A method for highly sensitive quantitative detection of inosine in RNA

Abstract

The present invention discloses a method for highly sensitive quantitative detection of inosine in RNA, and belongs to the field of biotechnology. The method of the present invention comprises the following steps: enzymatically digesting a cell sample to be tested or an RNA sample to be tested into a single nucleoside; performing a derivatization reaction on the digested nucleoside using CMCT; performing dispersed solid phase extraction on the derivatized product using CeO2; and performing LC-MS / MS analysis on the extracted sample. CMCT can selectively react with the NH group at the N1 position of inosine to generate I-CMCT, which can greatly improve the detection sensitivity of LC-MS / MS; CeO2 can selectively enrich I-CMCT and remove compounds such as CMCT that do not contain cis-hydroxyl groups. The method of the present invention is simple and efficient, has a mild reaction, is easy to operate, and has high sensitivity. It has great potential in determining endogenous modifications of trace RNA and a small number of cells (as low as a single cell), and is helpful for functional studies of RNA modifications in precious biological samples.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for quantitatively detecting inosine in RNA with high sensitivity. Background Art

[0002] Adenosine to inosine RNA editing is one of the most important modifications in RNA and was originally discovered in yeast tRNA. Inosine (I) is essentially a purine nucleoside produced by the enzymatic deamination of adenosine (A) at the C6 position. In eukaryotic mRNA, inosine is produced by the hydrolytic deamination of adenosine in the double-stranded region of RNA catalyzed by adenosine deaminases acting on RNA (ADARs), a process known as A-to-I RNA editing. Inosine preferentially pairs with cytidine (C) and is thus recognized as guanosine (G). At the same time, inosine can also pair with adenosine in a less stable manner, thus affecting transcript localization and translation accuracy.

[0003] Studies have shown that inosine is involved in the occurrence and development of many human diseases, including neurological diseases, cancer, and autoimmune diseases. Uncovering the function of inosine requires the development of sensitive and accurate methods to detect inosine in RNA. Liquid chromatography tandem mass spectrometry (LC-MS / MS) is often used to detect and quantify nucleic acid modifications. However, due to the low ionization level of inosine in LC-MS / MS analysis, the detection of inosine is often challenging, especially in single cell or trace RNA samples. Summary of the invention

[0004] The purpose of the present invention is to solve the problem that the existing LC-MS / MS cannot detect inosine in single-cell RNA or trace RNA with high sensitivity, and to provide a method for highly sensitive quantitative detection of inosine in RNA, that is, a simple and efficient, mild reaction, easy to operate, highly sensitive quantitative detection method that can be applied to single-cell (a few cells) RNA or trace RNA.

[0005] The method of the present invention is a method of combining N-cyclohexyl-N'-β-(4-methylmorpholine)ethylcarbodiimide p-toluenesulfonate (CMCT) derivatization with cerium dioxide (CeO2) dispersed solid phase extraction and LC-MS / MS. The present invention introduces easily ionized groups into inosine through a chemical derivatization strategy, thereby improving its detection sensitivity in LC-MS / MS analysis. The carbodiimide group in CMCT can selectively undergo addition reaction with the NH group at the N1 position of inosine. In addition, CMCT contains a positively charged quaternary ammonium group, so it is very easy to ionize, and ultimately the detection sensitivity of the derivatized product (I-CMCT) in LC-MS / MS analysis can be greatly improved. Excessive CMCT can affect the detection of I-CMCT in LC-MS / MS analysis. Cerium dioxide (CeO2) can selectively capture cis-hydroxy compounds. I-CMCT carries cis-hydroxyl groups, so CeO2 can be used to selectively enrich I-CMCT, while removing compounds such as CMCT that do not contain cis-hydroxyl groups.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for highly sensitive quantitative detection of inosine in RNA comprises the following steps:

[0008] (1) enzymatically digesting the cell sample or RNA sample to be tested into single nucleosides;

[0009] (2) using CMCT to derivatize the digested nucleosides;

[0010] (3) using CeO2 to perform dispersed solid phase extraction on the derivatized products;

[0011] (4) The extracted samples were subjected to LC-MS / MS analysis.

