CN114292914A - Visual RNA methylation detection method and application thereof - Google Patents
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Abstract
The invention discloses a visual RNA methylation detection method and application thereof, wherein a ring structure is formed at two ends of a methylation site based on a specific probe PP, and then the ring structure is respectively complementarily paired with a FIP sequence, a BIP sequence and an SLP sequence, and then methylation and non-methylation identification is realized according to the amplification condition. Compared with the traditional detection method, the detection is more sensitive, the detection limit is 100amol, the content condition of the methylation sites in the sample to be detected can be qualitatively and quantitatively detected, complex instruments and equipment are not needed, the detection cost is low, the detection time is short, the result can be effectively judged through visualization, and the method has high application value.
Description
Technical Field
The invention belongs to the field of gene detection, and particularly relates to a visual RNA methylation detection method and application thereof.
Background
Gene expression is regulated by DNA and RNA modifications. Various types of 100 RNA modifications have been discovered to date. Wherein, N6 methyl adenosine (m)6A) Is the most abundant post-transcriptional type of eukaryotic mRNA and long noncoding (lncRNA). m is6A is also present in rRNA, tRNA, microRNA, circRNA and snorRNA. Wherein m is6A has been demonstrated to be significantly upregulated in a variety of tumor tissues, with potential utility as a clinical diagnostic.
In the related art, m is detected6The most commonly used method for A modification is high throughput sequencing, e.g., m with massively parallel sequencing6A-specific methylated RNA immunoprecipitation (MeRIP-seq) and photosensitive Cross-Linked conjugation m6A sequencing (PA-m)6A-seq), bothThe method depends to a great extent on m6Antibody A immunoprecipitation (m)6a-IP). These sequencing methods can recognize m abundant in RNA6A site, but obtaining m6There are limitations in the analysis of information about the base and position of the A modification. And others developed for m6The single-resolution detection technique a has the requirements of limitation in conversion application or the need of a specific testing instrument, and the detection is time-consuming and inconvenient.
Therefore, aiming at the problems, a detection method capable of realizing visual judgment of RNA methylation is developed for the follow-up research m6A regulation of the biological behavior of mRNA and detection of m6A has extremely important significance as a tumor clinical marker.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a visual RNA methylation detection method and application thereof, the detection method is based on that a specific probe PP forms a ring structure at two ends of a methylation site, then the ring structure is complementary and paired with a FIP sequence, a BIP sequence and an SLP sequence respectively to realize constant temperature amplification, and then the identification of methylation and non-methylation is realized according to the amplification condition, the method is simple and rapid, the visualization can be realized, and the application value is extremely high.
In a first aspect of the invention, a set of RNA methylation detection probes is provided.
According to a first aspect of the invention, in some embodiments of the invention, the RNA methylation detection probe comprises: specific probes PP, FIP sequence, BIP sequence and SLP sequence.
In some preferred embodiments of the invention, the specific probe PP comprises, in order: a sequence A complementary-paired to one end of the methylation detection site, a sequence of region F1c, a sequence of region F2c, a region SHSc, and a sequence B complementary-paired to the other end of the methylation detection site.
The sequence A and the sequence B are combined with a sequence to be detected, in which a methylation detection site is located, respectively to form a ring structure.
In the inventionIn some preferred embodiments, the nucleotide sequence of the specific probe PP is:
or
Or
Wherein, the 5' end of the specific probe PP is also modified with a phosphate group Phos.
Only the bold sections are complementarily paired with the 3 'and 5' sequences of the methylation detection site, respectively, so that the specific probe PP forms a loop structure. The underlined section alone is the F1c region, the underlined and italicized section is the F2c region, and the underlined and bold section is the SHS c region. The specific probe PP is single-stranded DNA, and the total length is recommended to be not more than 100 bases so as to reduce the probability of secondary structure formation.
SEQ ID NO.1 is directed to human 18S rRNA A1832. SEQ ID No.6 is directed to NANOG mRNA A1095. SEQ ID NO.8 is directed to LncRNA-MALAT 1A 2515.
The specific probes PP (targeting non-methylation sites) used for comparison are respectively shown as SEQ ID NO.2 (corresponding to SEQ ID NO.1), SEQ ID NO.7 (corresponding to SEQ ID NO.6) and SEQ ID NO.9 (corresponding to SEQ ID NO. 8).
