CN114894916A

CN114894916A - Method for detecting RNA capping efficiency

Info

Publication number: CN114894916A
Application number: CN202210351807.4A
Authority: CN
Inventors: 滕以刚; 袁灿灿; 王小敏; 郭帅; 王雪琪
Original assignee: Yisheng Biotechnology Shanghai Co ltd
Current assignee: Yisheng Biotechnology Shanghai Co ltd
Priority date: 2022-04-02
Filing date: 2022-04-02
Publication date: 2022-08-12
Also published as: WO2023185947A1

Abstract

The invention discloses a method for detecting RNA capping efficiency, which comprises the following steps: designing deoxyribozymes according to RNA sequences to be detected, cutting RNA molecules to be detected by using the deoxyribozymes, detecting the molecular weight and relative proportion of the cut RNA to be detected, and determining the capping efficiency. The invention creatively utilizes the DNAzyme to cut the RNA to be detected and determines the capping efficiency, realizes the quantitative detection of the RNA capping efficiency, can avoid radioactive pollution, and has simple operation and low cost.

Description

Method for detecting RNA capping efficiency

Technical Field

The invention belongs to the technical field of biology, and relates to a method for detecting RNA capping efficiency.

Background

Ribonucleic acid (RNA) is an important macromolecule in the transmission of all biogenetic information, and plays a variety of regulatory roles in organisms, and RNA research has become one of the most popular fields in biological research in recent years. For example, mRNA-based therapies, which are more accurate in practical applications and can be used for personalized therapy, can avoid complex production problems compared to the production of therapeutic proteins in patients; mRNA as a drug also has much fewer side effects than gene therapy, which is a method of altering DNA to produce a permanent irreversible effect. Therefore, mRNA treatment has become one of the hottest drug research directions, is expected to be an effective method for treating various diseases, and has good development prospect.

Effective mRNA therapy requires efficient delivery of mRNA to a patient and efficient synthesis of the corresponding protein in vivo. To optimize mRNA delivery and in vivo protein production, proper capping is typically required at the 5' end of the construct to prevent mRNA degradation while promoting protein translation. Therefore, efficient detection of capping efficiency is of paramount importance, however, currently available capping efficiency detection methods utilize radioisotope labels, which are only qualitatively detectable and not quantitatively detectable, and are insufficient to assess RNA quality and associated in vivo safety and efficacy, and at the same time researchers have begun to develop detection methods that do not rely on isotope labeling.

CN112626177A discloses a method for rapidly and quantitatively detecting RNA capping efficiency, which comprises the following steps: s1, synthesizing uncapped RNA through in vitro transcription and removing template DNA; s2, capping the RNA; s3, performing monophosphorylation treatment on the RNA obtained in the S1 and S2 steps: dephosphorylating by alkaline phosphatase, purifying RNA, and adding monophosphate to the 5' end of RNA by polynucleotide kinase; s4, removing RNA subjected to monophosphatase treatment by using monophosphatase, and setting a control group which is not treated by the monophosphatase; s5, detecting running glue, quantitatively measuring the brightness of the RNA band treated by the monophosphatase (recorded as N) and the brightness of the RNA band of a control group (recorded as N), and calculating the capping efficiency of the RNA as follows: (N/N). times.100%.

CN105209633A discloses a quantitative evaluation of messenger RNA capping efficiency, which is mainly characterized in that an anti-m 7G specific antibody is combined with a cap structure, a second antibody is combined with an m 7G antibody, and a corresponding signal is measured through ELISA, so that the RNA capping efficiency is quantified.

CN105051213A discloses a quantitative assessment of messenger RNA capping efficiency by first performing selective degradation with nucleases, then separating the capped and uncapped fragments by chromatography, determining the relative amounts of the capped and uncapped fragments, and further quantifying the RNA capping efficiency.

