WO2023115495A1

WO2023115495A1 - Method for measuring mrna capping efficiency using mass spectrometry

Info

Publication number: WO2023115495A1
Application number: PCT/CN2021/141002
Authority: WO
Inventors: 张海燕; 王宇涵; 马庆伟
Original assignee: 北京毅新博创生物科技有限公司
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2023-06-29

Abstract

A method for rapidly measuring an mRNA capping efficiency by using a time-of-flight mass spectrometry technology, especially for in-vitro synthesized mRNA. The method comprises providing uncapped mRNA and capped mRNA, and using time-of-flight mass spectrometry technology to perform relative quantification on the capping efficiency of the mRNA. It is the first time that the use of time-of-flight mass spectrometry technology to implement a method for measuring an mRNA capping efficiency is proposed, operations are rapid and simple, and a measurement for trace samples can also be performed by means of the mass spectrometry technology, such that a relatively high measurement sensitivity can be achieved.

Description

Method for detecting mRNA capping efficiency by mass spectrometry

technical field

The invention belongs to the field of molecular biology detection, and relates to a method for quickly detecting mRNA capping efficiency by using time-of-flight mass spectrometry technology.

Background technique

The processing modification of cellular messenger RNA (mRNA) includes: forming a cap structure at the 5' end, adding PolyA to the 3' end, splicing to remove introns and methylation.

The 5' end of mature eukaryotic mRNA has m ⁷ GPPPN structure, called methyl guanosine cap. It is catalyzed by RNA triphosphatase, mRNA guanylyltransferase, mRNA (guanine-7) methyltransferase and mRNA (nucleoside-2') methyltransferase.

Different methyl groups can form 3 cap types: 0, 1, 2. Guanosine is linked to the 5' end of RNA by a 5'-5' triphosphate bond. When C at position 7 of G is methylated to form m ⁷ GPPPN, this cap structure is called "cap 0". present in single-celled organisms. If the 2'-O position of the first nucleotide in the RNA is also methylated, m ⁷ GPPPNm, called "cap 1", is ubiquitous. If the 2'-O positions of the 1st and 2nd nucleotides of the RNA are both methylated (the 2nd position must be A), m ⁷ GPPPNmNm is formed, which is called "cap 2", and the secondary structure rarely occurs. The complexity of the eukaryotic hat is closely related to the degree of biological evolution.

The cap structure at the 5' end of mRNA is necessary for translation initiation, providing signals for ribosomes to recognize mRNA, and assisting ribosomes to combine with mRNA, enabling translation to start from AUG. The cap structure can increase the stability of mRNA and protect mRNA from the attack of 5'-3' exonuclease.

In 2020, the outbreak of the new crown epidemic ignited the enthusiasm of pharmaceutical companies for the RNA track. With the approval of domestically produced COVID-19 inactivated, adenovirus and recombinant protein vaccines, the progress of mRNA vaccines has become the focus of attention. At the same time, the arrival of the post-epidemic era has also made people full of infinite reverie about the future of RNA therapy.

mRNA therapy is becoming an increasingly important approach for the treatment of a variety of diseases. Effective mRNA therapy requires efficient delivery of the mRNA to the patient and efficient production of the protein encoded by the mRNA in the patient. To optimize mRNA delivery and protein production in vivo, usually proper capping is required at the 5' end of the construct, which prevents degradation of mRNA and facilitates translation of mRNA, therefore, the accuracy of capping efficiency is critical for determining mRNA therapeutic applications The quality is particularly important.

At present, the commonly used detection method of capping efficiency is LC-MS method. LC-MS detection requires enzyme digestion, which is cumbersome to operate. Difficult to promote in the field of clinical application. Therefore, in the field of clinical application, it is urgent to establish a fast, accurate, and sensitive detection method for mRNA capping efficiency, so as to provide sufficient basis for determining the quality of mRNA therapeutic applications and the quality research of mRNA vaccines.

