CN114250268A

CN114250268A - Product for detecting capping efficiency of mRNA sample and application

Info

Publication number: CN114250268A
Application number: CN202111593526.1A
Authority: CN
Inventors: 张海燕; 马庆伟; 刘静祎
Original assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Current assignee: Beijing Clin Bochuang Biotechnology Co Ltd
Priority date: 2021-12-23
Filing date: 2021-12-23
Publication date: 2022-03-29

Abstract

The invention provides a detection kit for rapidly detecting mRNA capping efficiency by using a time-of-flight mass spectrometry technology, and particularly relates to detection of in-vitro synthesis quality of mRNA and a nucleic acid vaccine thereof. The detection kit of the invention comprises a standard sample and a calibration internal standard for providing uncapped mRNA and capped mRNA, and time-of-flight mass spectrometry technology is used to relatively quantify the capping efficiency of mRNA. The invention firstly provides a method for detecting the mRNA capping efficiency by using the time-of-flight mass spectrometry technology, the operation is quick and simple, and the mass spectrometry technology can be used for detecting trace samples, so that the detection sensitivity is higher.

Description

Product for detecting capping efficiency of mRNA sample and application

Technical Field

The invention belongs to the field of molecular biological detection, and relates to a product for rapidly detecting mRNA capping efficiency by using a time-of-flight mass spectrometry technology and application thereof.

Background

Processing modifications of cellular messenger rna (mrna) include: the 5 'end forms a cap structure, the 3' end is added with PolyA, and the intron is removed by splicing and methylation.

Mature eukaryotic mRNA has m at its 5' end⁷GPPPN structure, known as methylguanosine cap. It is formed 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 the RNA via a5' -5' triphosphate linkage. When C at position 7 of G is methylated to form m⁷In GPPPN, this cap structure is referred to as "cap 0". Present in unicellular organisms. If the 2' -O position of the 1 st nucleotide of the RNA is also methylated to form m⁷Gppnm, known as "cap 1", is ubiquitous. If the 2' -O position of the 1 st and 2 nd nucleotides of the RNA is methylated (the 2 nd position must be A), m is formed⁷GPPPNmNm, referred to as "cap 2", has little occurrence of substructure. The complexity of eukaryotic caps is closely related to the degree of biological evolution.

The mRNA5' end cap structure is necessary for translation initiation, provides a signal for ribosomes to recognize the mRNA, and aids in ribosome binding to the mRNA, allowing translation to begin from AUG. The cap structure can increase the stability of mRNA and protect mRNA from attack by 5'-3' exonuclease.

In 2020, the outbreak of new crown epidemic situation ignited the enthusiasm of medicine enterprises for RNA racetrack. With the successive approval of domestic new corona inactivated, adenoviral and recombinant protein vaccines, the progress of mRNA vaccines has become a focus of attention. At the same time, the arrival of the post epidemic era has also dredged the future of RNA therapy.

mRNA therapy is becoming an increasingly important method for treating a variety of diseases. Effective mRNA therapy requires efficient delivery of mRNA to a patient and efficient production of the protein encoded by the mRNA in the patient. To optimize mRNA delivery and in vivo protein production, proper capping is usually required at the 5' end of the construct, which can prevent degradation of the mRNA and facilitate translation of the mRNA, and therefore, the accuracy of the capping efficiency is particularly important for determining the quality of the mRNA therapeutic application.

The commonly used capping efficiency detection method at present is an LC-MS method. The detection by the LC-MS method needs enzyme digestion, and the operation is complicated. Is difficult to be popularized in the field of clinical application. Therefore, the clinical application field urgently needs to establish a rapid, accurate and sensitive mRNA capping efficiency detection method, and provides sufficient basis for determining the quality of mRNA treatment application and the quality research of mRNA vaccine.

Chinese patent application CN201480010108.7 "quantitative assessment of messenger RNA capping efficiency" discloses a method for measuring mRNA capping using ELISA. The method comprises the following steps: mRNA is synthesized in vitro to provide samples containing capped and uncapped RNA. Because the method uses conventional treatment, although the characteristic spectrum of the RNA can be represented to a certain extent, the to-be-detected object contains other molecules capable of being ionized, and the obtained spectrum is essentially the spectrum set of the various molecules, the information quantity of the spectrum which needs to be treated and compared is overlarge, and the characteristic of the spectrum is low due to the fact that the to-be-detected molecule is too huge, so that the method is only suitable for a specific substance and cannot be popularized to detection of other large quantities of substances.

