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WO2023115495A1 - Procédé de mesure de l'efficacité du coiffage des arnm par spectrométrie de masse - Google Patents

Procédé de mesure de l'efficacité du coiffage des arnm par spectrométrie de masse Download PDF

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Publication number
WO2023115495A1
WO2023115495A1 PCT/CN2021/141002 CN2021141002W WO2023115495A1 WO 2023115495 A1 WO2023115495 A1 WO 2023115495A1 CN 2021141002 W CN2021141002 W CN 2021141002W WO 2023115495 A1 WO2023115495 A1 WO 2023115495A1
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Prior art keywords
mrna
mass spectrometry
detection
cap1
cap0
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Chinese (zh)
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张海燕
王宇涵
马庆伟
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Beijing Yixin Bochuang Biological Technology Co Ltd
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Beijing Yixin Bochuang Biological Technology Co Ltd
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Priority to PCT/CN2021/141002 priority Critical patent/WO2023115495A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • 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.
  • the processing modification of cellular messenger RNA includes: forming a cap structure at the 5' end, adding PolyA to the 3' end, splicing to remove introns and methylation.
  • RNA triphosphatase RNA triphosphatase
  • mRNA guanylyltransferase mRNA guanylyltransferase
  • mRNA (guanine-7) methyltransferase
  • mRNA (nucleoside-2') methyltransferase mRNA triphosphatase
  • 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.
  • 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.
  • 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 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.
  • the cap species of the final purified mRNA product was determined by HPLC/MS method.
  • 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.
  • RNA spot blot method 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.
  • ion drift tube ion dirt tube
  • 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.
  • 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 .
  • 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.
  • various companies tend to detect oligonucleotide fragments with smaller molecular weights when detecting target sites.
  • 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.
  • PDE phosphodiesterase
  • 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.
  • MALDI-TOF MS matrix-assisted laser desorption ionization time-of-flight mass spectrometry
  • 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.
  • 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'.
  • the matrix is a composite matrix comprising an acidic component including but not limited to formic acid, acetic acid and citric acid.
  • 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.
  • 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:
  • Sample preparation Dilute the samples to appropriate concentrations, and add molecular weight calibration internal standards of corresponding final concentrations.
  • Step (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.
  • 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 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).
  • nucleic acid sequence (SEQ ID NO: 1) of the internal standard is 5'-CTTGTAAGTTCATTACCTGTATAATTC-3'
  • the matrix is a composite matrix comprising an acidic component including, but not limited to, formic acid, acetic acid, and citric acid.
  • 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.
  • NanoDrop ND-2000 nucleic acid detector uses NanoDrop ND-2000 nucleic acid detector to measure the concentration of mRNA in step (1).
  • the mass spectrometer is a MALDI TOF MS mass spectrometer.
  • 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.
  • the mRNA sample is an mRNA vaccine, including new coronary pneumonia, hepatitis B mRNA vaccine, influenza mRNA vaccine, HPV mRNA vaccine, etc.
  • 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.
  • the present invention has the following advantages:
  • 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.
  • Sensitivity the present invention uses techniques such as mass spectrometry detection, so its detection sensitivity is very high.
  • the data analysis required by the present invention is simple, only need to observe the spectrogram, and no complex bioinformatics analysis is required.
  • 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.
  • 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.
  • 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 molecular weight calibration internal standard was added at the corresponding final concentration.
  • 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.
  • purification refers to the processing steps used to reduce the influence of other substances in the system to be tested on subsequent reactions.
  • 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.
  • alkaline phosphatase 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.
  • 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.
  • 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.
  • Embodiment one synthesize mRNA in vitro
  • 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.
  • 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'.
  • 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.
  • 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;
  • 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.
  • the detection peaks 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.
  • Da characteristic molecular weights
  • the detection peaks 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%.
  • the detection peaks 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%.
  • the detection peaks 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 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.

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Abstract

Un procédé de mesure rapide d'une efficacité de coiffage d'ARNm à l'aide d'une technologie de spectrométrie de masse à temps de vol, en particulier pour un ARNm synthétisé in vitro. Le procédé comprend la mise à disposition d'un ARNm non coiffé et d'un ARNm coiffé, et l'utilisation de la technologie de spectrométrie de masse à temps de vol pour effectuer une quantification relative de l'efficacité de coiffage de l'ARNm. C'est la première fois que l'on propose d'utiliser la technologie de spectrométrie de masse à temps de vol pour mettre en œuvre un procédé de mesure de l'efficacité de coiffage d'un ARNm. Le fonctionnement est rapide et simple, et une mesure pour des échantillons à l'état de traces peut également être effectuée au moyen de la technologie de spectrométrie de masse, afin qu'une sensibilité de mesure relativement élevée puisse être obtenue.
PCT/CN2021/141002 2021-12-23 2021-12-23 Procédé de mesure de l'efficacité du coiffage des arnm par spectrométrie de masse Ceased WO2023115495A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025011385A1 (fr) * 2023-07-12 2025-01-16 上海奥浦迈生物科技股份有限公司 Sonde pour mesurer le taux de coiffage d'un échantillon d'arnm, kit et son procédé d'utilisation

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WO2014144039A1 (fr) * 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Caractérisation de molécules d'arnm
CN105051213A (zh) * 2013-03-14 2015-11-11 夏尔人类遗传性治疗公司 信使rna加帽效率的定量评估
CN108366604A (zh) * 2015-09-21 2018-08-03 垂林克生物技术公司 用于合成5’-加帽rna的组合物和方法
CN109562153A (zh) * 2016-08-07 2019-04-02 诺华股份有限公司 mRNA介导的免疫方法
CN112626177A (zh) * 2019-10-09 2021-04-09 华中科技大学 一种快速定量检测rna加帽效率的方法
CN112714795A (zh) * 2019-08-23 2021-04-27 新英格兰生物实验室公司 酶促rna加帽方法
CN113166737A (zh) * 2018-10-04 2021-07-23 新英格兰生物实验室公司 提高转录的rna的加帽效率的方法和组合物

Patent Citations (7)

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Publication number Priority date Publication date Assignee Title
CN105051213A (zh) * 2013-03-14 2015-11-11 夏尔人类遗传性治疗公司 信使rna加帽效率的定量评估
WO2014144039A1 (fr) * 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Caractérisation de molécules d'arnm
CN108366604A (zh) * 2015-09-21 2018-08-03 垂林克生物技术公司 用于合成5’-加帽rna的组合物和方法
CN109562153A (zh) * 2016-08-07 2019-04-02 诺华股份有限公司 mRNA介导的免疫方法
CN113166737A (zh) * 2018-10-04 2021-07-23 新英格兰生物实验室公司 提高转录的rna的加帽效率的方法和组合物
CN112714795A (zh) * 2019-08-23 2021-04-27 新英格兰生物实验室公司 酶促rna加帽方法
CN112626177A (zh) * 2019-10-09 2021-04-09 华中科技大学 一种快速定量检测rna加帽效率的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025011385A1 (fr) * 2023-07-12 2025-01-16 上海奥浦迈生物科技股份有限公司 Sonde pour mesurer le taux de coiffage d'un échantillon d'arnm, kit et son procédé d'utilisation

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