Chinese patent application CN201480010108.7 "Quantitative Evaluation of Messenger RNA Capping Efficiency" discloses a method for measuring mRNA capping by ELISA. Including: mRNA synthesis in vitro, providing samples containing capped RNA and uncapped RNA. Because this method uses conventional processing, although it can characterize the characteristic map of the RNA to a certain extent, because the analyte contains other molecules that can be ionized, the obtained map is essentially the same as that of the above-mentioned various molecules. Spectrum collection, so the amount of spectral information that needs to be processed and compared is too large, and the characteristic of the spectrum is low due to the large number of molecules to be detected. It is only applicable to a specific substance and cannot be extended to the detection of a large number of other substances.

Chinese patent application CN201980073219 "Method and composition for purifying messenger RNA" discloses a method for purifying mRNA which involves precipitating mRNA synthesized by in vitro transcription process (IVT) in a buffer containing denaturing salt in combination with a reducing agent, and then Capture of the precipitated mRNA and dissolving the captured mRNA in solution to obtain purified mRNA removes impurities from the messenger RNA preparation synthesized by large-scale IVT. Wherein, the cap species of the final purified mRNA product was determined by HPLC/MS method. However, this method needs to be combined with silver-stained gel image method and CE electrophoresis method for comparative analysis, and the whole process is tedious and time-consuming.

In summary, there are currently methods for determining the cap type and efficiency of mRNA products, mainly including ELISA method, HPLC chromatography, electrophoresis method, luciferase method and RNA spot blot method, etc. These methods can better determine the cap type , to evaluate the capping efficiency, but there are generally defects such as cumbersome process, time-consuming and labor-consuming, and expensive reagents.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, referred to as MALDI-TOF MS) technology is a mass spectrometry analysis technology that came out in the late 1980s and developed rapidly. Its mass analyzer is an ion drift tube (ion dirt tube). The ions generated by the ion source are collected first, and all ion velocities in the collector become 0. After being accelerated by a pulse electric field, they enter the field-free drift tube. Flying to the ion receiver at a constant speed, the greater the ion mass, the longer it takes to reach the receiver; the smaller the ion mass, the shorter the time it takes to reach the receiver. According to this principle, ions of different masses can be separated according to the mass-to-charge ratio, and the molecular mass and purity of biological macromolecules such as peptides, proteins, nucleic acids, and polysaccharides can be accurately detected. It has high accuracy, strong flexibility, and large throughput. , short detection cycle, and high cost performance.

The theoretical basis of MALDI-TOF MS detection of nucleic acid fragments is that there are quality differences between the four nucleotides, the basic unit of genetic material DNA. For example, the molecular weights of ddAMP, ddCMP, ddGMP, and ddTMP are 271.2 Da and 247.2 Da, respectively. Da, 287.2Da, 327.1Da (ddTMP is modified), the minimum molecular weight difference between them is 16Da, which can be completely distinguished by mass spectrometry. Mass spectrometry can be used to detect base mutations or polymorphic sites (SNPs), insertions/deletions (InDels), methylation sites, gene quantification, copy number variation (copy number variation, CNV) and other types of DNA changes .

In addition, based on MALDI-TOF MS, some nucleic acid detection methods have been developed, such as the hME and iPLEX methods of Agena Company in the United States, the GOOD assay method of Bruker Company in Germany, and the RFMP method of GeneMatrix Company in South Korea. In order to improve the resolution of mass spectrometers, various companies tend to detect oligonucleotide fragments with smaller molecular weights when detecting target sites. For example, the RFMP method uses multiplex PCR for sites containing single nucleotide polymorphisms (SNPs). The product is subjected to restriction enzyme digestion to produce oligonucleotide fragments of about 2000-4000 Da for detection, while the GOOD assay method uses phosphodiesterase (Phosphodiesterase, PDE) to cut the oligonucleotide fragments containing SNP sites into 1000-Da Small fragments around 2000 Da are detected. However, the above methods inevitably have problems such as complicated operation and long time consumption.

Contents of the invention

Based on the defects or deficiencies in the above-mentioned technologies, one of the principles of the present invention is to provide a method for rapidly detecting mRNA capping efficiency by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the first time. Synthetic mRNA is characterized and analyzed, including uncapped and capped mRNA, and relative quantification of mRNA capping efficiency.

