A method for detecting microRNA epigenetic modifications based on a two-step probe hybridization purification strategy combined with liquid chromatography-mass spectrometry

Abstract

The present invention establishes a method for simultaneously detecting multiple epigenetic modifications of microRNA. Based on the two-step probe hybridization purification of microRNA, enzymatic hydrolysis is performed, and then UPLC-MS / MS nucleoside analysis and detection are performed, thereby realizing the quantitative detection of multiple epigenetic modifications of microRNA at the same time. The method has the characteristics of strong resistance to matrix interference, high detection sensitivity, low false positive rate, accurate quantification, and can simultaneously perform qualitative and quantitative analysis on microRNA epigenetic modifications.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry detection, and specifically relates to a microRNA epigenetic modification detection method based on liquid chromatography tandem mass spectrometry combined with a two-step probe hybridization purification strategy. Background Art

[0002] RNA modification is an important part of epigenetics, affecting almost all life processes in which RNA participates. It has attracted a lot of attention since its discovery. MicroRNA plays an important role in regulating gene expression and has good application prospects in the early diagnosis and treatment of diseases such as cancer and environmental toxicology. In recent years, studies have found that 6-methyladenosine (m6-methyladenosine) exists in mammalian microRNA. 6 A), 5-methylcytosine (5-methylcytosine, m 5 C), 7-methylguanidine (m 7 These modifications affect the production and function of microRNAs, and the content of microRNAs in cancer tissues and normal tissues is significantly different. The importance of microRNA epigenetic modifications has gradually become prominent. However, due to the lack of suitable detection methods, microRNA epigenetic modifications have not been widely studied, and there is still controversy about whether microRNA sequences contain certain modifications.

[0003] Liquid chromatography tandem mass spectrometry (LC-MS / MS) is used for RNA modified nucleoside analysis. It has the advantages of high sensitivity, strong recognition of modification types and accurate quantification. It is widely used for characterization of RNA overall modification, as well as discovery and quantitative analysis of modifications in tRNA and rRNA. Due to the low content of microRNA and high sequence similarity, existing methods cannot obtain microRNA from cells and tissues in the quantity and purity required by the LC-MS / MS method, resulting in limited application of this method in the characterization of microRNA modifications.

[0004] There are two commonly used hybridization forms in the literature for microRNA hybridization purification methods. One is that RNA samples are hybridized with complementary sequence probe molecules in a solution, and then separated by combining with solid phase carriers such as magnetic beads. We call this liquid phase hybridization. The other is that complementary sequence probe molecules are first fixed on the surface of carriers such as magnetic beads, and then mixed with RNA samples for hybridization reaction, which is called solid phase hybridization. Although we have optimized the hybridization process, washing conditions, etc. in many aspects, the anti-interference ability of using liquid phase hybridization method or solid phase hybridization method alone cannot meet the requirements of LC-MS / MS nucleoside analysis.

[0005] "3'-Terminal 2'-O-methylation of lung cancer miR-21-5p enhances itsstability and association with Argonaute 2" ［1］ The authors reported that a two-step purification scheme of liquid phase hybridization purification plus complementary sequence protection enzymatic hydrolysis can be used to obtain highly pure target microRNAs, and this method was combined with LC-MS / MS to successfully detect the A in miR-21-5p. m Modification. We tried to use this purification method to extract and purify microRNAs containing other modifications, but due to the low recovery rate, it could not be used for quantitative characterization of microRNA modifications. This purification method involves multiple steps of enzymatic hydrolysis, which is not only cumbersome, but also has a narrow window of optimal experimental conditions, and the results are easily affected by the operation. 《A Novel Method to Purify Small RNAs from Human Tissues for Methylation Analysis by LC-MS / MS》 ［2］ The report stated that when the researchers operated the method under optimal conditions, the microRNA recovery rate obtained was only about 2%.

[0006] Application and Prospect of Liquid Chromatography-Mass Spectrometry in the Study of Nucleic Acid Epigenetic Modification ［3］ It was reported that there are two main analytical strategies for nucleic acid modification detection based on LC-MS / MS: one is the accurate quantitative analysis using nucleosides as targets; the other is based on a certain oligonucleotide sequence to identify and characterize the modification sites therein, but it mainly detects rRNA, tRNA and mRNA modifications. There are no reports on the accurate quantitative analysis of microRNA epigenetic modifications using LC-MS / MS.

[0007] DNA / RNA modification analysis based on liquid chromatography-mass spectrometry ［4］Reported: Established a LC-ESI-MS / MS-based method for detecting m 6 A content method, no report on the accurate quantitative analysis of microRNA epigenetic modifications by LC-MS / MS

[0008] Accurate determination of microRNA epigenetic modifications is the basis for studying the mechanism of microRNA modification and its further application. Therefore, there is an urgent need to develop reliable methods for characterizing microRNA modifications. However, existing technologies have not yet found methods for accurate quantitative analysis of multiple microRNA modified nucleosides simultaneously.

[0009] References

[0010] [1]Liang H , Jiao Z , Rong W ,et al.3'-Terminal 2'-O-methylation of lung cancer miR-21-5p enhances its stability and association with Argonaute2.[J].Oxford Academic, 2020(13).DOI:10.1093 / nar / gkaa504.

[0011] [2]Yang, R.; Li, J.; Wu, Y.; Jiang, X.; Qu, S.; Wang, Q.; Liang, H.;

[0012] [3] He Weiwei, Xu Hua, Qu Minmin, Zhang Yajiao, Xu Bin, & Xie Jianwei. (2021). Application and prospect of liquid chromatography-mass spectrometry in the study of nucleic acid epigenetic modification. Chinese Journal of Pharmacology and Toxicology, 035(005), 321-335.

