CN110055284A - One kind being based on PspCas13b-Alkbh5 single-gene specificity m6A modifies edit methods - Google Patents
One kind being based on PspCas13b-Alkbh5 single-gene specificity m6A modifies edit methods Download PDFInfo
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Classifications
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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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
Abstract
The invention discloses use PspCas13b combination m6A demethylase Alkbh5 targets the application of mRNA demethylation modification.The present invention carries out demethylation modification research shows that PspCas13b-Alkbh5 fusion protein can target target gene (such as CYB5A, CTNNB1) mRNA by specific gRNA, and then regulates and controls destination gene expression.m6A RNA immunoprecipitation experiment and m6The site A quantitative PCR is the result shows that PspCas13b-Alkbh5 fusion protein can effectively reduce the m of intracellular target gene mRNA6A methylation level, while not finding miss target phenomenon temporarily.The present invention is using PspCas13b as means, in conjunction with m6A demethylase Alkbh5 realizes the demethylation modification of targeting mRNA for the first time, and regulate and control the expression of specific purpose and application based on this, it breaches in the past based on the specific protein expression regulation for changing DNA hereditary information and realizing, the new therapeutic modality and therapeutic strategy with important potentiality is provided for the treatment of various diseases in the future, has good application prospect.
Description
Technical field
The invention belongs to single-gene mRNA to modify editor field, specifically, the present invention relates to a species specificity editor is single
Gene mRNA m6The method of A modification.
Background technique
CRISPR(clustered regularly interspaced short palindromic repeats)/Cas
(CRISPR-associated) system is distinctive acquired immune system in some bacteriums and archeobacteria, this system passes through
The RNA of particular sequence is guided, the exogenous DNA of specificity cutting degradation.CRISPR/Cas system can be divided into three types, wherein II
Type CRISPR/Cas system is transformed into the tool of genome targeting editor since its composition is simple.And pass through engineer
And RNA is transcribed in vitro, the sgRNA (single guide RNA) with guiding function can be synthesized, sgRNA guides Cas egg
White specificity cutting target DNA sequence.By the transformation to Cas albumen, under the guidance of sgRNA, CRISPR/Cas system exists
A variety of purposes can be reached in research, for example, gene is cut, is modified, silencing, knockout, adjustment expression etc. operation.
CRISPR/Cas has become the powerful of gene editing and research, has wide application prospect.
CRISPR/Cas9 is specific recognition target DNA and carries out gene editing to it at present, to realize gene expression
The common method of regulation.Then, research find to belong to Cas family Cas13 albumen can specific recognition mRNA and to its into
Row cutting degradation.By a series of research improve, discovery PspCas13b can specific recognition RNA but missing RNA enzyme cut work
Property.Therefore, PspCas13b starts the concern for causing people as the albumen tool of selectively targeted mRNA.It has now been found that
The fusion protein that PspCas13b and other function albumen are built into can realize the function of specificity editor RNA, to open target
Mark the new paragon of rna editing.
mRNA m6A methylation modification is the highest modification of abundance on mRNA, and regulation mRNA includes transcription, shearing, stablizes
Property, enter and leave core, the multiple biological functions such as translation.Have now been found that mRNA m6A modification by " writer " methylase,
" reader " identifies that a series of albumen such as enzyme and " eraser " demethylase realize dynamic regulation.Alkbh5 is m6A's goes first
Base enzyme, the m of mRNA can be lowered by being overexpressed Alkbh5 in the cell6A modification is horizontal.However, there has been no specific needles at present
To the demethylation method of target gene mRNA.
In view of m6A has important regulating and controlling effect, present invention combination PspCas13b/gRNA to the biological behaviour of mRNA
System, constructs PspCas13b-Alkbh5 fusion protein, and designs gRNA for target gene, experimental verification its can specificity
The methylation modification of target gene mRNA is removed, to provide a kind of based on PspCas13b-Alkbh5 single-gene specificity m6A
Modify edit methods.
Summary of the invention
The present invention provides a kind of simple, efficient and special target gene mRNA m6A modifies edit methods.