CN201910951836 discloses a method for rapidly and quantitatively detecting RNA capping efficiency, which comprises first performing monophosphorylation treatment on RNA, then removing the RNA after the monophosphorylation treatment with monophosphatase, setting a control group not treated with monophosphatase, and quantitatively determining band brightness (denoted as N) of the RNA treated with monophosphatase and band brightness (denoted as N) of the RNA of the control group by electrophoretic detection, wherein (N/N) × 100% is RNA capping efficiency.

Compared with an isotope method, the method has the advantage of being capable of quantitatively determining RNA capping efficiency, but is still complex and tedious to operate, high in difficulty and difficult to widely popularize and apply.

In conclusion, how to provide a simple, efficient and quantitative method for detecting RNA capping efficiency is one of the problems in the field of mRNA treatment.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the method for detecting the RNA capping efficiency, which can avoid radioactive pollution, has simple operation and low cost and can quantitatively detect the RNA capping efficiency.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for detecting RNA capping efficiency, the method comprising:

designing deoxyribozymes according to RNA sequences to be detected, cutting RNA molecules to be detected by using the deoxyribozymes, detecting the molecular weight and relative proportion of the cut RNA to be detected, and determining the capping efficiency.

In the invention, the deoxyribozyme (DNAzyme) is a single-stranded DNA fragment with a catalytic function, has high catalytic activity and structure recognition capability, and has the following advantages: (1) compared with Ribozyme (Ribozyme), the DNA molecule is more stable and is not easy to damage; (2) the molecular weight is relatively small, the artificial synthesis and modification are easy, and the cost is low; (3) the precision of the method for pairing the substrate with the substrate is high, other base sequences except a catalytic part can be changed according to the base sequence of the substrate, and the RNA cleavage efficiency is higher than that of Ribozyme; (4) because the detection range of the mass spectrum to the length of the mRNA is preferably less than 100nt, the background generated by the cut long-fragment RNA and the uncut RNA is very low, the cut mixture can be directly detected on a computer, the RNA to be detected is cut by using the deoxyribozyme, the capping efficiency is determined, the quantitative detection of the RNA capping efficiency is realized, the radioactive pollution can be avoided, the operation is simple, and the cost is low.

Preferably, the method of detection of the molecular weights and relative proportions comprises HPLC-MS or LC-MS.

In the invention, the cut RNA to be detected is directly loaded on a computer and is detected by an HPLC-MS method, or the 5' end of the cut mRNA can be detected by LC-MS after the cut RNA to be detected is purified.

In the invention, the capping efficiency can be measured in a semi-quantitative mode or a quantitative mode, the capping efficiency can be measured in the form of an ultraviolet absorption spectrum of a liquid phase (LC) or a TIC spectrum, and the capping efficiency can be calculated according to the abundance of mass spectrum molecules or directly by using an LC analysis method.

Preferably, the capping method of the RNA to be detected comprises enzymatic capping or cap analogue transcriptional co-capping.

In the present invention, enzymatic capping (e.g., vaccinia virus capping enzyme, 2' -O methyltransferase, etc.) may be used, and also transcription co-capping with a cap analog (e.g., ARCA, Cleancap, etc.) may be used.

In the present invention, a deoxyribozyme capable of cleaving RNA is suitable for use in the method of the present invention.

Preferably, the deoxyribozymes include 10-23 DNAzymes and/or 8-17 DNAzymes.

Preferably, the cutting method comprises the following steps:

the RNA to be detected and the deoxyribozyme were mixed and annealed, and then a buffer was added to carry out the reaction.

Preferably, the molar ratio of the RNA to be detected to the deoxyribozyme is 1 (1-100), including but not limited to 1:2, 1:3, 1:4, 1:5, 1:6, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:92, 1:95, 1:98 or 1: 99.

Preferably, the annealing conditions are:

pre-denaturing at 40-100 ℃ (41 ℃, 42 ℃, 43 ℃, 45 ℃, 46 ℃, 48 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 92 ℃, 94 ℃, 96 ℃ or 98 ℃) for 4-6 min;

40 to 100 ℃ (for example, 41 ℃, 42 ℃, 43 ℃, 45 ℃, 46 ℃, 48 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, 90 ℃, 92 ℃, 94 ℃, 96 ℃ or 98 ℃) denaturation for 8 to 12sec, and the temperature is reduced to 20 to 30 ℃ in a circulation way at-0.1 to-0.3 ℃/deg.

