length can reduce the cost of synthesizing the probe.

[0052] The two probes are mixed with the mRNA to be detected and annealed to form an mRNA-probe structure containing a DNA-RNA hybrid structure. The DNA-RNA hybrid structure in the mRNA-probe structure is further cut using a heat-stable RNase H enzyme, thereby severing the phosphodiester bond located at the cleavage site and derived from the mRNA to be detected. After the above-mentioned cutting, the mRNA-probe structure in the cutting system is optionally denatured and unwound before liquid chromatography-mass spectrometry analysis, or liquid chromatography-mass spectrometry analysis is directly performed and denaturation and unwound are achieved using liquid phase conditions, both of which can achieve simultaneous detection of mRNA 5' capping rate and 3' Poly A.

[0053] The beneficial effects of the present application will be further explained in detail below with reference to specific embodiments.

[0054] Example 1 Firefly luciferase mRNA capping rate and Poly A distribution one-step combined enzyme digestion experiment

[0055] The firefly luciferase mRNA (PolyA designed length 80A) was detected by one-step combined enzyme digestion. The analysis results are as follows Figure 1 shown.

[0056] The sequence of the firefly luciferase mRNA containing PolyA (SEQ ID NO: 2) is:

[0057]

[0058] Sample preparation process:

[0059] Pipette approximately 100 pmol of sample (sample volume ≤ 50 μL; if less than 50 μL, add nuclease-free water to 50 μL) into a centrifuge tube. Add 6 μL of RNase H Reaction Buffer. Use the mixed sample to dissolve 0.5 nmol of the capping rate analysis probe and 0.5 nmol of the Poly A distribution analysis probe. Then, add 5 μL of Thermostable RNase H enzyme (Thermo, EN0202) to the sample, mix thoroughly, and incubate in a metal bath at 50°C, 350 rpm for 30 ± 5 minutes before testing.

[0060] The sequence of the capping rate analysis probe is SEQ ID NO: 3, and the sequence of the Poly A distribution analysis probe is SEQ ID NO: 4.

[0061] SEQ ID NO: 3: mUmGmGmGmGdAdCdCdAmGmAmAmGmAmA.

[0062] SEQ ID NO: 4: mCmUmAmGmCdTdCdCdAmGmGmGmUmGmU.

[0063] The "m" before A, C, G or U indicates a 2'-methoxy modification of the ribonucleotide; and the "d" before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide.

[0064] The design principles of capping rate analysis probes and Poly A distribution analysis probes are well known to those skilled in the art, and those skilled in the art can flexibly adjust the sequences of the two probes according to the target mRNA.

[0065] Sample analysis method:

[0066] Mobile phase

[0067] Mobile phase A: 1% HFmIP, 0.1% DIEA, 1 μM EDTA in H2O.

[0068] Mobile phase B: 0.075% HFmIP, 0.0375% DIEA, 1 μM EDTA in 35% ACN. The UPLC analysis method is shown in Tables 3 and 4.

[0069] Table 3 UPLC analysis parameters

[0070] Flow / flow rate (mL / min) 0.3 Injection Volume / Injection Volume (μL) 10 Column Temperature (℃) 75 Sample Room Temperature / Sample Room Temperature (℃) 10 Detection Wavelength (nm) 260 Data collection rate (Hz) 20 Run Time / Run Time (min) 20

[0071] Table 4 UPLC analysis elution program

[0072]

[0073] The MS analysis method is shown in Table 5.

[0074] Table 5 MS analysis method

[0075]

[0076]

[0077] Example 2

[0078] 1. The capping rate of firefly luciferase mRNA (SEQ ID NO: 5) was detected using existing technology. The results are as follows: Figure 2 shown.

[0079] The experimental steps are as follows:

[0080] 1) Dissolve 1 nmol of capping probe (SEQ ID NO: 3) in 6 μL of RNaseH Buffer and 6 μL of ultrapure water. Then, mix 6 μL of the dissolved probe and 4 μL of RNaseH Buffer with 50 μL of mRNA sample (same as in Example 1, approximately 100 pmol) (total volume 60 μL).