[0013] [4] Huang Wei. (2016). DNA / RNA modification analysis based on liquid chromatography-mass spectrometry. (Doctoral dissertation, Wuhan University). Summary of the invention

[0014] Problems to be solved by the invention:

[0015] In view of the problems existing in the prior art, the present invention has established a method for simultaneously detecting multiple epigenetic modifications of microRNA. Based on the two-step probe hybridization purification of microRNA, enzymatic hydrolysis and then UPLC-MS / MS nucleoside analysis and detection are performed, thereby realizing the quantitative detection of multiple epigenetic modifications of microRNA at the same time. The method has the characteristics of strong resistance to matrix interference, high detection sensitivity, low false positive rate and accurate quantification, and can simultaneously perform qualitative and quantitative analysis on multiple epigenetic modifications of microRNA.

[0016] In the first aspect of the present invention, a preliminary scheme for two-step probe hybridization purification of microRNA was established.

[0017] In summary: microRNA is first purified by liquid phase hybridization and then by solid phase hybridization.

[0018] 1. Preliminary scheme for microRNA liquid phase hybridization purification

[0019] ① Add biotin-containing DNA complementary sequence probe to the extracted microRNA sample, the reaction system is 0.5×SSC buffer solution, and the reaction volume is 60μL;

[0020] ② After gentle mixing, place in a 50℃ water bath for 15h to perform hybridization reaction;

[0021] ③ Prepare 0.1 mg of streptavidin affinity magnetic beads, wash thoroughly and redisperse with 20 μL 0.5×SSC;

[0022] ④ Add the magnetic beads to the hybridization reaction system, mix well, and react at room temperature for 30 minutes;

[0023] ⑤Separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0024] ⑥ Add 200 μL of rinsing solution containing 2×SSC and 0.2% tween20, wash at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0025] ⑦ Add 200 μL of rinsing solution containing 2×SSC, rinse at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0026] ⑧Add 200 μL of rinsing solution containing 0.5×SSC, mix well and incubate at 37°C for 10 min, separate by magnetic stand, discard the supernatant and retain the magnetic beads;

[0027] ⑨ Add 200 μL of enzyme-free ultrapure water, rinse at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0028] ⑩ Add 45-60 μL of enzyme-free ultrapure water, mix well, and incubate at 80°C for 5 min. Immediately place on a magnetic rack for separation, aspirate the supernatant, and transfer it to a clean enzyme-free centrifuge tube for the next experiment.

[0029] 2. Preliminary scheme for microRNA solid phase hybridization purification

[0030] ① Prepare 0.1 mg of streptavidin affinity magnetic beads, wash thoroughly and redisperse with 60 μL 0.5×SSC;

[0031] ② Add the DNA complementary sequence probe containing biotin and place in a mixer to react at room temperature for 30 minutes;

[0032] ③Separate the magnetic beads, discard the supernatant, and wash the magnetic beads three times with 0.5 × SSC buffer solution;

[0033] ④ Mix the microRNA sample with the magnetic beads bound to the complementary DNA sequence probe, keeping the reaction system at 0.5×SSC and the volume at 60-80 μL;

[0034] ⑤ After gentle mixing, place in a 50℃ water bath for 4 hours, take out and shake every hour to keep the magnetic beads dispersed;

[0035] ⑥After the reaction is completed, immediately place it on a magnetic stand for separation, discard the supernatant, and retain the magnetic beads;

[0036] ⑦ Add 200 μL of rinsing solution containing 2×SSC and 0.2% tween20, wash at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0037] ⑧Add 200 μL of rinsing solution containing 2×SSC, rinse at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0038] ⑨Add 200 μL of rinsing solution containing 0.5×SSC, mix well and incubate at 37°C for 10 min, separate by magnetic stand, discard the supernatant and retain the magnetic beads;

[0039] ⑩Add 200 μL of enzyme-free ultrapure water, rinse at room temperature for 10 min in a homogenizer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads;

[0040] ⑪Add 20 μL of enzyme-free ultrapure water, mix well, and incubate at 80°C for 5 min. Immediately place on a magnetic rack for separation, aspirate the supernatant, and transfer it to a clean enzyme-free centrifuge tube for the next experiment.

[0041] In the second aspect of the present invention, a preliminary scheme for enzymatic hydrolysis of microRNA samples after two-step probe hybridization purification was established.

[0042] 1. Add 1 μL of Mix enzyme and 2 μL of 10x enzymatic buffer to the purified microRNA sample, perform enzymatic hydrolysis in a 37°C metal bath, and then heat to 70°C for 10 min to terminate the reaction.

[0043] 2. The enzymatic hydrolysis product was diluted to 60 μL with ultrapure water for UPLC-MS / MS analysis.

[0044] In the third aspect of the present invention, a preliminary scheme for simultaneous quantitative detection of multiple modified nucleosides of microRNA by UPLC-MS / MS was established.