Preferably, the annealing conditions are as shown in table 1.

TABLE 1

Step (ii) of	Temperature of	Time	Cooling/Each cycle	Number of cycles
					Pre-denaturation	85℃	5min
Gradient cooling	85℃	10sec	-0.1 ℃/cycle	600
						25℃	5min
Preservation of	4℃	-

Preferably, the buffer contains 5-300 mM Tris-HCl (which may be, for example, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 15mM, 20mM, 50mM, 100mM, 120mM, 150mM, 200mM, 220mM, 230mM, 250mM, 280mM, 290mM, 292mM, 295mM, 296mM or 298mM) and 0.5-300 mM MgCl ₂ (e.g., it may be 0.6mM, 0.7mM, 0.8mM, 0.9mM, 1mM, 11mM, 12mM, 15mM, 20mM, 50mM, 100mM, 120mM, 150mM, 200mM, 220mM, 230mM, 250mM, 280mM, 290mM, 292mM, 295mM, 296mM, or 298 mM).

Preferably, the buffer contains 10mM Tris-HCl and 50mM MgCl ₂ 。

Preferably, the pH of the buffer is 4-10, including but not limited to 5, 6, 7, 8 or 9.

Preferably, the reaction temperature is 30-40 ℃ (for example, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35, 36 ℃, 37 ℃, 38 ℃ or 39 ℃) and the time is 1-3 h (for example, 1.2h, 1.4h, 1.5h, 1.6h, 1.8h, 2h, 2.3h, 2.5h, 2.6h, 2.7h, 2.8h or 2.9 h).

As a preferred technical scheme, the method for detecting the RNA capping efficiency comprises the following steps:

(1) designing deoxyribozymes according to the RNA sequences to be detected;

(2) mixing RNA to be detected with deoxyribozyme and performing pre-denaturation at 80-90 ℃ for 4-6 min; denaturation at 80-90 ℃ for 8-12 sec, circularly cooling to 20-30 ℃ at the speed of-0.1-0.3 ℃/min, adding a buffer solution, and reacting for 1-3 h at 30-40 ℃;

(3) and (3) detecting the molecular weight and relative proportion of the reaction products in the step (2) and determining the capping efficiency.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively utilizes the DNAzyme to cut the RNA to be detected and determines the capping efficiency, realizes the quantitative detection of the capping efficiency of the RNA, can avoid radioactive pollution, and has simple operation and low cost.

Drawings

FIG. 1A is a schematic diagram of the principle of RNA cleavage by 10-23 DNAzyme;

FIG. 1B is a schematic diagram showing the principle of RNA cleavage with 8-17 DNAzyme;

FIG. 2 is a PAGE gel diagram of RNA cleaved by 10-23 and 8-17 DNAzymes;

FIG. 3A is a TIC map of full-length RNA;

FIG. 3B is an HPLC profile of full-length RNA;

FIG. 3C is a mass spectrum of the capped full-length RNA;

FIG. 3D is a mass spectrum of uncapped full-length RNA;

FIG. 4 is an HPLC chromatogram of DNAzyme cleaved RNA;

FIG. 5A is a mass spectrum of capped RNA after DNAzyme cleavage;

FIG. 5B is a mass spectrum of uncapped RNA after DNAzyme cleavage.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not specify particular techniques or conditions, and are to be construed in accordance with the description of the art in the literature or with the specification of the product. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

The invention establishes a method for rapidly and quantitatively detecting RNA capping efficiency through research, and the specific process comprises the following steps:

firstly, designing DNAzyme according to an RNA sequence to be detected; then, using DNAzyme to cut RNA molecules at fixed points; finally, the reaction mixture is directly loaded on a machine, and the size and relative proportion of the molecular weight of the sheared RNA product are detected by using an HPLC-MS or LC-MS method, so that the capping efficiency is determined. When RNA is capped, capped RNA and RNA which is not successfully capped exist in the system due to the difference of RNA capping efficiency, after RNA is subjected to site-specific shearing by using specific DNAzyme, the capped RNA and the uncapped RNA in the system generate the difference of molecular weights through HPLC-MS or LC-MS analysis, namely the capped RNA increases the size of a cap, and the capping efficiency is obtained according to the relative ratio of the capped RNA and total RNA (the capped RNA and the uncapped RNA).

