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WO2025050466A1 - Désoxyribozyme et procédé de détection de l'efficacité de coiffage d'arnm - Google Patents

Désoxyribozyme et procédé de détection de l'efficacité de coiffage d'arnm Download PDF

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Publication number
WO2025050466A1
WO2025050466A1 PCT/CN2023/124253 CN2023124253W WO2025050466A1 WO 2025050466 A1 WO2025050466 A1 WO 2025050466A1 CN 2023124253 W CN2023124253 W CN 2023124253W WO 2025050466 A1 WO2025050466 A1 WO 2025050466A1
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dnazyme
mrna
rna
deoxyribozyme
capping
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Chinese (zh)
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孙秀莲
赵晟
孙叶
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Nanjing Hongming Biotechnology Co Ltd
Ubrigene Suzhou Biosciences Co Ltd
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Nanjing Hongming Biotechnology Co Ltd
Ubrigene Suzhou Biosciences Co Ltd
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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • 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

Definitions

  • the invention relates to the field of biotechnology, and in particular to a deoxyribozyme and a method for detecting mRNA capping rate.
  • Effective mRNA therapy requires the effective delivery of mRNA to the patient and the effective synthesis of the corresponding protein in vivo.
  • appropriate capping is usually required at the 5' end of the construct to prevent mRNA degradation and promote protein translation. Therefore, effective detection of capping efficiency is crucial.
  • the earliest capping detection method was to label the cap structure with radioactive isotopes and then perform qualitative or quantitative analysis.
  • the radioactive labeling method has high sensitivity, but requires the use of isotopes, has certain safety hazards and isotope contamination risks, requires special protection measures, and is not easy to promote. Therefore, researchers have also begun to develop detection methods that do not rely on isotope labeling.
  • CN114894916A discloses a method for quantitatively detecting RNA capping rate, wherein a deoxyribozyme is designed according to the RNA sequence to be detected, the deoxyribozyme is used to cut the RNA molecule to be detected, the molecular weight and relative proportion of the RNA to be detected after cutting are detected, and the capping efficiency is determined.
  • Deoxyribozyme is a single-stranded DNA molecule with catalytic activity.
  • a well-known deoxyribozyme family is the "10-23" deoxyribozyme (DNAzyme (10-23)), which specifically recognizes, complementarily binds and cuts the target sequence of RNA molecules through catalytic activity, thereby losing or reducing the activity of the cut RNA molecules.
  • DNAzyme (10-23) has a core catalytic domain of 15 nucleotides, flanked by two substrate binding domains (I and II). According to the Watson-Crick rule, 10-23 DNAzyme binds to the RNA substrate through base pairing via substrate binding domains I and II. The deoxyribozyme is used to cut the RNA to be tested, and its molecular weight is measured to determine whether the capping is successful, and the RNA capping efficiency can be quantitatively determined by calculating the abundance of different molecular weights.
  • This method has high sensitivity and does not require the use of radioactive labels, so there is no need to worry about radioactive contamination. However, this existing method does not further optimize and screen DNAzyme (10-23), and low cutting efficiency is often encountered in actual use.
  • RNaseH RNA-based ribozymes
  • Ribozyme RNA-based ribozymes
  • Ribozyme and protein nucleases such as RNaseH
  • RNaseH protein nucleases
  • Ribozyme is prone to degradation, resulting in the production of invalid RNA small fragment impurities, and RNaseH often contains other non-specific nuclease residues during the preparation process.
  • this type of mass spectrometry-based method requires the use of expensive equipment such as eHPLC-MS or LC-MS to detect the cut RNA, and the cost of use and maintenance is very high. Therefore, how to design a detection method that is simpler to operate and lower in cost is still a problem that needs to be solved in this field.
  • the present invention provides a method for detecting RNA capping efficiency, comprising the following steps:
  • the lengths of the two binding arms of the deoxyribozyme DNAzyme (10-23) are not less than 11 nt, and preferably, can be independently selected from 11, 12, 13, 14 and 15 nt.
  • the RNA is mRNA.
  • the RNA fragment is 26 nt in length.
  • whether the RNA fragment has the cap structure is determined by molecular weight differences.
  • the cap structure is located at the 5' end of the RNA.
  • the cap structure is selected from Cap O, Cap I and Cap II type structures.