[0045] UPLC conditions: Acquity UPLC BEH C18 reverse phase column (2.1 mm × 100 mm, 1.7 μm particle size, Waters); mobile phase A was ultrapure water or acetic acid-ultrapure water (the volume ratio of acetic acid to ultrapure water was: 0.1%, 0.05%, 0.02%, 0.01%, 0.005%) or acetic acid-ammonium acetate (NH4Ac) aqueous solution (0.1% acetic acid + 2 mM ammonium acetate; 0.01% acetic acid + 0.5 mM ammonium acetate) or ammonium acetate aqueous solution (10 mM ammonium acetate) or ammonium bicarbonate aqueous solution (2 mM NH4HCO3), mobile phase B was 100% acetonitrile, gradient elution; injection volume was 10 μL; the gradient elution conditions were as follows:

[0046] 0~3.0min, 0% B, 0.25mL / min,

[0047] 3.0~6.0min, 0~10.0% B, 0.25mL / min,

[0048] 6.0~10.0min, 10.0%~50.0% B, 0.25mL / min,

[0049] 10.0~12.0min, 100.0% B, 0.25mL / min;

[0050] Mass spectrometry conditions: ion source is Unispray; working in positive ion mode; data acquisition is multiple reaction monitoring (MRM) mode; nitrogen is used for nebulization and desolvation; source temperature is 150°C, desolvation temperature is 400°C, cone gas flow is 150 L / h, desolvation gas flow is 800 L / h, nebulizer gas flow is 7.0 Bar, collision gas is high-purity nitrogen, and collision gas flow is 0.15 mL / min.

[0051] In the fourth aspect of the present invention, the preliminary solutions of the first, second and third aspects are optimized.

[0052] 1. Liquid phase hybridization and solid phase hybridization respectively compared three probe schemes

[0053] For liquid phase hybridization, the recovery rate of P1 probe is the highest, and P2 and P3 decrease in turn, that is, as the length of the spacer arm increases, the extraction efficiency decreases. Therefore, the spacer arm between the complementary sequence of the DNA probe and biotin selected for liquid phase hybridization is AA, that is, the P1 series probe, the sequence is shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6;

[0054] For solid phase hybridization, the recovery rate of P3 probe is the highest, and P2 and P1 decrease in turn, that is, as the length of the spacer arm decreases, the extraction efficiency decreases. The spacer arm between the complementary sequence of the DNA probe used in solid phase hybridization and biotin is AAAAAA, that is, the P3 series probe, the sequence is shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQID NO.13, SEQ ID NO.14.

[0055] 2. Screening of the amount of probe molecules used

[0056] It was finally determined that the optimal amount of probe molecules was 2.5~20pmol.

[0057] 3. Comparison of the anti-interference ability and recovery rate of simple liquid phase hybridization purification, simple solid phase hybridization purification and two-step hybridization purification (liquid phase hybridization purification first, then solid phase hybridization purification)

[0058] Results Compared with the method of using only liquid phase hybridization or solid phase hybridization purification, the two-step probe hybridization purification method had significantly improved anti-interference ability, and its recovery rate was higher than 50%, which was much higher than the recovery rate of the purification method reported in the literature (less than 2%). Finally, the two-step hybridization purification scheme was determined.

[0059] 4. Optimization of enzymatic hydrolysis time

[0060] The enzymatic hydrolysis time was determined to be 120 minutes through experiments.

[0061] 5. Screening and optimization of liquid chromatography mobile phase A in the UPLC-MS / MS method

[0062] The two mobile phases A were compared with 0.01% HAc and 2mM NH4HCO3. The results showed that these two mobile phases had different effects on A m , G m 、m 6 A.m 7 G、m 5 C all have good applicability, good linearity, and sensitive methods. However, for G, U, Cm , U m The method of adding NH4HCO3 to the mobile phase has better linearity and higher sensitivity. Therefore, 2mM NH4HCO3 was comprehensively selected as mobile phase A.

[0063] On the basis of the above optimization, the optimal two-step hybridization purification scheme, the optimal enzymatic hydrolysis scheme and the UPLC and mass spectrometry conditions of UPLC-MS / MS were determined, thereby determining a scheme for analyzing and detecting microRNA modified nucleosides based on two-step probe hybridization purification of microRNA, enzymatic hydrolysis, and then UPLC-MS / MS.

[0064] In a fifth aspect of the present invention, the quantitative accuracy of microRNA modified nucleosides is verified by using the detection method of the present invention.

[0065] Verification results: The ratio of nucleoside signals has a good linear relationship with the microRNA modification ratio, indicating that this method can be used to identify mRNAs in microRNAs. 6 A and m 5 Quantitative assessment of C content and ratio.

[0066] In a sixth aspect of the present invention, the detection method of the present invention is used for quantitative characterization of cell microRNA modification for spike verification

[0067] Verification results: The detection method of the present invention has high sensitivity and accurate quantitative detection, and can be used for quantitative characterization of cellular microRNA modified nucleosides.

[0068] The present invention has the following beneficial effects:

[0069] The present invention establishes a method for analyzing and detecting nucleosides based on two-step probe hybridization purification, enzymatic hydrolysis, and then UPLC-MS / MS, which achieves the improvement of the specificity and recovery rate of the microRNA sample pretreatment step, and provides a highly sensitive and quantitatively accurate detection method for the simultaneous detection of multiple epigenetic modifications of microRNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 , the relationship between probe spacer arm length and hybridization mode and microRNA recovery rate, among which,

[0071] L is liquid phase hybridization, S is solid phase hybridization, and P1, P2, and P3 are the corresponding miR-21-5p complementary DNA probes;

[0072] Figure 2 , the effect of probe usage on microRNA extraction;

[0073] Figure 3, Comparison of anti-interference ability between liquid phase hybridization purification and two-step hybridization purification (using 10-fold single-base mismatch sequence content for evaluation);

[0074] Figure 4 , the anti-interference ability evaluation of the two-step hybridization purification method, in which the sample is a mixed modified microRNA sample,

[0075] a is m 6 A,

[0076] b is m 5 C,

[0077] c is G m ,

[0078] d is U m ;

[0079] Figure 5 , recovery rate of target microRNA in two-step hybridization purification;

[0080] Figure 6 , enzymatic hydrolysis time and m 5 C mass spectrum peak area relationship, where

[0081] a is 40 fmol of miR-10a-m 5 C, UPLC-MS / MS determination of m 5 The peak area of ​​C reaches its peak in the 80-120 minute range.