The present invention is directed to target gene mRNA m using PspCas13b-Alkbh5/gRNA specificity6A modification is compiled
Volume, specifically includes the following steps:
(1) for the gRNA design and synthesis of target mRNA；
(2) m is carried out to purpose mRNA using PspCas13b-Alkbh5 fusion protein6A modification editor；
(3) m is carried out to treated mRNA6A abundance, mRNA expression and biological function are verified；
(4) Targeted-control that specific mRNA is expressed using PspCas13b-Alkbh5/gRNA.
In preferred embodiments, the method the step of in (1), the 5 ' ends of the gRNA are specific sequence,
It can be with template complementary pairing；3 ' the ends of the gRNA are that PspCas13b/Cas13b identifies sequence；
In preferred embodiments, the method the step of in (2), the PspCas13b-Alkbh5 fusion protein packet
Fusion combination containing a variety of Cas13b ease variants and Alkbh5 protein variant；
In preferred embodiments, the method the step of in (2), the PspCas13b-Alkbh5 fusion protein with
Single or multiple gRNA are added in reaction system jointly, identify target mRNA, PspCas13b-Alkbh5 fusion protein with gRNA
In conjunction with gRNA, and by PspCas13b-Alkbh5 fusion protein to the m on target mRNA6A carries out demethylation modification；
In preferred embodiments, the method the step of in (3), the m6A level verification includes pair
The verifying of PspCas13b-Alkbh5/gRNA system effectiveness and the various biological functions of mRNA after treatment are tested
Card；
In preferred embodiments, described to use PspCas13b-Alkbh5/gRNA the method the step of in (4)
System carries out Targeted-control to specific mRNA, including expression regulation, stability regulation, translation efficiency regulation, enters and leaves nuclear capability
Regulation etc..
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention
It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.
Detailed description of the invention
Fig. 1: PspCas13b-Alkbh5 targeting mRNA specificity editor m6A modifies schematic diagram；
Fig. 2: PspCas13b-Alkbh5 fusion protein and its variant schematic diagram；
Fig. 3: PspCas13b-Alkbh5 fusion protein and its modification A lkbh5-PspCas13b fusion protein can reduce carefully
M intracellular6A methylation modification is horizontal；
The m of Fig. 4: CYB5A mRNA6A decorating site schematic diagram
Fig. 5: m6The site A quantifies the gRNA tune that qPCR prompt PspCas13b-Alkbh5 fusion protein combines targeting CYB5A
Control the m of its mRNA6A modification is horizontal；
Fig. 6: m6A RIP-PCR prompts PspCas13b-Alkbh5 fusion protein that the gRNA of targeting CYB5A is combined to regulate and control
The m of CYB5A mRNA6A modification is horizontal；
Fig. 7: RT-PCR prompt PspCas13b-Alkbh5 fusion protein combines the gRNA of targeting CYB5A to regulate and control its mRNA table
Up to level；
The m of Fig. 8: CTNNB1 mRNA6A decorating site schematic diagram；
Fig. 9: m6The site A quantitative PCR prompts PspCas13b-Alkbh5 fusion protein to combine the gRNA tune for targeting CTNNB1
Control the m of its mRNA6A modification is horizontal；
Figure 10: Western blot prompts PspCas13b-Alkbh5 fusion protein to combine the gRNA for targeting CTNNB1mRNA
Regulate and control the protein expression level of β-catenin；
Figure 11: pGL3-5 ' UTR plasmid schematic diagram；
Figure 12: luciferase reporter gene experiment prompt PspCas13b-Alkbh5 fusion protein combines targeting CTNNB1
The gRNA of 5 ' UTR regulates and controls luciferase expression
Figure 13: luciferase reporter gene experiment prompt PspCas13b-Alkbh5 fusion protein combines targeting CTNNB1
The gRNA regulation luciferase mRNA expression of 5 ' UTR
Embodiment
Present invention be described in more detail with reference to the accompanying drawing.
1 PspCas13b-Alkbh5/gRNA system of embodiment, which realizes CYB5A mRNA demethylation and regulates and controls it, expresses water
It is flat.
1 experimental material and method
1.1 main agents and instrument
HEK293T cell strain is by this laboratory conservation or uses other routine experiment control laboratories and cell bank source.Tire
Cow's serum is purchased from Gbico company, and lipofectamine, siRNA blank control and si-GPR30 are limited purchased from sharp rich biotechnology
Company, G-1 and remaining reagent are Sigma company (analyzing pure or more).Cell Counting Kit-8 kit is purchased from same
Benevolence chemical company fluorescence microscope is purchased from Olympus company, fluorescence quantitative PCR instrument480II is public purchased from Roche
Department, laser confocal microscope LSM710 are purchased from Zeiss company.