The second principle of the present invention is that, in order to solve the mass spectrometry detection spectrum shift, the present invention adds an internal standard during the detection process, and further calibrates the molecular weight of the target substance to be tested.

The third principle of the present invention is that in the process of mass spectrometry detection, in order to avoid the interference of some ions in the substance to be tested, the present invention purifies the substance to be tested, thereby improving the detection accuracy.

Therefore, the first object of the present invention is to provide a mass spectrometry detection kit for MALDI TOF-MS detection of mRNA fragments synthesized in vitro, including mRNA standard sample composition, internal standard, purification reagent, mass spectrometry matrix and spotting chip and detection software, where

Described mRNA standard sample composition comprises:

The nucleic acid sequence of the internal standard has a molecular weight of 8214.4Da, and SEQ ID NO: 1 is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'.

In one embodiment, wherein the matrix is a composite matrix comprising an acidic component including but not limited to formic acid, acetic acid and citric acid.

In another embodiment, the chip is a microarray chip dedicated to time-of-flight mass spectrometry, and its material includes but not limited to stainless steel, diamond, single crystal silicon, and quartz crystal.

In other embodiments, the software is BioExplore software, whose copyright number is Ruan Zhu Deng Zi No. 136879, registration number 2009SR10700.

The second object of the present invention is to provide a method for detecting mRNA sample capping efficiency through the above-mentioned detection kit, comprising the following steps:

(1) Sample preparation: Dilute the samples to appropriate concentrations, and add molecular weight calibration internal standards of corresponding final concentrations.

(2) Purification: Purify the sample obtained in step (1) to obtain a high-purity sample and avoid the influence of impurities such as salt ions on subsequent detection.

(3) Spotting: Spot the purified substance obtained in step (2) on the target chip containing the matrix, and the analyte directly forms a crystalline mixture with the matrix.

(4) Detection by mass spectrometer: put the target plate that has been sampled into the mass spectrometer for detection.

(5) Data analysis: compare and analyze the spectrum obtained in step (4) with the pre-established mass spectrometry characteristic peak model by computer software, so as to obtain the capping efficiency of the mRNA sample to be tested; wherein,

The characteristic peak model of the mass spectrum includes the characteristic peak 7364.9205m/z corresponding to the target fragment of the uncapped mRNA (uncap), the characteristic peak 7645.0256m/z corresponding to the target fragment of the capped mRNA (cap0), and the capped mRNA The characteristic peak corresponding to the target fragment of (cap1): 7659.0412m/z.

In one embodiment, the mRNA capping efficiency calculation formula of step (5) is:

Cap1 capping rate = Area(cap1)/Area(cap0+cap1+uncapped);

Cap0 capping rate=Area(cap0)/Area(cap0+cap1+uncapped).

In another embodiment, wherein the nucleic acid sequence (SEQ ID NO: 1) of the internal standard is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'

In one embodiment, the matrix is a composite matrix comprising an acidic component including, but not limited to, formic acid, acetic acid, and citric acid. In another specific embodiment, the chip is a microarray chip dedicated to time-of-flight mass spectrometry, and its material includes but not limited to stainless steel, diamond, single crystal silicon, and quartz crystal.

In another embodiment, use NanoDrop ND-2000 nucleic acid detector to measure the concentration of mRNA in step (1).

In a specific embodiment, the mass spectrometer is a MALDI TOF MS mass spectrometer.

In one embodiment, the software is the BioExplore software researched and developed by the inventor himself, whose copyright number is Ruan Zhu Deng Zi No. 136879 and registration number 2009SR10700.

In any of the above embodiments, wherein the mRNA sample is an mRNA vaccine, including new coronary pneumonia, hepatitis B mRNA vaccine, influenza mRNA vaccine, HPV mRNA vaccine, etc.

In any of the above embodiments, the method is widely used in the field of mRNA detection as a non-diagnostic application, and provides sufficient basis for determining the quality of mRNA therapeutic applications and the quality research of mRNA vaccines.

technical effect

Compared with the prior art, the present invention has the following advantages:

1. For the first time, the present invention proposes to use time-of-flight mass spectrometry to detect mRNA capping efficiency, especially for mRNA vaccine capping efficiency, which has extremely high biological value.