In some preferred embodiments of the invention, the FIP sequence comprises, in order: a F1c region sequence and a F2 region sequence, wherein the F2 region is complementary to the F2c region sequence.
In some preferred embodiments of the invention, the nucleotide sequence of the FIP sequence is:
in the FIP sequence, the underlined part is the F1c region, which is completely identical to the F1c region in the specific probe PP; the bold part is the F2 region, which is reverse complementary to the F2c region in the specific probe PP.
In some preferred embodiments of the invention, the BIP sequence comprises, in order: the B1c region sequence and the B2 region sequence.
In some preferred embodiments of the invention, the nucleotide sequence of the BIP sequence is:
in the BIP sequence, the underlined part is the B1c region; the bold part is region B2.
In some preferred embodiments of the present invention, the SLP sequence comprises in order: a sequence of region B1c, a sequence of region B2, a sequence of region B1 and a sequence of region SHSc.
In some preferred embodiments of the invention, the SLP sequence has the nucleotide sequence:
in the SLP sequence, only the underlined part is a B1c region and is completely consistent with the B1c region sequence in the BIP sequence; the underlined and bolded part is the B2 region, which is identical to the B2 region sequence in the BIP sequence; the underlined and italicized part is the B1 region, reverse complementary to the B1c region in SLP or BIP sequences; only the thickened part is the SHSc area, and the SHSc area is completely consistent with the sequence of the SHSc area in the specific probe PP.
In a second aspect of the invention, there is provided the use of an RNA methylation detection probe according to the first aspect of the invention in the detection of RNA methylation.
Wherein the RNA comprises lncRNA, lincRNA, rRNA and mRNA.
In a third aspect of the present invention, there is provided a use of the RNA methylation detection probe of the first aspect of the present invention in the preparation of an RNA methylation detection product.
According to a third aspect of the invention, in some preferred embodiments of the invention, the product comprises a detection reagent and a detection kit.
In a fourth aspect of the present invention, a method for detecting RNA methylation is provided, which comprises the following steps:
(1) mixing RNA to be detected with the specific probe PP in the first aspect of the invention for cyclization to obtain a reaction product 1;
(2) mixing the reaction product 1 with Bst 2.0DNA polymerase and SplintR ligase to carry out ring closure to obtain a reaction product 2;
(3) mixing the reaction product 2 with the FIP sequence, the BIP sequence, the SLP sequence and the Bst 2.0DNA polymerase, carrying out isothermal amplification, and judging whether the RNA locus to be detected contains methylation modification according to the amount of the amplification product.
According to a third aspect of the present invention, in some preferred embodiments of the present invention, the cyclization system in step (1) is:
in some preferred embodiments of the present invention, the reaction conditions of step (1) are: the reaction is carried out at 90-98 ℃ for 0.5-1.5 minutes, at 75-85 ℃ for 1-3 minutes, at 65-75 ℃ for 1-3 minutes, at 55-65 ℃ for 1-3 minutes, at 45-55 ℃ for 1-3 minutes, at 35-45 ℃ for 1-3 minutes, and at 25-35 ℃ for 5-10 minutes.
In some more preferred embodiments of the present invention, the reaction conditions of step (1) are: 95 ℃ for 1 minute, 80 ℃ for 1 minute, 70 ℃ for 1 minute, 60 ℃ for 1 minute, 50 ℃ for 1 minute, 40 ℃ for 1 minute, and 30 ℃ for 10 minutes.
According to a third aspect of the present invention, in some preferred embodiments of the present invention, the reaction system in step (2) is:
| |
8μL |
| Bst 2.0DNA polymerase | 0.05U |
| SplintR ligase | 0.5U |
| ATP | Final concentration 10nM |
| dNTP | |
| DEPC water | Make up to 10 mu L |
In some preferred embodiments of the present invention, the reaction conditions of step (2) are: reacting at 35-40 deg.C for more than 20min, and at 65 deg.C for more than 10 min to inactivate the enzyme, and terminating the reaction.
In some more preferred embodiments of the present invention, the reaction conditions of step (2) are: 30 minutes at 37 ℃ and 10 minutes at 65 ℃.
