Quality detection method for mRNA capping rate and PolyA distribution

Abstract

The present invention provides a method for quality detection of mRNA capping rate and PolyA distribution. Among them, the above quality detection method includes: a) mixing the mRNA to be detected, a capping rate analysis probe and a PolyA distribution analysis probe to obtain an mRNA-probe structure; b) digesting the mRNA-probe structure with a thermostable RNaseH enzyme to obtain 5' digestion fragments and 3' digestion fragments; c) detecting the 5' digestion fragments and 3' digestion fragments by liquid chromatography-mass spectrometry, obtaining the capping rate from the 5' digestion fragments and obtaining the PolyA distribution from the 3' digestion fragments, so as to realize the simultaneous detection of the 5' capping rate and 3' PolyA of mRNA. It can solve the problems of many steps and long time required for detecting the capping rate and PolyA distribution of mRNA in the prior art, and is applicable to the field of mRNA quality detection.

Description

Technical Field

[0001] The present invention relates to the field of mRNA quality detection. Specifically, it relates to a method for detecting the capping rate and PolyA distribution of mRNA. Background Art

[0002] The detection of the capping rate and PolyA distribution of mRNA is a common detection in mRNA quality detection. Since a complete mRNA molecule is often composed of more than 1000 nt of nucleotides, it is impossible to directly analyze mRNA molecules of this size for mass spectrometry analysis. In the existing detection of the capping rate, ribonuclease H (RNaseH) is generally selected during the sample preparation process. The RNaseH enzyme can selectively recognize and cleave the phosphodiester bond of RNA in the RNA:DNA hybrid double strand, and the enzymatic digestion yields smaller and definite 5'-end fragments. In the existing technology for the detection of PolyA distribution, ribonuclease T1 (RNase T1) is selected for enzymatic digestion during the sample preparation process. RNase T1 can specifically cleave single-stranded RNA at the G residue. 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] The existing detection of the capping rate and PolyA distribution based on LC-MS has no correlation in sample preparation, and the corresponding detections are completed through two independent experiments. Summary of the Invention

[0004] The main object of the present invention is to provide a method for detecting the quality of the capping rate and PolyA distribution of mRNA, so as to solve the problems of many steps and long time required for detecting the capping rate and PolyA distribution of mRNA in the existing technology.

[0005] To achieve the above object, according to the first aspect of the present invention, a method for detecting the quality of mRNA is provided. The above quality detection method includes: 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) using a thermostable RNaseH enzyme to digest the mRNA-probe structure to obtain 5'-digested fragments and 3'-digested fragments; c) using liquid chromatography-mass spectrometry to detect the 5'-digested fragments and 3'-digested fragments, obtaining the capping rate from the 5'-digested fragments and obtaining the PolyA distribution from the 3'-digested fragments, thereby realizing the simultaneous detection of the 5'-capping rate and 3'-PolyA of mRNA; wherein, 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 sequence in the 5'-direction of the 3'-PolyA structure of the mRNA to be detected.

[0006] Further, 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.

[0007] Further, 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 buffer of the thermostable RNaseH enzyme to obtain an mRNA-probe structure; mixing the mRNA-probe structure with 1-10 μL of the thermostable RNaseH enzyme, and incubating at 40-60 °C for 25-35 min to obtain 5'-digested fragments and 3'-digested fragments.

[0008] Further, 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 buffer of the thermostable RNaseH enzyme to obtain an mRNA-probe structure; mixing the mRNA-probe structure with 5 μL of the thermostable RNaseH enzyme, and incubating at 50 °C for 25-35 min to obtain 5'-digested fragments and 3'-digested fragments.

[0009] Further, the chromatographic conditions in liquid chromatography-mass spectrometry are 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 is an aqueous solution containing 1 wt% HFmIP, 0.1 wt% DIEA, and 1 μM EDTA; mobile phase B is a 35% (v / v) acetonitrile-aqueous 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] Further, the mass spectrometry conditions in liquid chromatography-mass spectrometry are: full scan, anion mode, mass range 400 Da - 5000 Da.

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

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

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

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

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

[0018] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

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

[0020] Figure 2 Shows the LC-MS result diagram of using RNase H alone to detect the capping rate in Example 2 of the present invention.

[0021] Figure 3 Shows the LC-MS result diagram of using RNase T1 alone to detect the PolyA distribution in Example 2 of the present invention.

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

[0023] Figure 5 Shows the LC-MS result diagram of using RNase H alone to detect the capping rate in Example 4 of the present invention.