2. Sensitivity: the present invention uses techniques such as mass spectrometry detection, so its detection sensitivity is very high.

3. Simple and safe: simple and safe operation;

4. The data analysis required by the present invention is simple, only need to observe the spectrogram, and no complex bioinformatics analysis is required.

5. The present invention is low in cost, does not require fluorescent labels, and reduces system complexity and signal interpretation errors caused by the addition of fluorescent chemical probes.

6. High autonomy, using self-developed instruments, reagents, chips and analysis software.

Description of drawings

Figure 1: In the mass spectrum model constructed in Example 2, the characteristic peak corresponding to the uncap target fragment (7364.9205Da): 7364.9205m/z.

Figure 2: In the mass spectrum model constructed in Example 2, the characteristic peak corresponding to the cap0 target fragment (7645.0256Da): 7645.0256m/z.

Figure 3: In the mass spectrum model constructed in Example 2, the characteristic peak corresponding to the target fragment of cap1 (7659.0412Da): 7659.0412m/z.

Figure 4: Mass spectrometry results of sample loading and detection of the mRNA vaccine sample N1 to be tested.

Figure 5: Mass spectrometry results of sample loading and detection of the mRNA vaccine sample N2 to be tested.

Figure 6: Mass spectrometry results of sample loading and detection of the mRNA vaccine sample N3 to be tested.

Figure 7: Mass spectrometry results of sample loading and detection of the mRNA vaccine sample N4 to be tested.

Principles and Definitions

The invention provides a detection scheme for detecting the characteristic map of mRNA by using mass spectrometry detection technology, and then determining the capping efficiency of mRNA.

The principle is that:

During the sample preparation step, the molecular weight calibration internal standard was added at the corresponding final concentration.

In the process of mass spectrometry detection, the substance to be tested is spotted to the target sheet containing the matrix after purification, and is excited by a laser in a vacuum environment, and then passes through the flight tube to the detector. The time for different substances to pass through the flight tube is negatively correlated with their molecular weight, that is, the larger the molecular weight, the slower the flight speed and the later the time to reach the detector.

The term "internal standard" is used to solve the shift of the mass spectrometry detection spectrum and to calibrate the molecular weight of the target analyte.

The term "purification" refers to the processing steps used to reduce the influence of other substances in the system to be tested on subsequent reactions. There are two methods for purifying the PCR product of the present invention: one is to separate and discard the impurities, and the other is to inactivate the impurities. Among them, gel cutting purification, purification column, etc. are used to separate impurities by electrophoresis, purification column, etc., and recover relatively pure PCR products. It can be considered as the first purification method. This method is generally time-consuming and complicated to operate. When the sample size is large; the role of alkaline phosphatase is to degrade (also known as "digest") dNTP, so that it cannot continue to participate in PCR or single-base extension reaction as a substrate for DNA polymerase or single-base elongation enzyme, so as not to Interfering with subsequent reactions can be considered as the second purification method. It should be noted that the exonuclease ExoI alone does not play a role in purification. When it is mixed with alkaline phosphatase, its function is to pre-separate the single-stranded DNA (in the PCR product system after the reaction is completed, mainly the remaining PCR primers) are degraded into dNTPs, and the dNTPs are further degraded by alkaline phosphatase. Since the PCR primers are degraded and do not enter the final mass spectrometry step, it is not necessary to use PCR primers with protective bases if you plan to add ExoI exonuclease treatment to the purification step. In addition, since both exonuclease and alkaline phosphatase are inactivated by high temperature before the single-base extension step, it will not degrade the single-stranded extension primer, ddNTP, etc. Subsequent experiments had an impact.

The term "detection window" refers to the range that can be used to detect the molecular weight of nucleotides by mass spectrometry, and usually involves the design reference range of primers. Among them, when designing extension primers, for different specific sites, according to the sequence characteristics of the DNA region where these sites are located, and the genotype of the specific site, extension primers and extension products with different molecular weights can be designed to avoid different extension primers. There is interference between the product and the product due to the close molecular weight, so that the detection of multiple specific sites can be realized in a relatively wide detection window, such as 4000-9000Da.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually implemented according to conventional conditions.