[0082] b is 400 fmol of miR-10a-m 5 C, UPLC-MS / MS determination of m 5 The peak area of ​​C reaches its peak value in the 30-60 minute interval;

[0083] Figure 7 , 8 The linear fitting working curve of the modified nucleosides is shown in Figure 2, where

[0084] a is A m The linear fitting working curve of

[0085] b is G m The linear fitting working curve of

[0086] c is C m The linear fitting working curve of

[0087] d is U m The linear fitting working curve of

[0088] e is m 6 The linear fitting working curve of A,

[0089] f is m 7 The linear fitting working curve of G,

[0090] g is m 5 The linear fitting working curve of C,

[0091] h is m 6 A m Linear fitting working curve of

[0092] Figure 8 , m measured by UPLC-MS / MS 6 A Linear relationship between the ratio of modified nucleoside to unmodified nucleoside signals and the microRNA modification ratio;

[0093] Fig. 9 , m measured by UPLC-MS / MS 5 The linear relationship between the signal ratio of C-modified nucleosides and unmodified nucleosides and the microRNA modification ratio;

[0094] Fig.10 , cell small RNA samples spiked (1 fmol miR-21-m 6 A) Verification results, where

[0095] a is m 6 A content data, left: positive control; middle: cell small RNA sample; right: cell small RNA sample spiked (1fmol miR-21-m 6 A),

[0096] b Cellular small RNA sample and cell small RNA sample spiked with 1 fmol miR-21-m 6 A) m measured in the sample 6 A modification ratio;

[0097] Fig.11 , cell small RNA samples spiked (1 fmol miR-10a-m 5 C) Verification results, where

[0098] a is m 5 C content data, left: cell small RNA sample; right: cell small RNA sample spiked (1 fmol miR-10a-m 5 C),

[0099] b Cellular small RNA sample and cell small RNA sample spiked with 1 fmol miR-10a-m 5 C) m measured in the sample 5 C modification ratio. DETAILED DESCRIPTION

[0100] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0101] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0102] All materials, reagents, etc. in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0103] Materials and Reagents

[0104] Streptavidin affinity magnetic beads (SMB) and nucleic acid digestion mixed enzyme (Mix) were purchased from New England Biolabs (Ipswich, MA, USA). Tween 20 was purchased from Sigma-Aldrich (St. Louis, MO, USA), SSC buffer solution (20×, pH 7.4) was purchased from Beibo Biotechnology (Shanghai), chromatography-grade acetonitrile was purchased from J&K Technology Co., Ltd. (Beijing); LC / MS-grade acetic acid was purchased from Thermo Fisher Scientific (USA). RNA / DNAase-free ultrapure water was from Invitrogen (NY, USA) and was used in all experiments involving microRNA isolation and purification. Ultrapure water (18.2 MΩ.cm) was prepared by Milli-Q water purifier (USA) in other experiments; other reagents were purchased from Sinopharm Group (Beijing); all reagents were of analytical grade or higher purity.

[0105] In some experiments, open experiments involving sample and solution preparation and transfer were all conducted in a clean bench. The centrifuge tubes and pipette tips used in the experiments were all RNase-free products from Axygen (Corning, USA).

[0106] Oligonucleotide sequence:

[0107] The biotinylated single-stranded DNA probe sequences, artificially synthesized single-stranded RNA sequences (microRNA mimics), and artificially synthesized single-stranded RNA sequences containing methyl modifications were all ordered from ReboBio (Guangzhou) and used after purification by high-performance liquid chromatography. The sequences of each oligonucleotide are shown in Tables 1 to 3.

[0108] Table 1. Single-stranded DNA probe sequences

[0109]

[0110] Table 2. Artificially synthesized single-stranded RNA (microRNA mimics) sequences

[0111]

[0112] Table 3. Sequences of artificially synthesized single-stranded RNA containing methyl modifications (microRNA mimics containing modified nucleosides)

[0113]

[0114] Example 1: Two-step probe hybridization purification

[0115] 1. Liquid phase hybridization purification of microRNA

[0116] (1) Add 10 pmol of biotin-containing DNA complementary sequence probe P1 (see Table 1) to the extracted microRNA sample. The reaction system is 0.5 × SSC buffer solution and the reaction volume is 60 μL.

[0117] (2) After gentle mixing, place in a 50°C water bath for 15 hours to allow hybridization reaction.

[0118] (3) Prepare 0.1 mg of streptavidin affinity magnetic beads, wash thoroughly, and redisperse them in 20 μL 0.5 × SSC.

[0119] (4) Add the magnetic beads to the hybridization reaction system, mix well, and react at room temperature for 30 minutes.

[0120] (5) Separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0121] (6) Add 200 μL of rinse solution containing 2× SSC and 0.2% tween 20, wash at room temperature for 10 min in a mixer, separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0122] (7) Add 200 μL of rinsing solution containing 2×SSC, rinse at room temperature for 10 min in a mixer, separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0123] (8) Add 200 μL of rinsing buffer containing 0.5× SSC, mix well and incubate at 37°C for 10 min. Separate on a magnetic rack, discard the supernatant and retain the magnetic beads.