[0024] Figure 6The figure shows the LC-MS result diagram of detecting the PolyA distribution using RNase T1 alone in Example 4 of the present invention. Detailed implementation mode

[0025] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments 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 art, the quality attribute detection process of mRNA molecules often requires simultaneous monitoring of the capping rate and the PolyA distribution. Under the condition that the two experiments are carried out separately, the sample preparation of these two methods has no association, is time-consuming and has complex operations.

[0027] In the present application, the inventors attempt to develop a new quality detection method for the capping rate and PolyA distribution of mRNA, which can complete the detection of the capping rate and PolyA distribution in one pretreatment and detection, also known as the "one-step combined enzymatic digestion experiment". Based on this, a series of protection schemes of the present application are proposed.

[0028] In the first typical implementation mode of the present application, a quality detection method for the capping rate and PolyA distribution of mRNA is provided. The quality detection method includes: 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) using thermostable RNaseH enzyme to 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 the PolyA distribution to realize the quality detection of mRNA; wherein, 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 sequence in the 5'-direction of the 3'-PolyA structure of the mRNA to be detected.

[0029] In the prior art, the capping rate analysis is based on the enrichment of the probe with an enrichment tag after digestion by RNaseH enzyme, followed by chromatographic separation and mass spectrometry identification; the PolyA analysis process generally selects enzymatic digestion methods such as RNase T1 to hydrolyze mRNA, and then enriches the remaining PolyA part, followed by chromatographic separation and mass spectrometry identification. However, this method does not have the ability of site-specific digestion, and 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 makes innovations and optimizations on the basis of the prior art. For the sample preparation of the original two detections, Thermostable RNaseH is used for digestion. Thermostable RNaseH is used to digest specific sites where mRNA binds to the probe. By analyzing the capping rate of the probe, the capture of the 5'-end sequence in mRNA is achieved, and by analyzing the PolyA distribution of the probe, the capture of the 3'-poly(A) structure in mRNA is achieved.

[0031] The quality detection method of this application realizes the analysis of the capping rate and PolyA distribution simultaneously in one experiment for the first time. At the same time, it reduces the complexity and operation time in the analysis process, significantly improves the throughput, and avoids the enrichment process in the prior art. In this application, the capping rate analysis probe and the PolyA distribution analysis probe can respectively bind to the sequences located at the 5'-end sequence of the mRNA to be detected and the 5'-direction sequence of the 3'-PolyA, thereby forming the first and second cleavage sites. Then, the thermostable RNaseH enzyme is used to digest the two cleavage sites to achieve the cleavage of the 5'-end sequence and the PolyA sequence. Preferably, the first cleavage site is usually located at the position of the 5th - 40th nt at the 5'-end, and the second cleavage site is located at the position 1 - 20 nt upstream of the 5'-end of the first A in the PolyA structure. Those skilled in the art can flexibly design the sequences of the capping rate analysis probe and the PolyA distribution analysis probe according to the sequence of the mRNA to be detected. Further, the inventor improves the detection sensitivity of LC-MS for target molecules through the optimization of the mobile phase and gradient, realizes the purpose of simultaneously analyzing the capping rate and PolyA distribution in one experiment, with lower cost and higher efficiency, shortens the mRNA analysis and detection process and time, and realizes the sample preparation and on-machine analysis process of the capping rate and PolyA distribution of mRNA 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 can specifically degrade the RNA strand in the DNA:RNA hybrid double strand, generating oligonucleotides and mononucleotides with 3'-OH and 5'-phosphate ends; 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 cleave the phosphodiester bond of the RNA sequence in the RNA:DNA hybrid while keeping the DNA sequence intact. It still has high enzyme activity at temperatures above 65°C, with a half-life of several hours at 70°C and still about 30 minutes at up to 95°C. The high-temperature resistance of this enzyme enables heteroduplex RNA:DNA hybrid molecules to have higher specificity and more specifically cleave the RNA in the hybrid duplex at higher temperatures. The thermostable RNaseH has enzymatic properties similar to those of common RNaseH, but RNase H becomes inactivated at temperatures above 55°C.

[0034] Preferably, the above thermostable RNaseH enzyme is a protein having the amino acid sequence shown in SEQ ID NO: 1, or a protein having a homology of greater than or equal to 70% with SEQ ID NO: 1 and having the above thermostable and high-temperature resistant capabilities.

[0035] SEQ ID NO: 1:

[0036] MNPSPRKRVALFTDGACLGNPGPGGWAALLRFHAHEKLLSGGEACTTNNRMELKAAI EGLKALKEPCEVDLYTDSHYLKKAFTEGWLEGWRKRGWRTAEGKPVKNRDLWEALLLAM APHRVRFHFVKGHTGHPENERVDREARRQAQSQAKTPCPPRAPTLFHEEA.