According to a third aspect of the present invention, in some preferred embodiments of the present invention, the reaction system of the isothermal amplification in step (3) is:
in some preferred embodiments of the present invention, the reaction conditions of step (3) are: reacting for 30-120 min at 60-65 ℃.
In some more preferred embodiments of the present invention, the reaction conditions of step (3) are: reacting for 30-60 min at 65 ℃.
The visualized RNA methylation detection method in the present invention aims at detecting the amplification product of the circularization probe, and therefore, the identification of the amplification product can be performed using different detection techniques, such as nucleic acid gel electrophoresis detection, ultraviolet spectrophotometry detection, real-time fluorescence quantitative PCR detection, qualitative detection using a nucleic acid dye, visualized detection based on LAMP reaction, and the like.
According to a third aspect of the present invention, in some preferred embodiments of the present invention, the visual qualitative criterion is: compared with non-methylated sites, the amount of amplification products is obviously reduced, which indicates that the RNA to be detected contains methylated sites.
In some embodiments of the invention, visualization results using PAGE electrophoresis (fig. 9), DNA dyes such as golden view dye staining (fig. 10), and SYBR Green I dye staining (fig. 11) are also used. It can be found that the detection results of methylation and non-methylation can be visually distinguished by adopting different result presentation modes, for example, when the golden View dye is used for dyeing, the methylated modification is orange and more brown precipitates, the non-methylated modification is yellow and less brown precipitates, and the methylated modification has higher fluorescence intensity under ultraviolet light.
The invention has the beneficial effects that:
1. compared with the traditional detection method, the visual RNA methylation detection method provided by the invention has the advantages that the detection is more sensitive, the detection limit is 100amol methylated RNA in 10fmol specific RNA, the content condition of methylation sites in a sample to be detected can be qualitatively and quantitatively detected, complex instruments and equipment are not needed, the detection cost is low, the detection time consumption is short, and the application value is extremely high.
2. The detection method can use different result presentation modes, such as nucleic acid gel electrophoresis detection, ultraviolet spectrophotometry detection, real-time fluorescence quantitative PCR detection, qualitative detection adopting nucleic acid dye, visual detection based on LAMP reaction and the like, is flexible, and can effectively distinguish results through visualization, thereby being convenient and rapid.
Drawings
FIG. 1 is a schematic diagram of a visual RNA methylation detection method in an embodiment of the invention.
FIG. 2 shows the detection of 1832 m of 18S rRNA by the visual RNA methylation detection method according to the embodiment of the present invention6A methylation modification result.
FIG. 3 is in vitro m of ALKBH56A principle of demethylation reaction.
FIG. 4 is a graphical representation of the RNA methylation detection method of the present invention reflecting m by detecting methylation modification at A1095 of NANOG mRNA (A)6A Demethylase Activity results (B).
FIG. 5 shows the detection of m at position A2515 of LncRNA MALAT1 by the visual RNA methylation detection method in the example of the present invention6A methylation modification result.
FIG. 6 shows the results of the sensitivity of the visualized RNA methylation detection method by detecting different concentrations of methylated and unmethylated sites in the examples of the present invention.
FIG. 7 is a standard curve for visualizing the RNA methylation detection method in an embodiment of the invention.
FIG. 8 is a comparison of the results of visual RNA methylation detection method (A) and similar method (B) in an example of the invention.
FIG. 9 is a graph showing the result of the visualized RNA methylation detection method according to the embodiment of the present invention by using PAGE electrophoresis.
FIG. 10 is a graph showing the results of a visual RNA methylation detection method using a GoldenView dye staining in an embodiment of the invention.
FIG. 11 is a graph showing results of a visual RNA methylation detection method according to an embodiment of the present invention using SYBR Green I dye staining.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The experimental materials and reagents used are, unless otherwise specified, all consumables and reagents which are conventionally available from commercial sources.
Example 1 visual RNA methylation detection method for detecting m of 18S rRNA A18326A methylation modification
The specific detection steps are as follows:
(1) preparation of total RNA:
a549 cells were collected and cultured in DMEM containing 10% FBS on 5% CO2Culturing at 37 deg.C, extracting Total RNA from cells with E.Z.N.A.HP Total RNA Kit, and the extraction method is described in the specification.