1.2 cell recoveries and culture
The HEK293T cell strain in liquid nitrogen container will be frozen to take out, the fast melt in 37 DEG C of water-baths.Superclean bench
In, metastatic cells suspension is into 10mL centrifuge tube, addition 5mL DMEM culture medium, low-speed centrifugal 1000rpm × 5min, in abandoning
Clearly, it is added in the DMEM culture medium containing 10%FBS, is cultivated under the conditions of 5%CO2,37 DEG C.
The processing of 1.3 cells and transfection experiment
Adherent HEK293T cell is subjected to digestion resuspension, bed board to 6 orifice plates, 5x10 is added in every hole5Cell will be cultivated
Preculture is for 24 hours in the incubator for plate.When cell density reaches 50~60% convergence degree, using Lipofectamine 3000
(Invitrogen) it is transfected.Key step are as follows: dilute 1.5 μ g's without serum DMEM culture medium with 100 μ l
PspCas13b-Alkbh5 is overexpressed plasmid and 1ug gRNA, and 2ul/ug P3000 is added and mixes, and is free of serum with 100ul
DMEM culture medium dilution 1ug:0.75ul Lipofectamine3000 reagent simultaneously mix, plasmid dilution with
3000 dilution of Lipofectamine mixes gently, and the culture plate containing cell and culture solution is added after being incubated at room temperature 5min
In each hole.Cell is placed in 37 DEG C of CO2 incubator after cultivating 4~6h, removes the culture of the mixed liquor containing DNA-lipo3000
Base, Fresh cell culture medium of the replacement containing 10%FBS continue culture for 24 hours.
1.4 cell total rnas extract and Real-time PCR detects target mRNA
Intracellular total serum IgE is extracted according to specification using E.Z.N.A.HP Total RNA Kit.And the RNA of extraction is adopted
Reverse transcription is carried out with PrimeScript RT reagent Kit, 37 DEG C of reverse transcription reaction 15min obtain cDNA, then use
TaKaRa SYBR Premix Ex TaqTM kit carries out Real Time PCR analysis.Initial temperature is set as 65 DEG C；Temperature
Variation is set as 0.5 DEG C, terminates temperature and is set as 95 DEG C.Real Time PCR after reaction, confirms the expansion of Real time PCR
Increase curve and melt curve analysis, it is for statistical analysis to result using Δ Δ Ct method.Wherein Δ Ct=target gene Ct value-internal reference base
Because of Ct value, Δ Δ Ct=experimental group Δ Ct- control group Δ Ct, gene relative expression quantity=2- Δ Δ Ct.
1.5 m6A mass spectrum
The RNA of extraction is quantified, oligo dT and 200ug RNA combines 4hr at 4 DEG C, with separation and Extraction mRNA.It mentions
MRNA after taking successively carries out 2hr enzymatic hydrolysis with p1 nuclease and alkaline phosphatase.Equipped with Agilent
The ultra performance liquid chromatography tandem mass spectrometer of infinitylabPoroshell 120 EC-C18 (2.1 × 5mm) chromatographic column will be used
In the m of sample6The analysis of A and A content.Firstly, using mass spectrum to the single m of various concentration6The base of A and A is detected to build
Day-mark is bent.Then, the mRNA after enzymatic hydrolysis is analyzed by mass spectrometry, obtained peak area substitutes into mark song respectively and calculates A and m6A
Amount, analyze result m6The permillage ‰ of A/A indicates.
1.6 m6The site A quantifies qPCR
The m delivered according to Xiao (et al.2018)6It is fixed to carry out Qubit to the RNA of extraction for the site A quantifying PCR method
Amount carries out reverse transcription using Bst archaeal dna polymerase and SplintR ligase, and reverse transcription reaction obtains cDNA, then uses
TaKaRa SYBR Premix Ex TaqTM kit carries out Real Time PCR analysis.Initial temperature is set as 95 DEG C；95℃-
60 DEG C carry out 40 circulations, terminate temperature and are successively set as 95 DEG C, 60 DEG C, 95 DEG C, 4 DEG C.Real Time PCR after reaction,
Confirm the amplification curve and melt curve analysis of Real time PCR, it is for statistical analysis to result with the CT value after standardizing.