[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 PolyA 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 includes: mixing 50 - 200 pmol (including but not limited to 50, 75, 100, 125, 150, 175, 200 pmol) of 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 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 PolyA distribution analysis probe, and 2 - 10 μL (including but not limited to 2, 3, 4, 5, 6, 7, 8, 9 or 10 μL) of buffer of thermostable RNaseH enzyme to obtain an mRNA-probe structure; mixing the mRNA-probe structure with 1 - 10 μL (including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μL) of thermostable RNaseH enzyme, incubating 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 5'-digested fragments and 3'-digested fragments.

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

[0040] In a preferred embodiment, the chromatographic conditions in 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 isopropyl alcohol), 0.1 wt% DIEA (N,N-diisopropylethylamine) and 1 μM EDTA; mobile phase B is a 35% (v / v) acetonitrile - aqueous 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:

[0041] Using the above chromatographic conditions, it is possible to separate the mRNA fragments obtained by digestion with Thermostable RNaseH, and separate the fragments to be detected to achieve subsequent mass spectrometry detection.

[0042] In a preferred embodiment, the mass spectrometry conditions in liquid chromatography - mass spectrometry are: full scan, anion mode, mass range 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 - 20bp, including but not limited to 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20bp.

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

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

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

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

[0052] Mix the above two kinds of probes with the mRNA to be detected and anneal them to form an mRNA-probe structure, which contains a DNA-RNA hybrid structure. Further, use the thermostable RNaseH enzyme to cleave the DNA-RNA hybrid structure in the mRNA-probe structure, thereby cleaving the phosphodiester bond derived from the mRNA to be detected at the cleavage site. After the above cleavage, optionally, the mRNA-probe structure in the cleavage system is denatured and unwound and then subjected to liquid chromatography-mass spectrometry analysis, or directly subjected to liquid chromatography-mass spectrometry analysis and denatured and unwound using liquid phase conditions, both of which can achieve the simultaneous detection of the 5'-capping rate and 3'-Poly A of mRNA.

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

[0054] Example 1 One-step combined enzymatic digestion experiment for the capping rate and Poly A distribution of firefly luciferase mRNA

[0055] The one-step combined enzymatic digestion was carried out to detect the firefly luciferase mRNA (PolyA designed length 80A), and the analysis results are as Figure 1 shown.

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

[0057]

[0058] Sample preparation process:

[0059] Transfer approximately 100 pmol of the sample (sample volume ≤ 50 μL, make up to 50 μL with nuclease-free water if less than 50 μL) into a centrifuge tube, add 6 μL of RNase H Reaction Buffer, and use the mixed sample to dissolve 0.5 nmol of the capping rate analysis probe and 0.5 nmol of the PolyA distribution analysis probe. Then add 5 μL of Thermostable RNase H enzyme (Thermo, EN0202) to the sample, mix well, and incubate at 50 °C and 350 rpm in a metal bath for 30 ± 5 min for testing.

[0060] Among them, the sequence of the capping rate analysis probe is SEQ ID NO: 3, and the sequence of the PolyA distribution analysis probe is SEQ ID NO: 4.

[0061] SEQ ID NO: 3: mUmGmGmGmGdAdCdCdAmGmAmAmGmAmA.

[0062] SEQ ID NO: 4: mCmUmAmGmCdTdCdCdAmGmGmGmUmGmU.

[0063] Among them, m before A, C, G or U indicates a 2'-O-methyl modification of the ribonucleotide; d before A, C, G or T indicates that the nucleotide is a deoxyribonucleotide.

[0064] The design principles of the capping rate analysis probe and the PolyA distribution analysis probe 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 (μL) 10 Column Temperature (°C) 75 Sample Room Temperature (°C) 10 Detection Wavelength (nm) 260 Data collection rate (Hz) 20 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. Using the prior art, the capping rate of firefly luciferase mRNA (SEQ ID NO: 5) was detected, and the results are as Figure 2 shown.

[0079] The experimental steps are as follows:

[0080] 1) Add 6 μL of RNaseH Buffer and 6 μL of ultrapure water to 1 nmol of Capping probe (SEQ ID NO: 3) to dissolve it. Take 6 μL of the dissolved probe and 4 μL of RNaseH Buffer and mix them with 50 μL of mRNA sample (the same sample as used in Example 1, about 100 pmol). (Total system: 60 μL);

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

[0082] 3) After vortexing and mixing, place the sample in a thermostatic mixer (50 °C, 350 rpm) and incubate for 30 ± 5 min for measurement.