[0012] In step (1), the enzymatic digestion of the cell sample is to first lyse the cells at high temperature and then enzymatically digest them with at least one of nuclease and phosphodiesterase and alkaline phosphatase. The conditions for high temperature lysis are preferably incubated at 90°C to 95°C for 5 to 10 minutes, and the conditions for enzymatic digestion are preferably incubated at 37°C for 3 to 6 hours. The enzymes used for enzymatic digestion of the RNA sample are at least one of nuclease and phosphodiesterase and alkaline phosphatase, and the incubation is preferably at 37°C for 3 to 6 hours.

[0013] In step (2), the derivatization reaction is preferably carried out in a borate buffer solution with a CMCT concentration of 50 mM and a pH of 9.0, and the reaction conditions are preferably at 25-45° C. for more than 2 hours.

[0014] In step (3), the dispersed solid phase extraction is preferably adsorbed under weak alkaline conditions and eluted under weak acidic conditions.

[0015] The method for quantitatively detecting inosine in RNA with high sensitivity is applied to quantitatively detecting inosine in RNA, and can quantitatively detect inosine in RNA of a small number of cells (as low as a single cell) or in trace amounts (as low as 13 pg) of RNA.

[0016] A kit for highly sensitive quantitative detection of inosine in RNA comprises CMCT, CeO2, and an enzyme for enzymatically digesting RNA into single nucleosides.

[0017] The principle of the present invention is shown in Figure 1 , the present invention proposes a method for quantitative analysis of inosine in RNA based on CMCT derivatization. In this method, inosine in RNA undergoes an addition reaction with CMCT, and the reaction product is easily ionized. On the one hand, the introduction of easily ionized groups enhances the efficiency of electrospray ionization, thereby enhancing the detection sensitivity; on the other hand, the introduction of hydrophobic groups improves chromatographic separation. Excessive CMCT will affect the detection of I-CMCT in LC-MS / MS analysis. Cerium dioxide (CeO2) can selectively capture cis-hydroxyl compounds. I-CMCT carries cis-hydroxyl groups, so CeO2 can be used to selectively enrich I-CMCT, while removing compounds such as CMCT that do not contain cis-hydroxyl groups. The removal rate of CMCT can reach 99.7%. CMCT derivatization, supplemented by CeO2 dispersed solid phase extraction, combined with LC-MS / MS, can achieve highly sensitive quantitative detection of inosine in RNA, as low as single-cell accuracy.

[0018] The advantages and beneficial effects of the present invention are as follows:

[0019] 1. The present invention is simple and efficient, has a mild reaction, is easy to operate and has high sensitivity, and the reagents and enzymes used are commercially available and easy to obtain.

[0020] 2. The entire experimental cycle of the present invention is 6-8 hours, the process steps are standardized, and it is easy to promote.

[0021] 3. The derivatization reaction efficiency of CMCT and inosine involved in the present invention is as high as 93.4%.

[0022] 4. The derivatization reaction product of the present invention has good stability and will not degrade at least within 12 hours at room temperature.

[0023] 5. The present invention can be applied to the quantitative analysis of inosine in single cells.

[0024] 6. The present invention can achieve quantitative analysis and detection of inosine in samples as low as 13 pg total RNA.

[0025] 7. The present invention can be directly used for the quantitative analysis of inosine levels in different biological samples. It is a powerful tool for quantifying inosine levels in endogenous RNA and is helpful for the functional study of RNA modification in precious biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the sensitive determination of inosine in single cells by CMCT derivatization combined with LC-MS / MS analysis in the present invention.

[0027] Figure 2 Schematic diagram of the chemical derivatization reaction of CMCT and inosine in the present invention. (A) CMCT carries a carbodiimide group, which can selectively react with the NH group at the N1 position of inosine. (B) In LC-MS / MS analysis, the detection sensitivity of I-CMCT is greatly improved compared with natural inosine.

[0028] Figure 3 The test of CMCT derivatization reaction and the optimization of CMCT derivatization reaction conditions in the present invention. (A) Full scan mass spectrum of I-CMCT (primary spectrum). (B) Product ion mass spectrum of I-CMCT (secondary spectrum). (C) Schematic diagram of derivatization and dispersed solid phase extraction, capturing I-CMCT and removing excess CMCT. (D) Optimization of CMCT derivatization reaction conditions.