  • the present invention provides a deoxyribozyme DNAzyme (10-23), wherein the length of both binding arms is not less than 11 nt, preferably, can be independently selected from 11, 12, 13, 14 and 15 nt, more preferably, includes the sequence shown in any one of SEQ ID NO: 7, 8, 9, 10 and 11.
  • the present invention provides an mRNA capping efficiency detection kit, which comprises the above-mentioned deoxyribozyme DNAzyme (10-23).
  • Figure 1 Schematic diagram of the principle of 10-23 DNAzyme cleaving mRNA
  • FIG2 Capillary electrophoresis profile after cleavage of different cleavage sites in the 5′-UTR region of mRNA by DNAzyme (10-23) x-35;
  • Figure 3 Capillary electrophoresis profiles of DNAzyme (10-23)26-y with different arm lengths after cleavage of the 5'-UTR region of mRNA;
  • Figure 4 Capillary electrophoresis detection of mRNA cleavage using DNAzyme (10-23) 26-43 after different reaction times of mRNA enzymatic capping;
  • Figure 5 Capillary electrophoresis detection of mRNA cleavage using DNAzyme (10-23)26-43 after transcription and co-capping with mRNA cap analogs at different concentrations.
  • the term “about” or “approximately” is used to indicate that the value includes the error caused by the instruments and methods used in determining the value. As used herein, when used in reference to a specific listed value, the term “about” means that the value can vary by no more than 1% from the listed value. For example, the expression “about 100” includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
  • “Deoxyribozymes” or “deoxyribonucleases” or “DNAzymes” or similar expressions are DNA sequences that have catalytic activity. They consist of a catalytic core of approximately 15 nucleotides, flanked by short binding arms located to the left and right of the catalytic core. While the sequence of the catalytic core is fixed, the binding arms can be modified to specifically match almost any RNA target sequence.
  • Non-limiting examples of DNAzymes can be found in Usman et al., U.S. Pat. No. 6,159,714, the entire contents of which are incorporated herein by reference; Chartrand et al., 1995, NAR 23, 4092; Breaker et al., 1995, Chem. Bio.
  • the "10-23" DNAzyme motif is a special type of DNAzyme that was evolved using in vitro selection, as generally described in Joyce et al., U.S. Pat. No. 5,777,37. U.S. Patent No. 5,807,718 and Santoro et al., supra. Additional DNAzyme motifs may be selected using techniques similar to those described in these references and are therefore within the scope of the present invention.
  • Bases are the basic building blocks for the synthesis of nucleosides, nucleotides and nucleic acids.
  • A adenine
  • G guanine
  • C cytosine
  • T thymine
  • U uracil
  • the bases A, G, C and T are present in DNA, while A, G, C and U are present in RNA.
  • Each base pair contains a purine and a pyrimidine: A and T are paired through 2 hydrogen bonds, C and G are paired or Z is paired with P or S is paired with B through 3 hydrogen bonds.
  • “nt” refers to the number of nucleotides, and can also refer to the number of bases corresponding to the nucleotide.
  • nucleic acid molecules of the present invention refers to the ability of a nucleic acid to form hydrogen bonds with another RNA sequence by traditional Watson-Crick or other non-traditional types.
  • nucleic acid molecules of the present invention the free energy of binding of a nucleic acid molecule to its target sequence or complementary sequence is sufficient to allow the relevant function of the nucleic acid to be performed, such as enzymatic nucleic acid cleavage, ligation, isomerization, phosphorylation or dephosphorylation.
  • Complete complementarity means that all consecutive residues of a nucleic acid sequence will form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence.
  • RNA fragments herein refer to fragments produced after RNA (especially mRNA) is cleaved by DNAzyme DNAzyme (10-23), especially fragments related to the capping efficiency of interest.
  • the RNA fragment refers to the 5' end fragment including the 5' end base.
  • the capping is incomplete (the capping efficiency is less than 100%)
  • the RNA fragments produced after the treatment of DNAzyme DNAzyme (10-23) in the same sample may have a cap structure or not, that is, an RNA fragment with a cap structure and a corresponding RNA fragment without a cap structure are produced (the two are identical in nucleotide sequence, the difference is whether they have a cap structure).
  • “Binding arm”, “substrate binding arm”, “substrate binding domain”, “substrate binding region”, “homologous arm” or similar descriptions refer to a partial region of a DNAzyme that is capable of binding to a portion of its substrate or reporter gene through complementarity. Preferably, this complementarity is 100%, but it can be lower if necessary. For example, as few as 10 of the 14 bases can be base paired (see, for example, Werner and Uhlenbeck, 1995, Nucleic Acids Research, 23, 2092-2096; Hammann et al., 1999, Antisense and Nucleic Acid Drug Dev., 9, 25-31).