(2)m6And A, methylation detection:
mu.g of the total RNA (RNA amount to be detected not less than 100amol) obtained in the step (1) was mixed with 250fmol of the specific probe PP in a1 XCutSmart buffer system (as shown in Table 1, purchased from NEB), and the mixture was sequentially subjected to standing at 95 ℃ for 1 minute, 80 ℃ for 1 minute, 70 ℃ for 1 minute, 60 ℃ for 1 minute, 50 ℃ for 1 minute, 40 ℃ for 1 minute, and 30 ℃ for 10 minutes to allow the specific probe PP to bind to the methylation detection site, thereby forming a loop structure.
TABLE 1
| RNA to be tested | 0.1. mu.g (not less than 100amol) |
| Specific probe PP | 250fmol (not less than 50 fmol) |
| Potassium acetate | Final concentration 50mM |
| Tris-acetate，pH 7.9 | Final concentration 20mM |
| Magnesium acetate | Final concentration 10mM |
| BSA | |
| Total of | 8μL |
Wherein, for detecting m6The sequence of the specific probe PP of A is as follows:
as a control, the sequence of the specific probe PP corresponding to the non-methylated modification site (A) is:
wherein, the 5' end of the specific probe PP is also modified with a phosphate group Phos.
Only the bold sections are complementarily paired with the 3 'and 5' sequences of the methylation detection site, respectively, so that the specific probe PP forms a loop structure. The underlined section alone is the F1c region, the underlined and italicized section is the F2c region, and the underlined and bold section is the SHS c region. The specific probe PP is single-stranded DNA, and the total length does not exceed 100 bases so as to reduce the probability of secondary structure formation.
Then, the system shown in Table 2 was further added to the reaction system:
TABLE 2
| TABLE 1 systems | 8μL | |
| Bst 2.0DNA polymerase | 0.05U | |
| SplintR ligase | 0.5U | |
| ATP | Final concentration 10nM | |
| | Final concentration | 5. mu.M |
| DEPC water | Make up to 10 mu L |
Then the reaction is carried out at 37 ℃ for 30 minutes and 65 ℃ for 10 minutes, and the reaction product can be stored at 4 ℃ after the reaction is finished.
And mixing 2 mu L of the obtained reaction product according to a system shown in the table 3, placing the mixture in a fluorescence quantitative PCR instrument for reaction at 65 ℃ for 30-60 min, and reading a fluorescence signal every 30 seconds.
TABLE 3
| TABLE 2 reaction products | 2μL |
| FIP sequence | Final concentration 0.8. mu.M |
| BIP sequences | Final concentration 0.8. mu.M |
| SLP sequence | Final concentration 100nM |
| dNTP | Final concentration 1.4mM |
| MgSO4 | Final concentration 6mM |
| |
1× |
| Bst 2.0DNA polymerase | Final concentration 0.16U/. mu. |
| SYBR Green | |
| 1× | |
| Total of | 20μL |
Wherein, the nucleotide sequence of the FIP sequence is as follows:
in the FIP sequence, the underlined part is the F1c region, which is completely identical to the F1c region in the specific probe PP; the bold part is the F2 region, which is reverse complementary to the F2c region in the specific probe PP.
The nucleotide sequence of the BIP sequence is as follows:
in the BIP sequence, the underlined part is the B1c region; the bold part is region B2.
The nucleotide sequence of the SLP sequence is:
in the SLP sequence, only the underlined part is a B1c region and is completely consistent with the B1c region sequence in the BIP sequence; the underlined and bolded part is the B2 region, which is identical to the B2 region sequence in the BIP sequence; the underlined and italicized part is the B1 region, reverse complementary to the B1c region in SLP or BIP sequences; only the thickened part is the SHSc area, and the SHSc area is completely consistent with the sequence of the SHSc area in the specific probe PP.
Bst 2.0DNA polymerase, SplintR ligase, dNTPs and ATP were purchased from NEB.
The Isotermal Amplification buffer (from NEB) contained a final concentration of 20mM Tris-HCl, 10mM (NH)4)2SO4、50mM KCl、2mM MgSO4And 0.1%
20, pH 8.8. Of course, other conventional LAMP buffers may be used instead.
Instead of using fluorescent quantitative PCR, results can be visualized by PAGE electrophoresis, or by mixing with dyes (e.g., GoldeView, SYBR Green I).