2 results
In the present embodiment, HEK293T cell transfecting PspCas13b-Alkbh5 fusion protein or its modification A lkbh5-
PspCas13b, experiment discovery PspCas13b-Alkbh5 and Alkbh5-PspCas13b can lower the mRNA of intracellular totality
m6A is horizontal, and effect and independent transfection Alkbh5 are consistent (Fig. 3).In conjunction with the gRNA and PspCas13b- of targeting CYB5A mRNA
It is found after Alkbh5 cotransfection cells, the m of CYB6A mRNA6A methylation level obviously lowers (Fig. 4).m6The site A quantitative PCR knot
Fruit prompt, PspCas13b-Alkbh5/gRNA can effectively remove CYB5A mRNA m6Methylation modification (figure on A decorating site
5)。m6The whole m6A horizontal down-regulation (Fig. 6) of A-RIP-qPCR result confirmation CYB5A mRNA.Meanwhile CYB5A mRNA is carried out
Quantitative analysis, discovery eliminate m6The CYB5A mrna expression amount of A methylation modification significantly raises (Fig. 7).The above experimental result
Show using PspCas13b-Alkbh5/gRNA system can the demethylation of efficient targeting CYB5A modify, and regulate and control its mRNA
Expression.
2 PspCas13b-Alkbh5/gRNA System Approach CTNNB1 of embodiment, 5 ' UTR m6MRNA is expressed in A modification
It influences
1 experimental material and method
1.1 Western-Blotting analysis
Treated cell is collected, PBS is washed 2 times, and three decontamination cell pyrolysis liquids are added, place 15-20min on ice.It receives
Collect cell pyrolysis liquid to manage in EP, maximum (top) speed is centrifuged 20min, collects supernatant.Bradford method measures protein concentration.With every hole
20 μ g albumen applied sample amounts, voltage 80-120 V carry out SDS-PAGE electrophoretic separation.Electricity transfer under conditions of constant current 200mA, 90min
To the pretreated pvdf membrane of methanol.5% skimmed milk power (PBST preparation) closes pvdf membrane 2h.Antibody is detected with 1:1000 ratio
It is diluted with confining liquid, 4 DEG C of incubation pvdf membranes are stayed overnight.PBST wash pvdf membrane 10min × 3 time, be added HPR label goat-anti rabbit or
Sheep anti-mouse igg secondary antibody (1:5000, confining liquid dilution), is incubated at room temperature 1h.PBST washs pvdf membrane 10min × 3 time.ECL is added
Luminescent solution is exposed with X-ray.
The building of 1.2 dual-luciferase reporter systems
PGL3-basic plasmid with firefly luciferase reporter gene is purchased from Addgene company.Firefly fluorescence
Plain enzyme reporter gene upstream is sequentially inserted into the promoter sequence (1500bp) and 5 ' UTR sequences of CTNNB1, constructs pGL3-5 '
UTR plasmid.PGL3-5 ' UTR plasmid, Renilla luciferase reporter gene plasmid (as internal reference), PspCas13b-Alkbh5,
GRNA transfects HEK293T cell simultaneously.Transfection after r, according to luciferase reporter gene kit specification, is surveyed respectively for 24 hours
Determine the expression quantity of firefly luciferase (Fluc) and renilla luciferase (Rluc), 5 ' UTR are analyzed to the light of firefly with Fluc/Rluc
The expression of luciferin zymoprotein influences.
2 results
In the present embodiment, the m of CTNNB1 mRNA6A modification is present in 5 ' UTR regions (Fig. 8).HEK293T cell transfecting
PspCas13b-Alkbh5 fusion protein is substantially reduced first m of CTNNB1 mRNA6The site A (S1) methylation level (figure
9).Western blot interpretation of result prompt, β-catenin expressing quantity are eliminating m6It is significantly increased after A methylation modification
(Figure 10).We are further constructed CTNNB15 ' UTR to firefly luciferase reporter gene upstream (Figure 11).Double fluoresceins
Enzyme Reporter Gene Experiments discovery PspCas13b-Alkbh5/gRNA system significantly improves the expression quantity (figure of firefly luciferase
12), (Figure 13) is not made significant difference to the mRNA of luciferase expression.The experimental results showed that, utilize PspCas13b- above
Alkbh5/gRNA system can target the m of research 5 ' UTR of CTNNB16A modification promotes mRNA translation efficiency.