  • the sequence of these arms contained within the DNAzyme is intended to bind the DNAzyme and the substrate, such as RNA, together through complementary base pairing interactions.
  • the DNAzyme of the present invention may have binding arms that are continuous or discontinuous and may have different lengths.
  • the length of the binding arm is preferably greater than or equal to six nucleotides and is of sufficient length to stably interact with the target substrate.
  • the lengths of the left and right binding arms of the DNAzyme are either symmetric (i.e., each binding arm has the same length; for example, five and five nucleotides, or six and six nucleotides, or seven and seven nucleotides long) or asymmetric (i.e., the binding arms are different lengths; for example, six and three nucleotides; three and six nucleotides long; four and five nucleotides long; four and six nucleotides long; four and seven nucleotides long).
  • Cap structure refers to a chemical modification of either end of an oligonucleotide (see, e.g., Wincott et al., WO 97/26270, incorporated herein by reference). These terminal modifications can protect the nucleic acid molecule from exonuclease degradation and facilitate delivery and/or localization within a cell.
  • the cap may be present at the 5'-end (5'-cap) or the 3'-end (3'-cap) or may be present at both ends.
  • the 5'-cap is selected from the group comprising an inverted abasic residue (part), a 4',5'-methylene nucleotide, 1-( ⁇ -D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotide; ⁇ -nucleotide; modified base nucleotide; dithiophosphate bond; threofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide portion; 3'-3'-inverted abasic portion; 3'-2'-inverted nucleotide portion; 3'-2'-inverted nucleotide portion
  • the 3'-cap is selected from: 4',5'-methylene nucleotides; 1-( ⁇ -D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; 5'-aminoalkyl phosphates; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; L-nucleotides; ⁇ -nucleotides; modified base nucleotides; dithiophosphates; threofuranosyl nucleotides; No 3',4'-seco nucleotides; 3,4-dihydroxybutyl nucleotides;
  • the cap structure is 7-methylguanosine triphosphate m 7 Gppp (Cap O type); in other examples, the cap structure includes 7-methylguanosine triphosphate m 7 Gppp and 2'-OH methylation of the first nucleoside base or even 2'-OH methylation of the second nucleoside base (referred to as Cap I type and Cap II type, respectively).
  • Exemplary mRNA capping methods include enzymatic capping or cap analog transcriptional co-capping, etc.
  • the present invention utilizes screening and improvement of deoxyribozyme DNAzyme (10-23) to improve its efficiency in cutting mRNA molecules to be detected, and generates enough RNA capping fragment products or uncapping fragments after cutting to be clearly separated and detected by capillary electrophoresis, and quantified respectively, so that the mRNA capping rate can be quantitatively detected by a capillary electrophoresis instrument.
  • the method is simpler, has lower cost, and detection equipment is easier to obtain.
  • the present invention provides a method for detecting mRNA capping efficiency, the method comprising: designing a deoxyribozyme DNAzyme (10-23) x-y according to the RNA sequence to be detected, using the deoxyribozyme to cut the RNA molecule to be detected, and the cut detection RNA molecule is divided into a capped short fragment and an uncapped short fragment.
  • Capillary electrophoresis is used to detect the molecular weight of the short fragment cut from the RNA to be detected and the relative ratio of the two short fragments, and then the capping efficiency is determined, wherein x represents the number of nucleotides of the mRNA short fragment generated after cutting, y represents the number of nucleotides of the deoxyribozyme itself, and y is an integer greater than or equal to 37.
  • the RNA to be detected is relatively long in total length, has a large number of nucleotides, and has a large molecular weight.
  • the increase in molecular weight is negligible relative to the initial molecular weight and is difficult to distinguish using an instrument. Therefore, the capped end or the planned capped end is cut out to become a short fragment with a smaller molecular weight. The difference between the capped short fragment and the uncapped short fragment is increased, which is conducive to distinguishing and reading the capped or uncapped short fragments.
  • the present application uses DNAzyme (10-23) xy to cut the RNA to be detected to obtain a sufficient amount of capped or uncapped short fragments, and then uses the high sensitivity of capillary electrophoresis and the ability to perform high-precision single-base resolution to detect the short fragments cut from the RNA to be detected, and after performing single-base difference separation and detection on the displayed peaks of the capped or uncapped short fragments, calculates the peak integrated areas of the two short fragments, and then determines the capping efficiency, wherein the x represents the mRNA short fragment generated after cutting.