[0081] 2) Add 5 μL of Thermostable RNase H to the sample;

[0082] 3) After vortex mixing, the sample was placed in a thermomixer (50°C, 350 rpm) and incubated for 30 ± 5 min before testing.

[0083] 2. The distribution of PolyA of firefly luciferase mRNA (SEQ ID NO: 2) was detected using existing technology. The results are as follows: Figure 3 shown.

[0084] The experimental steps are as follows:

[0085] 1) Add 5 μL of RNase T1 to 50 μL of mRNA sample (the same sample used in Example 1, about 100 pmol).

[0086] (Total system 55μL)

[0087] 2) After vortex mixing, the sample was placed in a thermomixer (37°C, 350 rpm) and incubated for 30 ± 5 min before testing.

[0088] The above samples were detected using the same chromatographic method and mass spectrometry method as in Example 1.

[0089] Example 3: Detection of the capping rate and poly A distribution of the spike protein mRNA of the new coronavirus Delta strain using a one-step combined enzyme digestion experiment

[0090] 1. The Delta strain spike protein mRNA was detected using the method of Example 1 (one-step combined enzyme digestion experiment). The analysis results are as follows: Figure 4 shown.

[0091] The sequence of the Delta strain spike protein mRNA containing PolyA (SEQ ID NO: 5) is:

[0092]

[0093] The sequence of the capping rate analysis probe used is SEQ ID NO: 6, and the sequence of the Poly A distribution analysis probe used is SEQ ID NO: 7.

[0094] SEQ ID NO: 6: mUmUmCmUmUmAdCdTdCdTmUmCmUmUmUmUmC.

[0095] SEQ ID NO: 7: mCmAmCmUmCdAdGdAdCmUmUmUmAmU.

[0096] The "m" before A, C, G or U indicates a 2'-methoxy modification of the ribonucleotide; and the "d" before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide.

[0097] Example 4

[0098] 1. Use the optimized RNase H enzyme in the existing technology to digest the same Delta strain spike protein mRNA and detect the capping rate. The results are as follows: Figure 5 As shown (the experimental steps are the same as those in Example 2, step 1).

[0099] 2. The same Delta strain spike protein mRNA was digested with the optimized RNase T1 enzyme in the existing technology to detect the distribution of PolyA. The results are as follows: Figure 6 As shown (the experimental steps are the same as those in Example 2).

[0100] As can be seen from the experimental results of different mRNA molecules in Examples 1-4, the results of the one-step combined enzymatic digestion assay for capping efficiency and Poly A distribution are comparable to those of the capping efficiency and Poly A distribution assays performed separately, and are reproducible and have similar accuracy across different molecules. This demonstrates that the mRNA quality detection method described in this application is reliable and can meet the needs of routine testing.

[0101] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: applying the technical solution of the present invention, utilizing the above-mentioned mRNA quality detection method, and utilizing the thermostable RNaseH enzyme to simultaneously perform enzymatic digestion on the capping structure and PolyA structure in the mRNA to be detected, thereby obtaining the expected 5' digestion fragment (i.e., 5' end sequence) and 3' digestion fragment (i.e., 3' end sequence); further utilizing liquid chromatography-mass spectrometry (LC-MS) to detect the capping structure and PolyA structure in the expected 5' digestion fragment (i.e., 5' end sequence) and 3' digestion fragment (i.e., 3' end sequence), thereby realizing the simultaneous analysis of the capping rate and PolyA distribution of mRNA in one reaction system, thereby reducing the required experimental steps and time.