The experimental materials in the embodiment of the invention comprise:

1、

t7 HighYield RNA Synthesis Kit, Yeasen (next saint biotechnology (shanghai) gmbh), cat #: 10623;

2. GTP, Yeasen, cat #: 10132;

3. DNase 1 (DNase 1), Yeasen, cat No.: 10607;

4. vaccinia Capping Enzyme, Yeasen, cat #: 10615;

5. RRI (rnase inhibitor), Yeasen, cat No.: 10603;

6. SAM (S-adenosylmethionine), Yeasen, cat No.: 10619;

7. mRNA Cap 2' -O-Methyransferase, Yeasen, cat #: 10616, respectively;

8、

reagent GG, TriLink, cat # s: n-7133;

9. mRNA purification kit, Yeasen, cat #: 12603;

10、DEPC-H ₂ o, diethyl pyrocarbonate treated water;

11. tris (hydroxymethyl) aminomethane), Sigma, cat #: 252859, respectively;

12、MgCl ₂ sigma, cat #: m4880;

13. all DNAzymes of the present invention were synthesized by Shanghai Biotech Ltd.

The active center of 10-23DNAzyme enzyme consists of 15 deoxyribonucleotide sequences, namely 10-23 motif (motif) -5'-GGCTAGCTACAACGA-3', two ends of the active center are substrate binding regions, the length of the active center is generally 7-9 nt, the active center is specifically bound with target RNA through Watson-Crick base pairing, the sequence of the active center can be changed according to different substrate RNAs, a cutting site is positioned in a phosphodiester bond between unpaired purine and paired pyrimidine on an RNA molecule, and a cutting principle diagram is shown in FIG. 1A; 8-17 DNAzymes are similar to 10-23 DNAzymes except that they have a 12 base catalytic motif and core substrate requirement of AG, where A is unpaired and G forms a wobble pair, the cleavage scheme is shown in FIG. 1B.

Example 1

This example uses enzymatic preparation of capped RNA.

(1) In vitro transcription Synthesis of uncapped RNA

The purified linearized DNA is used as a transcription template (the nucleic acid sequence is shown as SEQ ID NO. 1), T7 RNA polymerase is combined with a specific T7 promoter on the template to start the in vitro transcription of RNA, so that the uncapped RNA is obtained, and the reaction system is shown as table 2.

TABLE 2

The reagents in the above table 2 were mixed well, incubated at 37 ℃ for 2h, digested at 137 ℃ for 15min with 2U DNase after the reaction was completed, and then purified by magnetic beads using the mRNA purification kit of Yeasen (cat # 12603), the detailed procedures are described in the mRNA purification kit of Yeasen.

SEQ ID NO.1：

ggaaataagagagaaaagaagagtaagaagaaatataagaccccggcgccgccaccatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtgataatagcagctggagcctcggtggcctagcttcttgccccttgggcctccccccagcccctcctccccttcctgcacccgtacccccgtggtctttgaataaagtctgagtgggcggcaaaaaaaaaaaaaaaaaaaaaaaaaaaaaagcatatgactaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa。

(2) Enzymatic capping preparation of capped RNA

Capped RNA was obtained by co-Capping the Vaccinium Capping Enzyme with mRNA Cap 2' -O-Methyransferase.

10 μ g of the RNA purified in (1) was denatured at 65 ℃ for 5min, immediately placed on ice for 5min, and then a capping reaction system was prepared as shown in Table 3.

TABLE 3

After the reagents in table 3 above were mixed well, incubation was performed at 37 ℃ for 1h, followed by magnetic bead purification to obtain capped RNA.