  • the number of nucleotides, y represents the number of nucleotides in the DNAzyme itself, and y is an integer greater than or equal to 37, that is, the length of both binding arms is not less than 11 nt, preferably, it can be any length from 11 to 20 nt.
  • a DNAzyme (10-23) that can distinguish between capped or uncapped short fragments by capillary electrophoresis can be designed.
  • DNAzyme is a single-stranded DNA fragment with catalytic function. It has high catalytic activity and structural recognition ability. DNAzyme has the following advantages: (1) As a DNA molecule, it is more stable than ribozyme and is not easily destroyed; (2) The molecular weight is relatively small, easy to synthesize and modify artificially, and the cost is much lower than protein nucleases (such as RNaseH) and ribozyme; (3) The pairing accuracy of its binding arm and substrate is high. In addition to the core catalytic sequence, other recognition base sequence parts can be changed according to the substrate base sequence. The target cleavage sites that can be screened are more and more flexible than the cleavage scheme of RNaseH. It is superior to RNaseH and ribozyme in terms of the comprehensive performance of RNA site-specific cleavage in mRNA capping test.
  • the cleavage position of the mRNA to be detected is selected at the 5'-capped end of the mRNA. In some embodiments, the cleavage position of the mRNA to be detected is selected within 100 bases of the 5'-capped end, such as the 5' end sequence of the cleavage range in the example of the present application is shown in SEQ ID NO: 1.
  • This range generally belongs to the 100 base UTR region of the 5'-end of the mRNA to be detected.
  • the mRNA prepared by in vitro synthesis often uses the 5'-UTR and 3'-UTR of the fixed sequence that has been verified to help stably express proteins, so the UTR regions of these mRNAs are often consistent. Therefore, the deoxyribozyme provided in this application has good cleavage universality for mRNAs using the same UTR region. For mRNAs with other different UTRs, efficient and available DNAzymes can also be quickly obtained according to the design principles and screening strategies provided in this application.
  • x is selected from any integer between 15 and 51. Specifically, x can be 15, 26, 47, or 51.
  • y is selected from any integer between 35 and 45. Specifically, y can be 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45.
  • the two arms of the DNAzyme are of equal length, and y can be 35, 37, 39, 41, 43, or 45; in some embodiments, the two arms of the DNAzyme are of unequal length.
  • the capping method of the mRNA to be detected includes enzymatic capping or cap analog transcriptional co-capping.
  • DNase I deoxyribonuclease 1
  • Tris-HCl Tris(hydroxymethyl)aminomethane
  • Invitrogen TM Tris(hydroxymethyl)aminomethane
  • LiCl precipitation solution (7.5 M), Invitrogen TM ;
  • High-resolution card holder (S1) product number C105202, Guangding Biotechnology (Jiangsu) Co., Ltd.;
  • This example uses in vitro transcription to synthesize uncapped mRNA. After linearizing the transcription template plasmid using restriction endonucleases, in vitro transcription of RNA is initiated by T7 RNA polymerase to obtain uncapped mRNA.
  • the reaction system is shown in Table 1. Transcription was performed using T7 High Yield RNA Transcription Kit, Vazyme (Nanjing Novogene Biotechnology Co., Ltd.), catalog number: DD4201, and the specific operation is referred to the instruction manual.
  • Example 2 Preparation of capped mRNA by two-step enzymatic capping method
  • the capped mRNA was prepared by a two-step enzymatic capping method.
  • Vaccinia Capping Enzyme (Vazyme, Catalog No.: 10615) and mRNA Cap 2'-O-Methyltransferase (Vazyme, Catalog No.: DD4110-PC-01) were used to co-cap the mRNA to obtain the capped mRNA.
  • This example uses the co-transcription method to prepare capped mRNA.
  • the in vitro transcription of RNA is initiated by T7 RNA polymerase.
  • T7 RNA polymerase According to the co-transcription reaction system in Table 3, additional cap analogs are added to obtain co-transcription capped mRNA.
  • T7 in vitro transcription reagent Cap GAG
  • HBP001510 for capping.
  • the final concentrations of Cap analog in the system were configured as 1, 2, 4, and 8 mM respectively.
  • DNAzyme (10-23) 26-43 was used to cleave the mRNA obtained in Example 3 after co-transcriptional capping with different cap analog concentrations.
  • the cleavage steps were referred to Example 4, and the capillary electrophoresis pattern was used for detection.
  • the capping efficiency was generally 50% when the AG cap analog was used for co-transcriptional capping.
  • the capped mRNA has an increased molecular weight of a Cap1 structure. Under the condition of the same mass of uncapped mRNA substrate, the capping efficiency increases with the increase of the cap analog concentration (0mM, 1mM, 2mM, 4mM, 8mM).