Chinese patent application CN201980073219 "method and composition for messenger RNA purification" discloses a method for purifying mRNA, which involves removing impurities from messenger RNA preparations synthesized by large-scale IVT by precipitating mRNA synthesized by in vitro transcription process (IVT) in a buffer comprising a combination of a denaturing salt and a reducing agent, then capturing the precipitated mRNA and dissolving the captured mRNA in a solution to obtain purified mRNA. Wherein the cap species of the final purified mRNA product is determined by HPLC/MS method. However, this method requires a comparative analysis combining silver staining gel image method and CE electrophoresis method, and the whole process is complicated and time-consuming.

In summary, the existing methods for determining the cap type and efficiency of mRNA products mainly include ELISA, HPLC, electrophoresis, luciferase, RNA spotting, and the like, which can better determine the cap type and evaluate the capping efficiency, but have the defects of complicated process, time and labor consumption, expensive reagents, and the like.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) technology is a mass spectrometry technology which is published and developed rapidly in the end of the 20 th century and the 80 th century. The mass analyzer is an ion drift tube (ion drift tube), ions generated by an ion source are firstly collected, the speed of all the ions in a collector is changed into 0, the ions enter a field-free drift tube after being accelerated by a pulse electric field and fly to an ion receiver at a constant speed, and the larger the mass of the ions is, the longer the time for the ions to reach the receiver is; the smaller the mass of the ions, the shorter the time it takes to reach the receiver. According to the principle, ions with different masses can be separated according to the mass-to-charge ratio, the molecular mass and the purity of biomacromolecules such as polypeptide, protein, nucleic acid, polysaccharide and the like can be accurately detected, and the method has the advantages of high accuracy, strong flexibility, large flux, short detection period and high cost performance.

The technology for detecting nucleic acid fragments by MALDI-TOF MS is based on the theory that the mass difference exists among four nucleotides which form the basic unit of genetic material DNA, such as the molecular weight of ddAMP, ddCMP, ddGMP and ddTMP which are 271.2Da, 247.2Da, 287.2Da and 327.1Da (wherein the ddTMP is modified), and the minimum molecular weight difference among the ddTMP is 16Da, which can be completely distinguished by mass spectrum. Many types of DNA changes, such as base mutations or Polymorphic Sites (SNPs), insertions/deletions (indels), methylation sites, gene quantification, and Copy Number Variation (CNV), can be detected using mass spectrometry.

In addition, some nucleic acid detection methods developed based on MALDI-TOF MS, such as hME and iPLEX methods by Agena, USA, GOOD assay method by Bruker, Germany, and RFMP method by GeneMatrix, Korea. In order to improve the resolution of mass spectrometers, the detection of target sites tends to detect oligonucleotide fragments with smaller molecular weights, for example, RFMP method detects oligonucleotide fragments of about 2000-4000 Da by restriction enzyme cleavage of multiplex PCR products containing Single Nucleotide Polymorphism (SNP) sites, and GOOD assay method detects oligonucleotide fragments containing SNP sites by Phosphodiesterase (PDE) cleavage into small fragments of about 1000-2000 Da. However, the above methods inevitably have problems of complicated operation, long time consumption, and the like.

Disclosure of Invention

Based on the defects or shortcomings of the technology, one principle of the invention is as follows: the method for rapidly detecting the mRNA capping efficiency by using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is provided for the first time, and can be used for efficiently identifying and analyzing mRNA synthesized in vitro, including uncapped mRNA and capped mRNA, and relatively quantifying the capping efficiency of the mRNA.

The second principle of the invention is that in order to solve the problem of spectrogram shift in mass spectrum detection, the invention adds an internal standard in the detection process to further calibrate the molecular weight of the target substance to be detected.

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

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

the mRNA standard sample composition comprises:

the internal standard has nucleic acid sequence with molecular weight of 8214.4Da and SEQ ID NO 1 of 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'.

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

In another embodiment, the chip is a time-of-flight mass spectrometry specific microarray chip made of materials including, but not limited to, stainless steel, diamond, single crystal silicon, quartz crystal.

In other embodiments, wherein the software is the BioExplore software, copyright number is Soft literary Density No. 136879, accession No. 2009SR 10700.