[0102] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for detecting the quality of mRNA capping rate and PolyA distribution, characterized in that: The quality detection method comprises: a) mixing the mRNA to be detected, the capping rate analysis probe and the PolyA distribution analysis probe to obtain an mRNA-probe structure; b) digesting the mRNA-probe structure with a thermostable RNase H enzyme to obtain a 5' digestion fragment and a 3' digestion fragment; c) detecting the 5' digested fragment and the 3' digested fragment by liquid chromatography-mass spectrometry, obtaining the capping rate from the 5' digested fragment, and obtaining the distribution of Poly A from the 3' digested fragment, so as to achieve simultaneous detection of the mRNA 5' capping rate and 3' Poly A; The capping rate analysis probe can specifically bind to the 5' end sequence of the mRNA to be detected, and the PolyA distribution analysis probe can specifically bind to the 5' direction sequence of the 3'-PolyA structure of the mRNA to be detected.

2. The quality inspection method according to claim 1, characterized in that: The molar ratio of the capping rate analysis probe to the mRNA to be detected is 1-10:1, and the molar ratio of the PolyA distribution analysis probe to the mRNA to be detected is 1-10:

1.

3. The quality inspection method according to claim 2, characterized in that: The quality detection method comprises: Mix 50-200 pmol of the mRNA to be detected, 0.2-1 nmol of the capping rate analysis probe, 0.2-1 nmol of the PolyA distribution analysis probe, and 2-10 μL of the thermostable RNase H enzyme buffer to obtain the mRNA-probe structure; The mRNA-probe structure is mixed with 1-10 μL of the thermostable RNase H enzyme, and incubated at 40-60° C. for 25-35 min to obtain the 5′ enzyme-cleaved fragment and the 3′ enzyme-cleaved fragment.

4. The quality inspection method according to claim 3, characterized in that: The quality detection method comprises: Mix 100 pmol of the mRNA to be detected, 0.5 nmol of the capping rate analysis probe, 0.5 nmol of the PolyA distribution analysis probe, and 6 μL of the heat-stable RNase H enzyme buffer to obtain the mRNA-probe structure; The mRNA-probe structure was mixed with 5 μL of the thermostable RNase H enzyme, and incubated at 50° C. for 25-35 min to obtain the 5′ enzyme-cleaved fragment and the 3′ enzyme-cleaved fragment.

5. The quality inspection method according to claim 1, characterized in that: The chromatographic conditions in the liquid chromatography-mass spectrometry are: chromatographic column: C18 chromatographic column; mobile phase: mobile phase A, mobile phase B; flow rate: 0.2-0.4 mL / min; column temperature 40-80° C.; detection wavelength 260 nm; injection volume: 10 μL; The mobile phase A is an aqueous solution containing 1 wt% HFmIP, 0.1 wt% DIEA and 1 μM EDTA; The mobile phase B is a 35% (v / v) acetonitrile-water solution containing 0.075 wt% HFmIP, 0.0375 wt% DIEA and 1 μM EDTA; Gradient elution, elution conditions are as follows: 。 6. The quality inspection method according to claim 1, characterized in that: The mass spectrometry conditions in the liquid chromatography-mass spectrometry are: Full scan, negative ion mode, mass range 400Da-5000Da.

7. The quality inspection method according to claim 1, characterized in that: The length of the capping rate analysis probe is 10-20 bp.

8. The quality inspection method according to claim 1, characterized in that: The length of the PolyA distribution analysis probe is 10-20 bp.

9. The quality inspection method according to claim 1 or 7, characterized in that: The capping rate analysis probe includes ribonucleotides and deoxyribonucleotides; Preferably, the 5' end and the 3' end of the capping rate analysis probe are both fragments consisting of the ribonucleotides, and the middle part is a fragment consisting of the deoxyribonucleotides.

10. The quality inspection method according to claim 1 or 8, characterized in that: The PolyA distribution analysis probe includes ribonucleotides and deoxyribonucleotides; Preferably, the 5' end and the 3' end of the PolyA distribution analysis probe are both fragments consisting of the ribonucleotides, and the middle part is a fragment consisting of the deoxyribonucleotides.

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