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Abstract

L'invention concerne un procédé de détection de l'efficacité de coiffage d'ARN. Le procédé comprend les étapes suivantes consistant à : 1) traiter l'ARN au moyen de la désoxyribozyme DNAzyme (10-23) pour générer un fragment d'ARN ayant une structure de coiffe et un fragment d'ARN correspondant sans structure de coiffe ; et 2) détecter une relation proportionnelle entre le fragment d'ARN avec la structure de coiffe et le fragment d'ARN correspondant au moyen d'une électrophorèse capillaire, de façon à déterminer l'efficacité de coiffage, les longueurs de deux bras de liaison du désoxyribozyme DNAzyme (10-23) n'étant pas inférieures à 11 nt. L'invention concerne le désoxyribozyme DNAzyme (10-23), et un kit de détection d'efficacité de coiffage d'ARN comprenant le désoxyribozyme DNAzyme (10-23).
PCT/CN2023/124253 2023-09-09 2023-10-12 Désoxyribozyme et procédé de détection de l'efficacité de coiffage d'arnm Pending WO2025050466A1 (fr)

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CN202311159046.3A CN116875658B (zh) 2023-09-09 2023-09-09 一种脱氧核酶及检测mRNA加帽率的方法
CN202311159046.3 2023-09-09

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CN117467742B (zh) * 2023-12-28 2024-04-05 南京鸿明生物科技有限公司 一种检测mRNA的polyA尾长的方法
WO2025226048A1 (fr) * 2024-04-24 2025-10-30 Green Cross Corporation Procédé d'évaluation de l'efficacité de coiffage en 5'
CN118291604B (zh) * 2024-06-06 2024-09-20 南京鸿明生物科技有限公司 一种用于小片段rna检测的标准品及制备方法

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US20160304938A1 (en) * 2013-12-30 2016-10-20 Curevac Ag Methods for rna analysis
US20170241971A1 (en) * 2014-04-28 2017-08-24 Juewen Liu Phosphorothioate dnazyme complexes and use thereof
CN114894916A (zh) * 2022-04-02 2022-08-12 翌圣生物科技(上海)股份有限公司 一种检测rna加帽效率的方法
WO2023282245A1 (fr) * 2021-07-05 2023-01-12 国立研究開発法人科学技術振興機構 Procédé de purification de nucléotides, dispositif de purification de nucléotides, réactif hydrophobe et substrat hydrophobe
CN115678968A (zh) * 2021-07-22 2023-02-03 仁景(苏州)生物科技有限公司 一种检测mRNA加帽效率的方法
CN116640833A (zh) * 2023-06-01 2023-08-25 中国医学科学院医学生物学研究所 一种利用毛细管电泳技术自动化批量检测mRNA加帽率的方法

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CN1382211A (zh) * 1999-08-04 2002-11-27 强生研究有限公司 用脱氧核酶治疗炎症性或恶性疾病
US20160304938A1 (en) * 2013-12-30 2016-10-20 Curevac Ag Methods for rna analysis
US20170241971A1 (en) * 2014-04-28 2017-08-24 Juewen Liu Phosphorothioate dnazyme complexes and use thereof
WO2023282245A1 (fr) * 2021-07-05 2023-01-12 国立研究開発法人科学技術振興機構 Procédé de purification de nucléotides, dispositif de purification de nucléotides, réactif hydrophobe et substrat hydrophobe
CN115678968A (zh) * 2021-07-22 2023-02-03 仁景(苏州)生物科技有限公司 一种检测mRNA加帽效率的方法
CN114894916A (zh) * 2022-04-02 2022-08-12 翌圣生物科技(上海)股份有限公司 一种检测rna加帽效率的方法
CN116640833A (zh) * 2023-06-01 2023-08-25 中国医学科学院医学生物学研究所 一种利用毛细管电泳技术自动化批量检测mRNA加帽率的方法

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