The second purpose of the invention is to provide a method for detecting the capping efficiency of an mRNA sample by using the detection kit, which comprises the following steps:

(1) sample preparation: the samples were diluted to the appropriate concentrations separately, each with the addition of the corresponding final concentration of molecular weight calibration internal standard.

(2) And (3) purification: and (3) purifying the sample obtained in the step (1) to obtain a high-purity sample, and avoiding the influence of impurities such as salt ions on subsequent detection.

(3) Sample application: and (3) spotting the purified substance obtained in the step (2) on a target plate containing a matrix, and directly forming a crystallization mixture by the substance to be detected and the matrix.

(4) Mass spectrometer detection: and (5) putting the well-spotted target plate into a mass spectrometer for detection.

(5) And (3) data analysis: comparing and analyzing the map obtained in the step (4) with a pre-established mass spectrum characteristic peak model through computer software, so as to obtain the capping efficiency of the mRNA sample to be detected; wherein the content of the first and second substances,

the mass spectrum characteristic peak model comprises a characteristic peak 7364.9205m/z corresponding to a target fragment of uncapped mRNA (uncap), a characteristic peak 7645.0256m/z corresponding to a target fragment of capped mRNA (cap0), and a characteristic peak corresponding to a target fragment of capped mRNA (cap 1): 7659.0412 m/z.

In one embodiment, the mRNA capping efficiency of step (5) is calculated as:

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 of the internal standard (SEQ ID NO:1) is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'

In one embodiment, the matrix is a composite matrix containing acidic components 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 the material thereof includes, but is not limited to, stainless steel, diamond, monocrystalline silicon, and quartz crystal.

In another embodiment, the concentration of mRNA is measured in step (1) using a NanoDrop ND-2000 nucleic acid detector.

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

In one embodiment, the software is the BioExplore software developed by the inventors, having copyright number Soft literary registration No. 136879, registration No. 2009SR 10700.

In any of the above embodiments, wherein the mRNA sample is an mRNA vaccine, including neocoronary pneumonia, hepatitis b mRNA vaccine, influenza mRNA vaccine, HPV mRNA vaccine, and the like.

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

Technical effects

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

1. the invention firstly proposes that the time-of-flight mass spectrometry technology is utilized to realize the detection of the mRNA capping efficiency, particularly the mRNA vaccine capping efficiency, and has extremely high biological value.

2. And (3) sensitivity: the invention uses mass spectrum detection and other techniques, so the detection sensitivity is high.

3. Simple and safe: the operation is simple and safe;

4. the data analysis required by the invention is simple, only a spectrogram is required to be observed, and complex bioinformatics analysis is not required.

5. The invention has low cost, does not need fluorescent labeling and reduces the system complexity signal interpretation error caused by adding the fluorescent chemical probe.

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

Drawings

FIG. 1: in the mass spectrum model constructed in example 2, the characteristic peak corresponding to the uncap target fragment (7364.9205Da) is as follows: 7364.9205 m/z.

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

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

FIG. 4: and (3) a sample loading detection mass spectrum result chart of the mRNA vaccine sample N1 to be detected.

FIG. 5: and (3) a sample loading detection mass spectrum result chart of the mRNA vaccine sample N2 to be detected.

FIG. 6: and (3) a sample loading detection mass spectrum result chart of the mRNA vaccine sample N3 to be detected.

FIG. 7: and (3) a sample loading detection mass spectrum result chart of the mRNA vaccine sample N4 to be detected.

Principles and definitions

The invention provides a detection scheme for detecting the characteristic spectrum of mRNA by using a mass spectrometry detection technology so as to determine the capping efficiency of the mRNA.

The principle is as follows:

in the sample preparation step, the corresponding final concentration of molecular weight correction internal standard was added.

In the mass spectrometric detection process, a substance to be detected is purified, spotted on a target sheet containing a matrix, excited by laser in a vacuum environment, and then passes through a flight tube to a detector. The time for different substances to pass through the flight tube is inversely related to their molecular weight, i.e. the higher the molecular weight, the slower the flight speed and the later the time to reach the detector.

The term "internal standard" is used for solving the problem of mass spectrum detection spectrogram shift and calibrating the molecular weight of a target substance to be detected.