The detection schematic is shown in fig. 1.
The results are shown in FIG. 2.
It can be found that human 18S rRNA A1832 (m)6A) And human 18S rRNA a1825(a) in fluorescence intensity. RT-qPCR results showed that the amplification rate for human 18S rRNA A1832 was slower using the probe in the above example, and the Ct value of the qPCR cycle number was significantly higher than that of A1825, which could indicate the presence of m at the A1832 site6And (C) modifying. The above experimental results show that the method in the above embodiment can effectively detect whether m exists on a specific site of a target RNA6And A is subjected to methylation modification.
Example 2 visual RNA methylation detection method detection m6A demethylase Activity
NANOG mRNA A1095 is a known, defined methylation m6A modification site and NANOG mRNA A1099 is a known defined unmethylated m6The modified site A in this example is HEK293T cell line (human kidney cells) containing the two sites as samples, and shows the actual detection effect.
The specific detection steps are as follows:
(1) preparation of total RNA:
HEK293T cells were harvested and cultured in DMEM with 10% FBS on 5% CO2At 37 ℃ ofAnd (4) culturing, and extracting Total RNA in cells by using an E.Z.N.A.HP Total RNA Kit, wherein the extraction method is shown in the specification.
(2)m6A demethylation reaction:
the total HEK293T RNA extracted in step (1) was added to 3. mu.M ALKBH5 in 25. mu.L buffer (containing 50mM Tris-HCl (pH 7.5), 300. mu.M. alpha. -ketoglutaric acid, 280. mu.M (NH)4)2Fe(SO4)2And 2mM L-vitamin C) at 25 ℃ for 1 hour. The reaction was then terminated by heating to 90 ℃ for 3 minutes.
A negative control was set (ALKBH 5 was mixed with EDTA and the above procedure was followed, and the mixing of the two inactivated ALKBH5 and lost methylase activity (as shown in FIG. 3)).
And (3) reusing the method in the step (1) to extract the total RNA in the experimental group and the negative control, and quantifying for later use.
(3)m6And A, methylation detection:
the detection method was the same as in example 1.
With the difference that for detecting m6The sequence of the specific probe PP of A is as follows:
the sequence of the probe PP specific to the corresponding non-methylated modification site (A) as a control is:
the results of the detection are shown in FIG. 4.
As a result, it was found that total RNA was treated with ALKBH5 to give total m6A methylation modification degree is reduced, and m at A1095 site of NANOG mRNA is detected by visual RNA methylation detection method in the above example6A modification level, RTThe qPCR result shows that after ALKBH5 treatment, the amplification speed of the probe targeting NANOG mRNA A1095 site is higher, the Ct value of the qPCR cycle number is smaller, and the m at the A1095 site is shown6The a modification was abolished by ALKBH5, as expected. When the A1099 site adjacent to A1095 of NANOG mRNA was detected, m did not exist at the A1099 site6The number of qPCR cycles of A methylation modification, ALKBH5 treatment and ALKBH5+ EDTA treatment are not obviously different. The above experiment results show that the visualized RNA methylation detection method in the above embodiment has accurate detection effect on RNA methylation, and can also be used for detecting m6The A demethylase ALKBH5 activity, e.g. at m6The ALKBH5 inhibitor is added in the A demethylation reaction process, and the application can be expanded to m6Screening of A demethylase inhibitor or screening of methylase inhibitor.
Example 3 visual RNA methylation detection method for detecting methylation modification of LncRNA-MALAT1 in HEK293T cells
LncRNA-MALAT 1A 2515 methylation site (m)6A) Is a characteristic feature of LncRNA-MALAT1, and whether the LncRNA-MALAT1 has methylation modification in HEK293T cells can be effectively reflected by detecting the site. As a control, the non-methylation site LncRNA-MALAT 1A 2511 of LncRNA-MALAT1 was selected.
The specific detection steps are as follows:
(1) preparation of a detection sample:
a549 cells were collected and cultured in DMEM containing 10% FBS on 5% CO2Culturing at 37 deg.C, extracting Total RNA from cells with E.Z.N.A.HP Total RNA Kit, and the extraction method is described in the specification.
(2)m6And A, methylation detection:
the detection method was the same as in example 1.