[0124] (9) Add 200 μL of enzyme-free ultrapure water, rinse in a mixer at room temperature for 10 min, separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0125] (10) Add 45-60 μL of enzyme-free ultrapure water, mix well, and incubate at 80°C for 5 min. Immediately separate the tube on a magnetic rack, aspirate the supernatant, and transfer it to a clean enzyme-free centrifuge tube for the next step of the experiment.

[0126] 2. MicroRNA solid phase hybridization purification

[0127] (1) Prepare 0.1 mg of streptavidin affinity magnetic beads, wash thoroughly, and redisperse in 60 μL 0.5× SSC.

[0128] (2) Add 10 pmol of biotin-containing DNA complementary sequence probe P3 and place in a mixer to react at room temperature for 30 min.

[0129] (3) Separate the magnetic beads, discard the supernatant, and wash the magnetic beads three times with 0.5×SSC buffer solution.

[0130] (4) Mix the RNA sample with the magnetic beads bound to the complementary DNA sequence probe, keeping the reaction system at 0.5 × SSC and the volume at 60-80 μL.

[0131] (5) After gentle mixing, place the beads in a 50°C water bath for 4 hours, taking them out and shaking them every hour to keep the beads dispersed.

[0132] (6) After the reaction is completed, immediately place it on a magnetic rack for separation, discard the supernatant, and retain the magnetic beads.

[0133] (7) Add 200 μL of rinse buffer containing 2× SSC and 0.2% tween 20, wash at room temperature for 10 min in a mixer, separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0134] (8) Add 200 μL of rinsing solution containing 2×SSC, rinse at room temperature for 10 min in a mixer, separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0135] (9) Add 200 μL of rinsing buffer containing 0.5× SSC, mix well and incubate at 37°C for 10 min. Separate on a magnetic rack, discard the supernatant and retain the magnetic beads.

[0136] (10) Add 200 μL of enzyme-free ultrapure water, rinse at room temperature for 10 min in a homogenizer, separate on a magnetic rack, discard the supernatant, and retain the magnetic beads.

[0137] (11) Add 20 μL of enzyme-free ultrapure water, mix well, and incubate at 80°C for 5 min. Immediately separate the tube on a magnetic rack, aspirate the supernatant, and transfer it to a clean enzyme-free centrifuge tube for the next step of the experiment.

[0138] Example 2: Enzymatic hydrolysis of purified miRNA samples

[0139] 1. Add 1 μL of Mix enzyme (New England Biolabs) and 2 μL of 10x enzymatic hydrolysis buffer to the purified microRNA sample, place in a 37°C metal bath for 2 h for enzymatic hydrolysis, and then heat to 70°C for 10 min to terminate the reaction.

[0140] 2. Dilute the enzymatic hydrolysis product to 60 μL with ultrapure water for liquid-mass analysis.

[0141] Example 3, UPLC-MS / MS analysis

[0142] Nucleosides were separated using an Acquity UPLC BEH C18 reverse phase column (2.1 mm × 100 mm, 1.7 μm particle size, Waters). Mobile phase A was 2 mM ammonium bicarbonate in water, and mobile phase B was 100% acetonitrile. The following gradient elution was used for UPLC separation:

[0143] 0~3.0min, 0% B, 0.25mL / min;

[0144] 3.0~6.0min, 0~10.0% B, 0.25mL / min;

[0145] 6.0~10.0min, 10.0%~50.0% B, 0.25mL / min;

[0146] 10.0~12.0min, 100.0% B, 0.25mL / min.

[0147] The injection volume was 10 μL.

[0148] The mass spectrometer ion source was Unispray, working in positive ion mode, data acquisition was in multiple reaction monitoring (MRM) mode, and nitrogen was used for nebulization and desolvation. The source temperature was 150°C, and the desolvation temperature was 400°C. The cone gas flow rate was 150L / h, the desolvation gas flow rate was 800L / h, and the nebulizer gas flow rate was 7.0Bar. High-purity nitrogen (99.999%) was used as the collision gas. The collision gas flow rate was 0.15mL / min. The acquisition parameters are shown in Table 4.

[0149] Table 4. Mass spectrometry acquisition parameters for target nucleosides

[0150]

[0151] Example 4: Screening of probe spacer arm length in two-step hybridization

[0152] The extraction, purification and recovery rates of target microRNAs were compared between three spacer arm probe schemes and two liquid and solid phase hybridization methods.

[0153] For liquid phase hybridization, the recovery rate using the P1 probe was the highest, followed by P2 and P3, that is, as the length of the spacer arm increased, the extraction efficiency decreased.

[0154] For solid phase hybridization, the recovery rate using P3 probe is the highest, followed by P2 and P1, that is, as the length of the spacer arm decreases, the extraction efficiency decreases.

[0155] The complementary sequence DNA probes used in liquid phase hybridization are P1 series probes, and the solid phase hybridization is P3 series probes to obtain a better recovery rate, thereby improving the overall recovery rate of the two-step hybridization scheme. Figure 1 shown.

[0156] Therefore, the spacer arm between the complementary sequence of the DNA probe used in liquid phase hybridization and biotin is AA, that is, the P1 series probes; the spacer arm between the complementary sequence of the DNA probe used in solid phase hybridization and biotin is AAAAAA, that is, the P3 series probes.