Claims (22)
1. a kind of eliminate target mRNA m using PspCas13b-Alkbh5/gRNA system6The method of A methylation modification, feature
It is, described method includes following steps: (1) purpose mRNA is identified using the gRNA for target mRNA；(2) it uses
PspCas13b-Alkbh5 fusion protein carries out demethylation processing to purpose mRNA；(3) m is carried out to treated mRNA6A
Abundance, mRNA expression and biological function are verified；(4) using PspCas13b-Alkbh5/gRNA system to specific mRNA
Carry out Targeted-control.
2. the method according to claim 1, wherein the 5 ' ends of the gRNA are targeting in the step (1)
The specific sequence of purpose mRNA, can be with template complementary pairing；Contain repetitive sequence (Direct in the 3 ' ends of the gRNA
repeats)。
3. according to the method described in claim 2, it is characterized in that, 5 ' the end-specificity sequences of the gRNA can be with target
5 ' the areas UTR of mRNA, include sub-district or the 3 ' area UTR complementary pairings at the code area CDS；3 ' the ends of the gRNA can form similar
In the mRNA secondary structure of hairpin structure, and can be by PspCas13b, PspCas13b-Alkbh5 fusion protein, active
Cas13b or the variants of other Cas13 protease identify and combine.
4. method according to claim 2, which is characterized in that the gRNA includes that artificial synthesized RNA and inside and outside are transcribed
RNA。
5. method according to claim 4, which is characterized in that if the gRNA uses T7DNA polymerase transcription in vitro
5 ' the ends of gRNA, gRNA require addition GG sequence, to be identified by T7DNA polymerase；If turned using T4DNA polymerase
Record increases the identification and transcription of T4DNA polymerase in the 5 ' ends of gRNA usually addition X base；If transcribing gRNA in vivo
(including Intracellular transcription) makes tanscription termination by holding addition U base in gRNA 3 '.
6. the method according to claim 1, wherein in the step (2), using PspCas13b-Alkbh5
Fusion protein, and single or multiple gRNA are added, specific recognition is carried out to target mRNA, and melted by PspCas13b-Alkbh5
Hop protein carries out demethylation processing to target mRNA.
7. according to the method described in claim 6, it is characterized in that, the PspCas13b-Alkbh5/gRNA system is living thin
In intracellular, living animal or external carry out.
8. according to the method described in claim 6, it is characterized in that, the PspCas13b-Alkbh5 fusion protein includes a variety of
The fusion of Cas13b ease variants and Alkbh5 protein variant is combined.
9. according to the method described in claim 8, it is characterized in that, the PspCas13b-Alkbh5 fusion protein includes a variety of
Alkbh5 merge form, as Alkbh5 PspCas15b N-terminal and/or C-terminal be formed by fusion protein, PspCas13b with
The formed fusion protein of Alkbh5 truncate, the formed fusion protein of PspCas13b and Alkbh5 mutant are marked with label
PspCas13b-Alkbh5 fusion protein etc..
10. method according to claim 8, which is characterized in that the PspCas13b-Alkbh5 fusion protein includes a variety of
The variant of Cas13b protease such as cuts mRNA single-stranded, the part or all of digestion activity of forfeiture, loses part or all of gRNA knowledge
The not variants such as the Cas13b such as activity variant and Cas13b protease truncate.
11. method according to claim 8, which is characterized in that the albumen of the PspCas13b-Alkbh5 fusion protein connects
Socket part point contains or not contain connection amino acid sequence (linker).
12. method according to claim 8, which is characterized in that the PspCas13b-Alkbh5 fusion protein contains or not
Contain nuclear signal peptide out (Nuclear export signal peptide, NES).
13. the method according to claim 1, wherein in the step (3), by PspCas13b-
The mRNA of Alkbh5/gRNA system processing, carries out m to it6A level, mRNA expression, mRNA biological function are tested
Card.