The term "purification" refers to a treatment step that serves to reduce the effect of other substances in the system being tested on subsequent reactions. The PCR product of the invention can be purified in two ways: firstly, the impurities are separated and discarded, and secondly, the impurities are inactivated. Wherein, gel cutting purification, purification column chromatography and the like are used for separating impurities through electrophoresis, purification column chromatography and the like, and relatively pure PCR products are recovered, which can be regarded as a first purification mode, and the mode generally consumes time and is complex to operate, particularly when the sample amount is large; alkaline phosphatase acts to degrade (also called "digest") dNTPs so that they do not continue to participate in the PCR or single base extension reaction as substrates for DNA polymerase or single base extension enzyme, thereby not interfering with the subsequent reaction, and can be considered a second mode of purification. It should be noted that ExoI alone does not play a role in purification, and when it is used in combination with alkaline phosphatase, it plays a role in degrading single-stranded DNA (mainly the remaining PCR primers in the PCR product system after completion of the reaction) into dNTPs in advance, and then the dNTPs are further degraded by the alkaline phosphatase. Since the PCR primers are degraded, the final mass spectrometric detection step is not entered, and therefore, if the ExoI exonuclease treatment is added to the planned purification step, PCR primers with protected bases do not need to be used. In addition, since both exonuclease and alkaline phosphatase are inactivated by high temperature before the single base extension step, it does not degrade the single-stranded extension primer, ddNTP, etc. added in the single base extension step, thus avoiding influence on the subsequent experiments.

The term "detection window" refers to the range of nucleotide molecular weights that can be used for mass spectrometric detection, and generally refers to the design reference range of primers. Wherein, when designing the extension primer, for different specific sites, according to the sequence characteristics of the DNA region where the sites are located and the genotype of the specific sites, the extension primer and the extension product with different molecular weights can be designed, so as to avoid the interference between the different extension primers and the products due to the proximity of the molecular weights, thereby realizing the detection of a plurality of specific sites in a relatively wide detection window, such as 4000-.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental methods in the following examples, which are not specified under specific conditions, are generally carried out under conventional conditions.

It should be noted that although the embodiments of the present invention present the mass spectrometric data below 50,000Da, the nucleic acid substances between 50,000 and 100,000Da can be detected, and therefore the application of the present invention to the mass spectrometric detection between 50,000 and 100,000Da is also included in the scope of the claimed invention.

Example one in vitro Synthesis of mRNA

The following mRNA fragments are synthesized in vitro by known methods known in the art, for example, molecular cloning guidelines (fourth edition) or the well-documented guidelines for molecular biology experiments (fifth edition), or by commercial companies.

TABLE 1mRNA fragment information Table

Example II method for detecting mRNA capping efficiency by constructing mass spectrum characteristic peak model

The nucleic acid sequence used as an internal standard was synthesized by Shanghai Czeri bioengineering, Inc., with a molecular weight of 8214.4Da and a SEQ ID NO of 5'-CTTGTAAGTTCATTACCTGTATAATTC-3' at 1.

The method for detecting mRNA capping efficiency by using the mRNA fragments and the internal standard comprises the following specific operation methods:

1. sample preparation

mRNA samples were diluted individually to the appropriate concentrations and then each was added with the corresponding final concentration of molecular weight correction internal standard.

2. Purification of

To each tube of mRNA samples, 15mg of resin was added and mixed by inversion for 5 minutes.

3. Spotting is carried out

Using a micropipette, 0.5ul of the purified product was pipetted and spotted onto the target.

4. On-machine detection and result interpretation

The purified product was detected by clinical time of flight mass spectrometry Clin-TOF-ii (MALDI-TOF MS manufactured by new-business biotechnology limited).

5. Interpretation of results

The Clin-TOF type time-of-flight mass spectrometer developed by the inventor is used for detecting the spotted target and judging the result.

6. The result of the detection

And (4) spotting the mRNA samples onto the same chip, and carrying out nucleic acid mass spectrometry detection.

The results of characteristic peaks (m/z) represented by the molecular weights of the target fragments corresponding to the positive targets are shown in FIGS. 1 to 3.