With the difference that for detecting m6The sequence of the specific probe PP of A is as follows:
the sequence of the probe PP specific to the corresponding non-methylated modification site (A) as a control is:
the results of the detection are shown in FIG. 5.
As a result, it was found that LncRNA-MALAT 1A 2515 (m)6A) And LncRNA-MALAT 1A 2511 (A). RT-qPCR results showed that the amplification rate for LncRNA-MALAT 1A 2515 was slower using the probe in the above example, with the Ct value for the qPCR cycle number significantly higher than that of A2511, thus indicating the presence of m at the LncRNA-MALAT 1A 2515 site6And (C) modifying. The above experimental results show that the method in the above embodiment can effectively detect whether m exists on a specific site of a target RNA6And A is subjected to methylation modification.
Sensitivity of visual RNA methylation detection method
An oligonucleotide sequence (oligo-1-A) was synthesized, which was specifically identified as:
subjecting the oligonucleotide sequence to m6A methylation modification (bold underlined) to give oligo-1-m6A. oligo-1-A and oligo-1-m6A was mixed in a ratio of 99:1 so that the degree of methylation was 1%.
With oligo-1-A and oligo-1-m6The mixture A was used as a sample, and the detection was carried out at a concentration of 10pmol, 1pmol, 100fmol, 10fmol, 1fmol, 100amol, or 10amol, respectively.
The specific detection method was the same as in example 1.
The difference is that the nucleotide sequence of the specific probe PP is as follows:
the results are shown in FIGS. 6 to 7.
Drawing a standard curve according to the detection result, and finding that the standard curve equation is as follows:
y＝3.512x-18.842；
wherein R is20.9946, the linear range is: 10 to 1 pmol. The detection limit was 100 amol. The visualized RNA methylation detection method provided by the embodiment of the invention has a good linear relation and can be used for quantitative analysis of methylated RNA.
Visual RNA methylation detection method and detection effect difference of similar method
oligo-1-A and oligo-1-m as in the above examples6A is a sample.
The detection samples were examined using the visual RNA methylation detection method of example 1 above and similar methods (see Yu, Xiao, Ye, Wang, Qian, & Tang, et al (2018), An electrophoresis and ligation-based qPCR amplification method for the radiolabeling-free detection of focus-specific n 6-methylation modifications, Angewandte Chemie.DOI: 10.1002/anie.201807942), respectively.
The detection results are shown in fig. 8.
It can be found that similar methods only detect differences in cycle values of about 1.2 RT-qPCR for methylated and unmethylated sites, but that differences in cycle values of 25.2 ± 2.55 RT-qPCR can be achieved using the visualized RNA methylation detection method in the above example. The difference is more obvious, and the detection sensitivity and accuracy are higher.
Visualization effect of visualization RNA methylation detection method
oligo-1-A and oligo-1-m as in the above examples6A is a sample.
The test sample was tested using the visual RNA methylation detection method described above in example 1. In order to embody the visualization of the method, different result display modes are adopted for qualitative determination.
The results of the electrophoresis by PAGE are shown in FIG. 9.
The results using the golden view dye are shown in fig. 10.
The results using SYBR Green I dye are shown in FIG. 11.
PAGE electrophoresis and methods of use of the dyes are performed with reference to conventional methods or kit instructions in the art.
It can be found that the detection results of methylation and non-methylation can be visually distinguished by adopting different result presentation modes, for example, when PAGE is used as a detection means, the more products are, the higher the intensity of the strip is, and thus the detection results of methylation and non-methylation can be distinguished; when using the golden view stain, the more the product, the lighter the color, the light orange/yellow (negative control orange), and the more brown precipitate under white light; under the ultraviolet light, the more products, the higher the fluorescence intensity, so that the detection results of methylation and non-methylation can be distinguished by the color under the white light and the ultraviolet fluorescence intensity; SYBR Green I staining distinguishes methylated from unmethylated measurements by product color (more product more yellow/yellow-Green (orange negative control)). The above results demonstrate that visual detection of RNA methylation and non-methylation modifications can be achieved using the visual RNA methylation detection method of example 1.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhongshan university
<120> visual RNA methylation detection method and application thereof
<130>
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Claims (10)
1. A set of RNA methylation detection probes, wherein the RNA methylation detection probes comprise:
a specific probe PP, which comprises the following components in sequence: a sequence A complementary paired with one end of the methylation detection site, a sequence of region F1c, a sequence of region F2c, a sequence of region SHSc, and a sequence B complementary paired with the other end of the methylation detection site;
the sequence A and the sequence B are combined with a sequence to be detected, in which a methylation detection site is located, respectively to form a ring structure; a FIP sequence comprising, in order: a F1c region sequence and a F2 region sequence, wherein the F2 region is complementary to the F2c region sequence;
a BIP sequence comprising in order: the B1c region sequence and the B2 region sequence;
an SLP sequence comprising, in order: a sequence of region B1c, a sequence of region B2, a sequence of region B1 and a sequence of region SHSc, wherein said region B1 is complementary to said region B1 c.