[0083] 2. Using the prior art, the PolyA distribution of firefly luciferase mRNA (SEQ ID NO: 2) was detected, and the results are as 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 as used in Example 1, about 100 pmol).

[0086] (Total system: 55 μL)

[0087] 2) After vortexing and mixing, place the sample in a thermostatic mixer (37 °C, 350 rpm) and incubate for 30 ± 5 min for measurement.

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

[0089] Example 3 One-step combined enzymatic digestion experiment for detecting the capping rate and Poly A distribution of the spike protein mRNA of the COVID-19 Delta strain

[0090] 1. Detect the spike protein mRNA of the Delta strain using the method of Example 1 above (one-step combined enzymatic digestion experiment), and the analysis results are as Figure 4 shown.

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

[0092]

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

[0094] SEQ ID NO: 6: mUmUmCmUmUmAdCdTdCdTmUmCmUmUmUmUmC.

[0095] SEQ ID NO: 7: mCmAmCmUmCdAdGdAdCmUmUmUmAmU.

[0096] Among them, m before A, C, G or U represents a 2'-O-methyl modification of the ribonucleotide; d before A, C, G or T represents that the nucleotide is a deoxyribonucleotide.

[0097] Example 4

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

[0099] 2. Use the optimized RNase T1 enzyme in the prior art to digest the same Delta strain spike protein mRNA, and detect the PolyA distribution. The results are as Figure 6 shown (the experimental steps are the same as 2) in Example 2).

[0100] From the experimental results of different mRNA molecules in Examples 1-4, it can be seen that the results of the one-step combined enzymatic digestion experiment of the capping rate and PolyA distribution are comparable to the results of the individual capping rate and PolyA distribution experiments, and are reproducible and have similar accuracy in different molecules. It shows that the mRNA quality detection method in this application is credible and can meet the needs of daily detection.

[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, using the above mRNA quality detection method, using a thermostable RNaseH enzyme to simultaneously digest the capping structure and PolyA structure in the mRNA to be detected, obtaining the expected 5' digestion fragment (i.e., the 5' end sequence) and 3' digestion fragment (i.e., the 3' end sequence); further using liquid chromatography-mass spectrometry (LC-MS) to detect the capping structure and PolyA structure in the expected 5' digestion fragment (i.e., the 5' end sequence) and 3' digestion fragment (i.e., the 3' end sequence), realizing the simultaneous analysis of the capping rate and PolyA distribution of mRNA in a single reaction system, reducing the required experimental steps and time.

[0102] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for quality detection of mRNA capping rate and PolyA distribution for non-diagnostic purposes, characterized in that, The quality detection method includes: 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) Using a thermostable RNaseH enzyme to 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 and the 3'-digested fragment, obtaining the capping rate from the 5'-digested fragment and the PolyA distribution from the 3'-digested fragment, thereby achieving simultaneous detection of the 5'-capping rate and 3'-PolyA of the mRNA; Among them, 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 sequence in the 5'-direction of the 3'-PolyA structure of the mRNA to be detected; Both the 5'-end and 3'-end of the capping rate analysis probe are fragments composed of ribonucleotides, and the middle part is a fragment composed of deoxyribonucleotides; Both the 5'-end and 3'-end of the PolyA distribution analysis probe are fragments composed of ribonucleotides, and the middle part is a fragment composed of deoxyribonucleotides.

2. The quality inspection method according to claim 1, wherein 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, wherein 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 buffer of the thermostable RNaseH enzyme to obtain the mRNA-probe structure; Mixing the mRNA-probe structure with 1-10 μL of the thermostable RNaseH enzyme, and incubating at 40-60 °C for 25-35 min to obtain the 5'-digested fragment and the 3'-digested fragment.

4. The quality inspection method according to claim 3, wherein 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 buffer of the thermostable RNaseH enzyme to obtain the mRNA-probe structure; Mixing the mRNA-probe structure with 5 μL of the thermostable RNaseH enzyme, and incubating at 50 °C for 25-35 min to obtain the 5'-digested fragment and the 3'-digested 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-aqueous solution containing 0.075 wt% HFmIP, 0.0375 wt% DIEA and 1 μM EDTA; Gradient elution was performed under the following conditions:

6. The quality inspection method according to claim 1, characterized in that, The mass spectrometry conditions in the liquid chromatography-mass spectrometry were as follows: full scan, anion mode, mass range 400 Da - 5000 Da.

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.

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