[0157] Example 5: Screening of probe molecule usage

[0158] When miR-21-me was used as the target RNA sequence and the addition amount of biotinylated complementary sequence probe molecules was 2.5, 5, 10, 15, and 20 pmol, respectively, there was no significant difference in the mass spectrometry signal of the extracted miR-21-me enzymatic hydrolysis product. Figure 2 When the probe molecule addition amount was 25pmol, the signal decreased to a certain extent. It may be that the affinity site of magnetic beads streptavidin was saturated at this time, resulting in some miR-21-me hybridized with the probe not being captured by the magnetic beads. Therefore, the amount of probe molecules used was selected to be 2.5-20pmol.

[0159] Example 6: Comparison of two-step liquid and solid phase hybridization purification with one-step liquid phase hybridization purification or one-step solid phase hybridization purification

[0160] The target microRNA sequence in the sample is extracted and purified by sequentially using a liquid phase hybridization purification method and a solid phase hybridization purification method, namely a two-step hybridization purification method.

[0161] The liquid phase and solid phase two-step hybridization purification method has better anti-interference ability than the simple liquid phase hybridization purification one-step method or solid phase hybridization purification method. Figure 3 shown.

[0162] When tested with 10-fold the amount of single-base mismatch sequences, the amount of single-base mismatch sequences detected in the two-step hybridization purification method was only 1 / 3 of that in the simple liquid phase hybridization purification method.

[0163] The anti-interference ability of the two-step hybridization scheme was evaluated using mixed modified microRNA samples. It was found that the modified nucleosides of other modified microRNAs other than the complementary sequence were basically undetectable. For probes with single base mismatches in the same family (Let-7a-5p and Let-7e-5p), the modified nucleosides content that could be detected was 10% of that of the probe with the completely complementary sequence. The results are shown in Figure 4Compared with the method using only liquid phase hybridization or solid phase hybridization purification, the anti-interference ability of this method is significantly improved. The recovery rate of the two-step hybridization purification method was evaluated, and the recovery rates of the five microRNA mimics were shown in Figure 5 The recovery rates were all higher than 50%, which was much higher than the recovery rates of purification methods reported in the literature (less than 2%).

[0164] Example 7: Screening and optimization of enzymatic hydrolysis time

[0165] For 40 fmol of miR-10a-m 5 C, UPLC-MS / MS determination of m 5 The peak area of ​​C reaches its peak in the 80-120 minute interval. Figure 6 (a);

[0166] For 400 fmol of miR-10a-m 5 C, UPLC-MS / MS determination of m 5 The peak area of ​​C reaches its peak value in the range of 30 to 60 minutes, and does not change much with the increase of enzymatic hydrolysis time. Figure 6 (b)

[0167] Therefore, the enzymatic hydrolysis time was selected as 120 min in subsequent experiments.

[0168] Example 8: Optimization of Liquid Chromatography Mobile Phase A

[0169] As optimization: The HPLC mobile phase A was optimized in the UPLC-MS / MS method. The tested mobile phase A items included: ultrapure water, acetic acid-ultrapure water (the volume ratio of acetic acid to ultrapure water was 0.1%, 0.05%, 0.02%, 0.01%, 0.005%); acetic acid-ammonium acetate (NH4Ac) aqueous solution (0.1% acetic acid + 2mM ammonium acetate; 0.01% acetic acid + 0.5mM ammonium acetate); ammonium acetate aqueous solution (10mM ammonium acetate); ammonium bicarbonate aqueous solution (2mM NH4HCO3). 100% acetonitrile was used as the mobile phase B.

[0170] The results showed that m 5 C as an example, using different concentrations of acetic acid-water as the mobile phase, as the proportion of acetic acid decreases, m 5 The retention time of C increased slightly, and the mass spectrum peak area of ​​m5C increased significantly. When pure water was used as the mobile phase, m 5 C has the largest mass spectrum peak area. However, pure water or extremely low concentration acetic acid-water solution as mobile phase has weak buffering capacity, is susceptible to matrix interference, and has poor retention time / peak area stability. Therefore, 0.01% HAc was selected as mobile phase A.

[0171] The applicability of the acetic acid-ammonium acetate system to nucleoside samples containing digestion buffer in the matrix was also investigated. However, even with the addition of 0.5 mM ammonium acetate (0.01% HAc + 0.5 mM NH4Ac), m 5 The signal intensity of C mass spectrometry was greatly reduced. Whether adding ammonium acetate to the acetic acid buffer system or directly using ammonium acetate-water solution as the mobile phase, the m 5 The mass spectrometry peak area of ​​C was about one order of magnitude lower than that of the acetic acid mobile phase system. This indicates that the acetic acid-ammonium acetate system cannot be used to reduce the detection m 5 In addition, ammonium bicarbonate is also used in LC-MS / MS mobile phases. It has been reported that the use of ammonium bicarbonate-water mobile phase can greatly reduce the matrix ionization suppression effect on 5-hydroxymethycytosine (5hmC). Therefore, the two mobile phases A, 0.01% HAc and 2mM NH4HCO3, were compared. The results showed that these two mobile phases had no significant effect on A m , G m 、m 6 A.m 7 G、m 5 C all have good applicability, good linearity, and sensitive methods. However, for G, U, C m , U m The method of adding NH4HCO3 to the mobile phase has better linearity and higher sensitivity. Therefore, 2mM NH4HCO3 was comprehensively selected as mobile phase A.

[0172] As an optimization: the UPLC-MS / MS nucleoside analysis method with the addition of NH4HCO3 in the mobile phase was evaluated. The fitting curves, linear ranges, linear equations and method detection limits of the eight modified nucleosides are shown in Figure 2. Figure 7 and Table 5. This method demonstrates that it has high sensitivity and accurate quantitative ability for m 6 A.m 5 C.m 7 G.A m , C m , G m The quantitative limit of analysis of various modified nucleosides can reach pM level (0.1~0.4fmol).