2. The RNA methylation detection probe of claim 1, wherein the nucleotide sequence of the RNA methylation detection probe is:
specific probe PP: 5'-TACGACTTTTACTTCCTCTATTTCGACACGACACGATTTGGAACTCTGCTCGACGGATTAAATAATACAGTCTGCCCACAACCTTTACCTACGGAAACCTTG-3', respectively;
or
5’-TTCACTCATCTTCACACTTTCGACACGACACGATTTGGAACTCTGCTCGACGGATTAAATAATACAGTCTGCCCACAACCTTTACTGAAATTGAGTAATATCAG-3’；
Or
5’-CCTTCACATTTTTCAAACTAAGCTACTTTTCGACACGACACGATTTGGAACTCTGCTCGACGGATTAAATAATACAGTCTGCCCACAACCTTTAATTACTTCCGTTACGAAAG-3’；
The 5' end of the specific probe PP is preferably modified with a phosphate group Phos.
3. The RNA methylation detection probe of claim 1 or 2, wherein the nucleotide sequence of the RNA methylation detection probe is:
FIP sequence: 5'-CGACACGACACGAAAAAATCCGTCGAGCAGAGTTCC-3', respectively;
the BIP sequence: 5'-ATCGTCGTGACTGTTTCCCTAACCCTAACCCTAACCC-3', respectively;
SLP sequence: 5'-ATCGTCGTGACTGTTTCCCTAACCCTAACCCTAACCCTTTCAGTCACGACGATTTTTAATACAGTCTGCCCACAACC-3' are provided.
4. Use of the RNA methylation detection probe of any one of claims 1 to 3 for RNA methylation detection.
5. Use of the RNA methylation detection probe of any one of claims 1 to 3 for preparing an RNA methylation detection product; the product comprises a detection reagent and a detection kit.
6. An RNA methylation detection method comprises the following steps:
(1) mixing RNA to be detected with the specific probe PP in the claim 2 for cyclization to obtain a reaction product 1;
(2) mixing the reaction product 1 with Bst 2.0DNA polymerase and SplintR ligase to carry out ring closure to obtain a reaction product 2;
(3) mixing the reaction product 2 with the FIP sequence, the BIP sequence, the SLP sequence and the Bst 2.0DNA polymerase described in claim 3, carrying out isothermal amplification, and judging whether the RNA locus to be detected contains methylation modification according to the amount of the amplification product.
7. The method for detecting RNA methylation according to claim 6, wherein the reaction conditions in step (1) are as follows: the reaction is carried out at 90-98 ℃ for 0.5-1.5 minutes, at 75-85 ℃ for 1-3 minutes, at 65-75 ℃ for 1-3 minutes, at 55-65 ℃ for 1-3 minutes, at 45-55 ℃ for 1-3 minutes, at 35-45 ℃ for 1-3 minutes, and at 25-35 ℃ for 5-10 minutes.
8. The method for detecting RNA methylation according to claim 6, wherein the reaction conditions in step (2) are as follows: reacting at 35-40 deg.C for more than 20min, and at 65 deg.C for more than 10 min to inactivate the enzyme, and terminating the reaction.
9. The method for detecting RNA methylation according to claim 6, wherein the reaction conditions in step (3) are as follows: reacting for 30-120 min at 60-65 ℃.
10. The method for detecting RNA methylation according to claim 6, wherein the qualitative criteria are:
compared with non-methylated sites, the amplification product is obviously reduced, which indicates that the RNA site to be detected contains methylated sites.
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