14. according to the method for claim 14, which is characterized in that the m of the mRNA6The detection of A level includes totality m6A water
The m of flat detection and specific mRNA6The detection of A level.
15. according to the method for claim 15, which is characterized in that the totality m6A level verification, including m6The inspection of A/A mass spectrum
It surveys, m6A RIP-PCR detection, m6A-seq sequencing detection etc.；The specific mRNAm6The detection of A level, including m6A RIP-PCR inspection
It surveys, specific site m6A RT-qPCR detection etc..
16. according to the method for claim 15, which is characterized in that the mRNA expression verifying, including cell or group
Knit mRNA-seq sequencing detection, the RT-qPCR detection of specific mRNA etc. of total mRNA.
17. according to the method for claim 14, which is characterized in that the mRNA biological function verification, including to total
The transcription rate of mRNA and/or specific mRNA, transcriptional efficiency, the shear efficiency of precursor RNA, mRNA stability, mRNA enter and leave core
The detection of ability, mRNA translation rate etc..
18. the method according to claim 1, wherein in the step (4), using PspCas13b-
Alkbh5/gRNA system carries out the regulation of specificity, including the regulation of expression regulation, stability, translation efficiency tune to target mRNA
Control, the regulation for entering and leaving nuclear capability etc..
19. according to the method for claim 19, which is characterized in that the specific regulation of the target mRNA can be by
PspCas13b-Alkbh5/gRNA system directly or indirectly regulates and controls mRNA m6A is horizontal and realizes.
20. according to the method for claim 20, which is characterized in that the direct regulation and control mode refers to PspCas13b-
Alkbh5/gRNA system directly carries out m to target mRNA6The processing of A demethylation is to realize the specific regulation of target mRNA；Institute
Stating indirect adjustments and controls expression way includes PspCas13b-Alkbh5/gRNA systemic effect in the upstream regulating genes of target mRNA
mRNAm6A, target mRNA promoter binding protein/translation GAP-associated protein GAP/stability GAP-associated protein GAP mRNAm6A, target mRNA table
Up to regulatory factor/albumen mRNA m6A, the mRNA m of target mRNA adjustment signal molecule6A etc., and then indirect adjustments and controls target
The special biological of mRNA.
21. a kind of eliminate target mRNA m using PspCas13b-Alkbh5/gRNA system6The method of A methylation modification, it is special
Sign is, the method it is built-up using method described in any one of claim 1 to 21.
22. a kind of eliminate target mRNA m using PspCas13b-Alkbh5/gRNA system6The method of A methylation modification, it is special
Sign is, the method is used for purposes selected from the group below: (i) PspCas13b-Alkbh5/gRNA system in business, industry or/
And the production of agricultural；(ii) PspCas13b-Alkbh5/gRNA system is in the exploitation of disease targets drug；(iii)
PspCas13b-Alkbh5/gRNA system is in the exploitation of prevention and treatment medical treatment aspect.
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| WO2021169980A1 (en) * | 2020-02-25 | 2021-09-02 | Shanghaitech University | Compositions and methods for detecting nucleic acid-protein interactions |
| CN111330009A (en) * | 2020-04-02 | 2020-06-26 | 南通大学 | Application of m6A modified related gene ALKBH5 in promotion of nerve axon injury repair |
| CN111471097B (en) * | 2020-04-08 | 2021-07-30 | 青岛市中心医院 | Application of interleukin 37 and test method for influence on methylation of lung cancer cell RNA m6A |
| CN111471097A (en) * | 2020-04-08 | 2020-07-31 | 青岛市中心医院 | Application of interleukin 37 and test method for influence on methylation of lung cancer cell RNA m6A |
| CN112126645B (en) * | 2020-09-11 | 2021-06-01 | 广州吉赛生物科技股份有限公司 | Ring RNA (ribonucleic acid) knocking-down method and application thereof |
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| CN113584135A (en) * | 2021-07-26 | 2021-11-02 | 中山大学 | Method for mixed sample detection of RNA modification and realization of accurate quantification |
| CN113584135B (en) * | 2021-07-26 | 2022-05-27 | 中山大学 | Method for mixed sample detection of RNA modification and realization of accurate quantification |
| WO2023142230A1 (en) * | 2022-01-25 | 2023-08-03 | 苏州大学 | Target for chronic pain treatment and use thereof |
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