It should be pointed out that although the mass spectrometry detection data below 50,000Da is presented in the examples of the present invention, nucleic acid substances between 50,000-100,000Da can also be detected, so the application of the present invention to the detection data between 50,000-100,000Da The mass spectrometry detection is also included in the scope of the claims of the present invention.

Embodiment one, synthesize mRNA in vitro

The following mRNA fragments were synthesized in vitro by known existing methods, such as "Molecular Cloning Experiment Guide" (Fourth Edition) or "Refined Molecular Biology Experiment Guide" (Fifth Edition), or by commercial companies.

Table 1. mRNA fragment information table

Embodiment 2. Method for constructing a mass spectrometry characteristic peak model to detect mRNA capping efficiency

The nucleic acid sequence used as an internal standard was synthesized by Shanghai Jierui Bioengineering Co., Ltd., with a molecular weight of 8214.4Da, and SEQ ID NO: 1 is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'.

Use the above mRNA fragments and internal standards to detect the mRNA capping efficiency method, the specific operation method is as follows:

1. Sample Preparation

Dilute the mRNA samples to appropriate concentrations, and then add molecular weight calibration internal standards of corresponding final concentrations.

2. Purification

Add 15 mg of resin to each tube of mRNA sample and mix by inversion for 5 minutes.

3. Spotting

Use a micropipette to draw 0.5ul of the purified product and apply it to the target slice.

4. On-board testing and result interpretation

The above purified product was detected by clinical time-of-flight mass spectrometer Clin-TOF-II (MALDI-TOF MS produced by Yixin Bochuang Biotechnology Co., Ltd.) mass spectrometer.

5. Interpretation of results

The Clin-TOF time-of-flight mass spectrometer developed by the inventor is used to detect and judge the results of the sampled target slices.

6. Test results

All the mRNA samples were spotted on the same chip for nucleic acid mass spectrometry detection.

The characteristic peak (m/z) represented by the molecular weight of the target fragment corresponding to the positive target, the results are shown in Figure 1-3.

Figure 1 is the characteristic peak corresponding to the target fragment (7364.9205Da) of uncapped mRNA (uncap): 7364.9205m/z;

Figure 2 is the characteristic peak corresponding to the target fragment (7645.0256Da) of the capped mRNA (cap0): 7645.0256m/z;

Figure 3 is the characteristic peak corresponding to the target fragment (7659.0412Da) of the capped mRNA (cap1): 7659.0412m/z;

In addition, there were no obvious miscellaneous peaks in all groups, and the baseline was stable;

There are no impurity peaks or <2 impurity peaks in the range of 3000-10000 Da in the spectrum.

Therefore, the mass spectrometry characteristic peak model of the mRNA sample detected by mass spectrometry was constructed.

Example 3. Detection of mRNA samples using mass spectrometry model

According to the method of Example 2, after sample processing and purification of 4 mRNA vaccine samples, the target slices after spotting were detected and the results were judged by the Clin-TOF time-of-flight mass spectrometer.

Calculate the capping rate of Cap1=Area(cap1)/Area(cap0+cap1+uncapped), and the corresponding capping rate of Cap0 is=Area(cap0)/Area(cap0+cap1+uncapped), the result is shown in Figure 4-7: The mass spectrometry results are shown in Figure 4-7:

For sample N1, the detection peaks (m/z) are respectively: 7364.9205, 7645.0256, and 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, and cap1 are respectively: 7364.9205, 7645.0256, and 7659.0412. According to the mass spectrum model established in Example 2, therefore The detection results were determined to be: uncap, cap0, cap1; the capping rate of Cap1 was 54.5%.

For sample N2, the detection peaks (m/z) are respectively: 7364.9205, 7645.0256, and 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, and cap1 are respectively: 7364.9205, 7645.0256, and 7659.0412, according to the mass spectrum model established in Example 2, so The detection results were determined to be: uncap, cap0, cap1; the capping rate of Cap1 was 73%.