[0173] Table 5. Detection linear range and linear equation of nucleosides and modified nucleosides

[0174]

[0175] Example 9: Verification of the accuracy of quantitative analysis of microRNA modified nucleosides

[0176] By using synthetic normal microRNA and microRNA containing methylated nucleosides, microRNA mixtures containing different modification ratios were prepared, and whether the ratio of modified nucleosides to unmodified nucleosides determined by the UPLC-MS / MS method was linearly related to the configured microRNA modification ratio was examined, so as to verify the quantitative ability of the method of the present invention.

[0177] 1. The detection method of the present invention is used for m in microRNA 6 Verification of the accuracy of quantitative analysis of A modifications

[0178] (1) Prepare miR-21-m at a concentration of 1 fmol / μL 6 A and miR-21-5p solution, the solvent is 0.5×SSC buffer.

[0179] (2) Prepare microRNA mixtures with different modification ratios: 1.25 μL, 2.5 μL, 5.0 μL, 7.5 μL, 10 μL, and 15 μL of miR-21-m 6 Solution A, add 98.75μL, 97.5μL, 95.0μL, 92.5μL, 90μL, 85μL of miR-21-5p solution respectively, mix well. At the same time, measure 100μL of miR-21-5p solution as the modified blank sample.

[0180] (3) According to the method of Examples 1-8, the microRNA mixture solution was subjected to two-step hybridization purification, enzymatic hydrolysis and UPLC-MS / MS analysis.

[0181] (4) m 6 The ratio of the mass spectrometry peak area of ​​A to A is related to the content of miR-21-m in the microRNA mixture. 6 A ratio was linearly fitted to examine the m 6 Is the A / A ratio linearly related to the modification ratio in the microRNA mixture?

[0182] Results: Among the tested microRNA mixtures, miR-21-m 6 A accounts for the proportion of total microRNA (miR-21-m 6 A and miR-21-m 6 The ratio of A+miR-21-5p) is 1.25%~15%. There are 6 A in the miR-21-5p sequence, and miR-21-m 6 There are 5 A's and 1 m in A 6 A, therefore, m 6 The ratio of A / A is 0.21%~2.6%. Figure 8As shown in Figure 2, within this range, the ratio of nucleoside signals has a good linear relationship with the microRNA modification ratio, indicating that the detection method of the present invention can be used for mRNA in microRNA. 6 Quantitative evaluation of A content and proportion.

[0183] 2. The detection method of the present invention is used for m in microRNA 5 Verification of the accuracy of quantitative analysis of C modifications

[0184] Refer to the steps (1)-(4) in 1 above to prepare the mixture containing different ratios of miR-10a-m 5 C and miR-10a-5p mixed microRNA samples were subjected to two-step hybridization purification, enzymatic digestion and UPLC-MS / MS analysis to investigate m 5 The linear relationship between C / C and microRNA modification ratio is due to miR-10a-m 5 All nucleosides in C are m 5 C, therefore, the ratio of modified nucleosides in the mixed sample is consistent with the ratio of modified microRNA. 5 C / C value and m 5 C / (m 5 C+C) value is close, m 5 C / C value instead of m 5 C / (m 5 C+C) value is used for quantitative characterization and evaluation. Fig. 9 As shown in Figure 2, within this range, the ratio of nucleoside signals has a good linear relationship with the microRNA modification ratio, indicating that the detection method of the present invention can be used for mRNA in microRNA. 5 Quantitative assessment of C content and ratio.

[0185] Example 10: Validation of the detection method of the present invention for detecting microRNA epigenetic modification in cells

[0186] 1. The detection method of the present invention is used to detect m in microRNA in cells 6 Verification of the accuracy of quantitative analysis of A modifications

[0187] To verify the sensitivity and quantitative accuracy of the detection method of the present invention, 1 fmol of synthetic miR-21-m 6 A sequence was then used for the two-step hybridization extraction and purification of miR-21-5p. After enzymatic digestion, miR-21-5p from the positive control sample, the cell small RNA sample, and the cell small RNA spiked sample was analyzed by UPLC-MS / MS to obtain the m 6 A content data and modification ratio data are shown in Fig.10, where a is the content data and b is the modification ratio data.

[0188] m was detected in miR-21-5p extracted from cellular small RNA spike samples 6 The A content is 0.44±0.04fmol, and the calculated m 6 The A / A ratio was 0.39%±0.04%, which was consistent with the theoretical calculated value (0.38%).

[0189] 2. The detection method of the present invention is used to detect m in microRNA in cells 5 Verification of the accuracy of quantitative analysis of C modifications

[0190] To verify the sensitivity and quantitative accuracy of the detection method of the present invention, 1 fmol of synthetic miR-10a-m 5 C sequence, followed by miR-10a-m 5 C Two-step hybridization extraction and purification experiment, miR-10a-m from positive control sample, cell small RNA sample and cell small RNA spike sample 5 C, m after enzymatic hydrolysis, analyzed by UPLC-MS / MS 5 C content data and modification ratio data are shown in Fig.11 , where a is the content data and b is the modification ratio data.

[0191] miR-10a-m extracted from cellular small RNA spike samples 5 m detected in C 5 The C content is 2.5 fmol, and the calculated m 5 The C / C ratio is 5.4%, which is consistent with the theoretical calculated value.

[0192] This result shows that the detection method established by the present invention can be used for the quantitative characterization of cellular microRNA modified nucleosides.