Example 2

This example uses the Co-transcription method to prepare capped RNA

The cap analogue was additionally supplemented in the co-transcription reaction system of table 4 on the basis of step (1) of example 1.

TABLE 4

After the reagents in the above table 4 are uniformly mixed, incubating at 37 ℃ for 2h, after the reaction is finished, adding 2U DNase to digest at 137 ℃ for 15min, and then performing magnetic bead purification to obtain capped RNA.

Example 3

This example uses DNAzyme to treat capped RNA.

(1) Annealing RNA with specific DNAzymes (10-23 DNAzymes and 8-17 DNAzymes), wherein the specific annealing reaction system and annealing program are shown in tables 5 and 6, the annealing program is pre-denaturation at 85 ℃ for 5min, maintaining at 85 ℃ for 10sec, then performing gradient cooling, cooling to-0.1 ℃ in each cycle, cooling to 25 ℃ after 600 cycles, maintaining for 5min, and setting an instrument to maintain at 4 ℃ after the reaction is finished.

TABLE 5

Components	Amount of addition	Final concentration
			RNA	24μM	0.8μM
DNAzyme(100μM)	3μL	10μM
			DEPC-H ₂ O	To27μL

TABLE 6

Step (ii) of	Temperature of	Time	Cooling/Each cycle	Number of cycles
					Pre-denaturation	85℃	5min
Gradient cooling	85℃	10sec	-0.1℃/cycle	600
						25℃	5min
Preservation of	4℃	-

(2) DNAzyme Reaction was activated and after completion of the Reaction, 3. mu.L of 10 XDNAzyme Reaction Buffer (containing 10mM Tris and 50mM MgCl) was additionally added ₂ ) And reacting at 37 ℃ for 2 h.

The results are shown in FIG. 2, which is a gel image of the PAGE of 10-23 and 8-17 DNAzymes after cleaving RNA, wherein lane 1 is full-length RNA, lane 2 is 10-23DNAzyme cleaved capped RNA, lane 3 is 10-23DNAzyme cleaved uncapped RNA, lane 4 is 8-17DNAzyme cleaved capped RNA, and lane 5 is 8-17DNAzyme cleaved uncapped RNA, indicating that RNA can be efficiently cleaved into small fragments by DNAzyme.

Example 4

This example utilizes HPLC-MS to detect capping efficiency.

DNAzyme treated samples were detected using LC-MS with HPLC conditions: c18 nano column, mobile phase a: 20mM trifluoroacetic acid, mobile phase B: 80% acetonitrile. The molecular weight of the fragment was measured by negative ion mode Q-TOF.

The capping rate is measured in the form of an LC ultraviolet absorption spectrum; or according to the abundance of mass spectrum molecules, when the RNA is capped, because of the difference of the capping efficiency of the RNA, capped RNA and RNA which is not successfully capped exist in the system, when the RNA is cut at fixed points by using specific DNAzyme, the capped RNA and the RNA which is not capped in the system generate the molecular weight difference through HPLC-MS analysis, namely the capped RNA increases the size of the cap, the capping efficiency is obtained according to the relative ratio of the capped RNA and the total RNA (the capped and the uncapped RNA), FIG. 3A is a TIC map of the full-length RNA, FIG. 3B is a HPLC map of the full-length RNA, FIG. 3C is a mass spectrum map of the capped full-length RNA, the RNA fragments are longer, the molecules are beyond the detection limit of the mass spectrum, FIG. 4 is an HPLC map of the RNA after DNAzyme cutting, arrows mark the peak areas of the DNAzyme, the capped RNA (the capped RNA), (the capped RNA) and the uncapped RNA (the total RNA) peak area can be calculated according to the ratio of the capped RNA peak area occupied by the capped RNA, the capping rate in the figure is 83.4%. FIG. 5A is a mass spectrum of capped RNA after DNAzyme cleavage, FIG. 5B is a mass spectrum of uncapped RNA after DNAzyme cleavage, and the molecular weight of RNA at the 5' end of capped RNA after DNAzyme treatment is 12558.78; after uncapped RNA is treated by DNAzyme, the molecular weight of RNA at the 5' end of the uncapped RNA is 12185.58, compared with uncapped RNA, the molecular weight of a Cap1 structure is increased, and the capping rate measured according to the ion abundance is 82.7%.