FIG. 1 shows the characteristic peaks corresponding to the target fragment (7364.9205Da) of uncapped mRNA (uncap): 7364.9205 m/z;

FIG. 2 shows the characteristic peaks corresponding to the target fragment (7645.0256Da) of the capped mRNA (cap 0): 7645.0256 m/z;

FIG. 3 is a characteristic peak corresponding to the target fragment (7659.0412Da) of the capped mRNA (cap 1): 7659.0412 m/z;

in addition, all groups had no significant miscellaneous peaks and the baseline was smooth;

no hybrid peak or <2 hybrid peaks in the spectrum in the range of 3000-10000 Da.

Therefore, a mass spectrum characteristic peak model of the mRNA sample detected by the mass spectrum is constructed.

EXAMPLE III detection of mRNA samples Using Mass Spectrometry model

According to the method of the second embodiment, 4 mRNA vaccine samples are subjected to sample treatment and purification, and then the spotted target piece is detected and judged by a Clin-TOF time-of-flight mass spectrometer.

Cap1 capping rate was calculated as Area (Cap1)/Area (Cap0+ Cap1+ uncapped), Cap0 capping rate was calculated as Area (Cap0)/Area (Cap0+ Cap1+ uncapped), and the results are shown in fig. 4-7: the mass spectrometry results are shown in FIGS. 4-7:

sample N1, the detection peaks (m/z) were: 7364.9205, 7645.0256, 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, cap1 are: 7364.9205, 7645.0256, 7659.0412, according to the mass spectrometric model established in example 2, thus determining the test results as: uncap, cap0, cap 1; cap1 was capped at 54.5%.

Sample N2, the detection peaks (m/z) were: 7364.9205, 7645.0256, 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, cap1 are: 7364.9205, 7645.0256, 7659.0412, according to the mass spectrometric model established in example 2, thus determining the test results as: uncap, cap0, cap 1; cap1 capping rate was 73%.

Sample N3, the detection peaks (m/z) were: 7364.9205, 7645.0256, 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, cap1 are: 7364.9205, 7645.0256, 7659.0412, according to the mass spectrometric model established in example 2, thus determining the test results as: uncap, cap0, cap 1; cap1 capping rate was 74.5%.

Sample N4, the detection peaks (m/z) were: 7364.9205, 7645.0256, 7659.0412, wherein the characteristic molecular weights (Da) of uncap, cap0, cap1 are: 7364.9205, 7645.0256, 7659.0412, according to the mass spectrometric model established in example 2, thus determining the test results as: uncap, cap0, cap 1; cap1 capping rate was 75.8%.

The peak areas were combined and finally shown in the following table.

Therefore, the target characteristic peak base lines in the images 4-7 are smooth, the signal-to-noise ratio is high, and the separation degree between adjacent signal peaks is high, so that the mass spectrometry can detect the uncap, cap0 and cap1mRNA characteristic peaks simultaneously, can obtain the detection result quickly and intuitively, and avoids the defects of objective enzyme digestion and the like and low resolution. The capping efficiency of the mRNA vaccine is high, and the biological value is high.

Sequence listing

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Claims

1. A mass spectrometric detection kit for detecting in vitro synthesized mRNA fragments by MALDI TOF-MS comprises an mRNA standard sample composition, an internal standard, a purification reagent, a mass spectrometric matrix, a sample application chip and detection software, wherein,

the mRNA standard sample composition comprises:

。

2. the mass spectrometry detection kit of claim 1, wherein the internal standard has a nucleic acid sequence of 5'-CTTGTAAGTTCATTACCTGTATAATTC-3' and a molecular weight of 8214.4 Da.

3. The mass spectrometry detection kit of claim 1 or 2, wherein the matrix is a composite matrix containing acidic components including formic acid, acetic acid and citric acid.

4. The mass spectrometry detection kit of claim 3, wherein the chip is a time-of-flight mass spectrometry microarray chip, and the material of the chip comprises stainless steel, diamond, monocrystalline silicon and quartz crystal.

5. The mass spectrometry detection kit of claim 4, wherein the software is BioExplore software having copyright number Soft literary registration No. 136879, accession No. 2009SR 10700.

6. Use of the test kit according to any of claims 1 to 5 for testing the quality of mRNA therapeutic applications, the quality of mRNA vaccines.

7. The use of claim 6, wherein the mRNA sample is an mRNA vaccine.

8. The use of claim 7, wherein the mRNA vaccines include neocoronary pneumonia, hepatitis B mRNA vaccines, influenza mRNA vaccines, HPV mRNA vaccines and the like.