[0193] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for simultaneously detecting multiple epigenetic modifications of microRNA, characterized in that: The modified nucleoside included in the multiple epigenetic modifications of the microRNA is A m , G m , C m , U m 、m 6 A.m 7 G、m 5 C.m 6 A m Any of the above, wherein the method comprises the following steps: (1) Liquid phase hybridization purification of microRNA ① Add 2.5~20pmol of biotin-containing DNA complementary sequence probe P1 to the extracted microRNA sample, the reaction system is 0.5×SSC buffer solution, the reaction volume is 60μL, The sequences of the biotin-containing DNA complementary sequence probe P1 series are shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6; ② After gentle mixing, place in a 50℃ water bath for 15h to perform hybridization reaction; ③ Prepare 0.1 mg of streptavidin affinity magnetic beads, wash thoroughly and redisperse with 20 μL 0.5×SSC; ④ Add the magnetic beads to the hybridization reaction system, mix well, and react at room temperature for 30 minutes; ⑤Separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑥ Add 200 μL of rinsing solution containing 2×SSC and 0.2% tween20, wash at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑦ Add 200 μL of rinsing solution containing 2×SSC, rinse at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑧Add 200 μL of rinsing solution containing 0.5×SSC, mix well and incubate at 37°C for 10 min, separate by magnetic stand, discard the supernatant and retain the magnetic beads; ⑨ Add 200 μL of enzyme-free ultrapure water, rinse at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑩Add 45-60 μL of enzyme-free ultrapure water, mix well, and warm in 80℃ for 5 min. After taking out, immediately separate on a magnetic rack, aspirate the supernatant, and transfer it to a clean enzyme-free centrifuge tube for the next experiment; (2) microRNA solid phase hybridization purification ① Prepare 0.1 mg of streptavidin affinity magnetic beads, wash thoroughly and redisperse with 60 μL 0.5×SSC; ②Add 2.5~20pmol of DNA complementary sequence probe P3 containing biotin, and place in a mixer to react at room temperature for 30 minutes. The sequences of the biotin-containing DNA complementary sequence probe P3 series are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, and SEQ ID NO.14; ③Separate the magnetic beads, discard the supernatant, and wash the magnetic beads three times with 0.5×SSC buffer solution; ④ Mix the microRNA sample with the magnetic beads bound to the complementary DNA sequence probe, keeping the reaction system at 0.5×SSC and the volume at 60-80 μL; ⑤ After gentle mixing, place in a 50℃ water bath for 4 hours, take out and shake every hour to keep the magnetic beads dispersed; ⑥After the reaction is completed, immediately place it on a magnetic stand for separation, discard the supernatant, and retain the magnetic beads; ⑦ Add 200 μL of rinsing solution containing 2×SSC and 0.2% tween20, wash at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑧Add 200 μL of rinsing solution containing 2×SSC, rinse at room temperature for 10 min in a mixer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑨Add 200 μL of rinsing solution containing 0.5×SSC, mix well and incubate at 37°C for 10 min, separate by magnetic stand, discard the supernatant and retain the magnetic beads; ⑩Add 200 μL of enzyme-free ultrapure water, rinse at room temperature for 10 min in a homogenizer, separate on a magnetic stand, discard the supernatant, and retain the magnetic beads; ⑪Add 20 μL of enzyme-free ultrapure water, mix well, and warm in 80℃ for 5 min. After taking out, immediately place it on a magnetic stand for separation, aspirate the supernatant, and transfer it to a clean enzyme-free centrifuge tube for the next experiment; (3) Enzymatic digestion of purified miRNA samples ① Add 1 μL of Mix enzyme and 2 μL of 10x enzymatic hydrolysis buffer to the purified microRNA sample, and perform enzymatic hydrolysis in a 37°C metal bath for 2 h, then heat the temperature to 70°C and bathe for 10 min to terminate the reaction; ② The enzymatic hydrolysate was diluted to 60 μL with ultrapure water for UPLC-MS / MS analysis; (4) UPLC-MS / MS analysis UPLC conditions: Acquity UPLC BEH C18 reverse phase chromatographic column, whose inner diameter, length, filler particle size and brand are 2.1 mm, 100 mm, 1.7 μm, Waters, respectively; mobile phase A is 2 mM ammonium bicarbonate aqueous solution, mobile phase B is 100% acetonitrile, gradient elution; injection volume is 10 μL; the gradient elution conditions are as follows: 0~3.0min, 0% B, 0.25mL / min, 3.0~6.0min, 0~10.0% B, 0.25mL / min, 6.0~10.0min, 10.0%~50.0% B, 0.25mL / min, 10.0~12.0min, 100.0% B, 0.25mL / min; Mass spectrometry conditions: ion source is Unispray; working in positive ion mode; data acquisition is multiple reaction monitoring MRM mode; nitrogen is used for nebulization and desolvation; source temperature is 150°C, desolvation temperature is 400°C, cone gas flow is 150 L / h, desolvation gas flow is 800 L / h, nebulizer gas flow is 7.0 Bar, collision gas is high-purity nitrogen, and collision gas flow is 0.15 mL / min.

2. The method of claim 1, wherein the modified nucleoside A m , G m , C m , U m 、m 6 A.m 7 G、m 5 C.m 6 A m The mass-to-charge ratios m / z of Q1 ions are 282, 298, 244, 259.1, 282.1, 298, 258, and 296.1, and the mass-to-charge ratios m / z of Q3 ions are 136, 152, 112, 113.1, 150.1, 166, 126, and 150.1, respectively.

3. The method according to claim 1, characterized in that The method can be used to quantitatively characterize epigenetic modifications of cellular microRNAs.

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