For sample N3, the detection peaks (m/z) are respectively: 7364.9205, 7645.0256, and 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, and cap1 are respectively: 7364.9205, 7645.0256, and 7659.0412, according to the mass spectrum model established in Example 2, so The determined detection results are: uncap, cap0, cap1; the capping rate of Cap1 is 74.5%.

For sample N4, the detection peaks (m/z) are respectively: 7364.9205, 7645.0256, and 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, and cap1 are respectively: 7364.9205, 7645.0256, and 7659.0412, according to the mass spectrum model established in Example 2, so The determined detection results are: uncap, cap0, cap1; the capping rate of Cap1 is 75.8%.

The area of each peak is integrated and finally shown in the table below.

It can be seen that the baseline of the characteristic peaks in Fig. 4-7 is smooth, the signal-to-noise ratio is high, and the separation between adjacent signal peaks is high, so the mass spectrometry method of the present invention can detect uncap, cap0, cap1 mRNA characteristic peaks simultaneously, and can Obtain detection results quickly and intuitively, and avoid the shortcomings of objective and low resolution methods such as enzyme digestion. Aiming at the capping efficiency of mRNA vaccines, it has extremely high biological value.

Claims

A kind of mRNA fragment composition that is used for MALDI TOF-MS detection capping efficiency, it comprises:
An internal standard sequence for detecting the mRNA fragment composition of capping efficiency according to claim 1, SEQ ID NO: 1 is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3', and the molecular weight is 8214.4Da.
A method for detecting mRNA capping efficiency, comprising the steps of:

(1) Sample preparation: Dilute the samples to appropriate concentrations, and add molecular weight calibration internal standards of corresponding final concentrations.

(2) Purification: Purify the sample obtained in step (1) to obtain a high-purity sample and avoid the influence of impurities such as salt ions on subsequent detection.

(3) Spotting: Spot the purified substance obtained in step (2) on the target chip containing the matrix, and the analyte directly forms a crystalline mixture with the matrix.

(4) Mass spectrometer detection: put the sampled target plate into the mass spectrometer for detection;

(5) Data analysis: compare and analyze the spectrum obtained in step (4) with the pre-established mass spectrometry characteristic peak model by computer software, so as to obtain the capping efficiency of the mRNA sample to be tested; wherein,

The characteristic peak model of the mass spectrum includes the characteristic peak 7364.9205m/z corresponding to the target fragment of the uncapped mRNA (uncap), the characteristic peak 7645.0256m/z corresponding to the target fragment of the capped mRNA (cap0), and the capped mRNA The characteristic peak corresponding to the target fragment of (cap1): 7659.0412m/z.
The method of claim 3, wherein the mRNA capping efficiency calculation formula of step (5) is:

Cap1 capping rate = Area(cap1)/Area(cap0+cap1+uncapped);

Cap0 capping rate=Area(cap0)/Area(cap0+cap1+uncapped).
The method of claim 4, wherein the nucleic acid sequence (SEQ ID NO: 1) of the internal standard is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'.
The method of claim 5, wherein the matrix is a composite matrix containing acidic components, the acidic components include but not limited to formic acid, acetic acid and citric acid, and the chip is a microarray chip dedicated to time-of-flight mass spectrometry, and its material includes but not limited to Stainless steel, diamond, monocrystalline silicon, quartz crystal.
The method of claim 6, wherein in step (1), utilize NanoDrop ND-2000 nucleic acid detector to measure the concentration of mRNA, and described mass spectrometer is MALDI TOF MS mass spectrometer.
The method of claim 7, wherein said software is BioExplore software, the copyright number of which is Ruan Zhu Deng Zi No. 136879, and the registration number is 2009SR10700.
The method according to any one of claims 4-8, wherein the mRNA sample is an mRNA vaccine, including new coronary pneumonia, hepatitis B mRNA vaccine, influenza mRNA vaccine, HPV mRNA vaccine, etc.
The method of any one of claims 3-8 is used as an application for non-diagnostic purposes, for detecting the quality of mRNA therapeutic applications and the quality of mRNA vaccines.