In conclusion, the DNAzyme is designed according to the RNA sequence to be detected, the DNAzyme is used for cutting RNA molecules at fixed points, and the size and relative proportion of molecular weight of the sheared RNA product are detected by using an HPLC-MS or LC-MS method, so that the capping efficiency is determined, the RNA capping efficiency is quantitatively detected, the radioactive pollution can be avoided, the operation is simple, and the cost is low.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Sequence listing

<110> Histo Histoste of next (Shanghai) Ltd

<120> a method for detecting RNA capping efficiency

<130> 2022-03-30

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1004

<212> DNA

<213> Artificial sequence

<400> 1

ggaaataaga gagaaaagaa gagtaagaag aaatataaga ccccggcgcc gccaccatgg 60

tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag ctggacggcg 120

acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc acctacggca 180

agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg 240

tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac atgaagcagc 300

acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc atcttcttca 360

aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac accctggtga 420

accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg gggcacaagc 480

tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag aagaacggca 540

tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag ctcgccgacc 600

actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc 660

tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc 720

tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagtgataat 780

agcagctgga gcctcggtgg cctagcttct tgccccttgg gcctcccccc agcccctcct 840

ccccttcctg cacccgtacc cccgtggtct ttgaataaag tctgagtggg cggcaaaaaa 900

aaaaaaaaaa aaaaaaaaaa aaaagcatat gactaaaaaa aaaaaaaaaa aaaaaaaaaa 960

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1004

Claims

1. A method for detecting RNA capping efficiency, the method comprising:

2. The method for detecting RNA capping efficiency according to claim 1, wherein the method for detecting molecular weight and relative proportion comprises HPLC-MS or LC-MS.

3. The method for detecting RNA capping efficiency according to claim 1 or 2, wherein the capping method of the RNA to be detected comprises enzymatic capping or cap analog transcription co-capping.

4. The method for detecting RNA capping efficiency according to any one of claims 1 to 3, wherein the DNAzyme comprises 10-23DNAzyme and/or 8-17 DNAzyme.

5. The method for detecting RNA capping efficiency according to any one of claims 1 to 4, wherein the cleavage is performed by:

6. The method for detecting RNA capping efficiency according to claim 5, wherein the molar ratio of the RNA to be detected to the DNAzyme is 1 (1-100).

7. The method for detecting RNA capping efficiency according to claim 5 or 6, wherein the annealing is performed under the following conditions:

pre-denaturation at 40-100 ℃ for 4-6 min;

the temperature is changed for 8-12 sec at 40-100 ℃, and the temperature is circularly reduced to 20-30 ℃ at the speed of-0.1 to-0.3 ℃.

8. The method for detecting RNA capping efficiency according to any one of claims 5 to 7, wherein the buffer contains 5 to 300mM Tris-HCl and 0.5 to 300mM MgCl ₂ ；

Preferably, the pH value of the buffer solution is 4-10.

9. The method for detecting RNA capping efficiency according to any one of claims 5 to 8, wherein the reaction temperature is 30 to 40 ℃ and the reaction time is 1 to 3 hours.

10. The method for detecting RNA capping efficiency according to any one of claims 1 to 9, wherein the method comprises the steps of:

(1) designing deoxyribozymes according to the RNA sequences to be detected;

(2) mixing the RNA to be detected with deoxyribozyme and performing pre-denaturation at the temperature of 80-90 ℃ for 4-6 min; denaturation at 80-90 ℃ for 8-12 sec, circularly cooling to 20-30 ℃ at the speed of-0.1-0.3 ℃/min, adding a buffer solution, and reacting at 30-40 ℃ for 1-3 h;