[0029] Figure 4 Detection of CMCT derivative reaction products under different collision voltages in the present invention. (A) Collision voltage is 11V. (B) Collision voltage is 13V. (C) Collision voltage is 21V. (D) Collision voltage is 35V.

[0030] Figure 5 Evaluation of the efficiency of the CMCT derivatization reaction in the present invention. (A) Chromatogram of inosine before derivatization. (B) Chromatogram of inosine after derivatization.

[0031] Figure 6 This is an evaluation of the stability of the CMCT-derived reaction products of the present invention.

[0032] Figure 7 This is the calibration curve for the quantification of inosine in RNA in the present invention.

[0033] Figure 8 This is the determination of inosine in RNA in the present invention.

[0034] Fig. 9 Determination of inosine in RNA of single cells and sleep-deprived mice in the present invention. (A) Schematic diagram of the analytical process for detecting inosine in RNA from single cells (or a few cells). (B) Extracted ion chromatograms for detecting inosine in RNA of single cells, 2 cells, and 4 cells. (C) Quantification of inosine in total RNA of different tissues of sleep-deprived mice (sleep-deprived, n=3) and control mice (control, n=3); two-sided unpaired t-test was used; **p<0.01. DETAILED DESCRIPTION

[0035] The method for highly sensitive quantitative detection of inosine in RNA provided by the present invention comprises the following steps:

[0036] (1) enzymatically digesting the cell sample or RNA sample to be tested into single nucleosides;

[0037] (2) using CMCT to derivatize the digested nucleosides;

[0038] (3) using CeO2 to perform dispersed solid phase extraction on the derivative products;

[0039] (4) The extracted samples were subjected to LC-MS / MS analysis.

[0040] In step (1), the enzymatic digestion step of the cell sample to be tested is as follows: a single cell (or a small number of cells) is added to a centrifuge tube, incubated at 90°C for 10 minutes, phosphodiesterase I (0.0125U), alkaline phosphatase (0.75U) and 2μL 10× buffer (500mM Tris–HCl, 10mM MgCl2, 100mM NaCl, 10mM ZnSO4, pH 7.0) are added, water is added to make up the total volume to 20μL, and enzymatic digestion is performed at 37°C for 3 to 4 hours.

[0041] The enzymatic digestion system of the RNA sample to be tested is: RNA sample, 2 μL 10× buffer (500 mM Tris-HCl, 10 mM MgCl2, 100 mM NaCl, 10 mM ZnSO4, pH 7.0), 0.5 μL S1 nuclease (180 U / μL), 0.5 μL phosphodiesterase I (0.25 U / μL) and 0.25 μL alkaline phosphatase (30 U / μL), add water to make the total volume 20 μL. The mixture was incubated at 37°C for 3 to 4 hours.

[0042] In step (2), the conditions for the derivatization reaction of nucleosides are as follows: CMCT previously dissolved in acetonitrile is added to a borate buffer (concentration of 50 mM, pH 9.0), and the reaction product of step (1) is added (if a single cell or a trace amount of RNA is analyzed, the enzymatic hydrolysis in step (1) does not need to be processed to prevent loss; if a large number of cells or a large amount of RNA is analyzed, chloroform extraction can be used to remove the enzyme used for enzymatic hydrolysis). The final concentration of the borate buffer in the reaction system is 25 mM, and the final concentration of CMCT is 50 mM. After thorough mixing, the mixture is shaken at a constant temperature of 30° C. for 2 hours at a rotation speed of 850 rpm.

[0043] In step (3), the steps of using CeO2 to perform dispersed solid phase extraction on the derivative product are as follows: 300 mg CeO2 is washed in a centrifuge tube with 0.5 mL 1% formic acid aqueous solution, 0.5 mL water and 0.5 mL borate buffer (50 mM) in sequence. Vortex for 2 minutes each time, centrifuge at 13000 rpm for 2 minutes and discard the supernatant. The mixture after CMCT derivatization in step (2) is centrifuged at low speed until there are no droplets on the tube wall, diluted with 1 mL borate buffer (50 mM), then added to the washed CeO2 and vortexed vigorously for 4 minutes. After centrifugation at 13000 rpm for 4 minutes, the supernatant is discarded, and the CeO2 obtained by capturing I-CMCT is washed twice with 1 mL water for 3 minutes each time. Then, 0.5 mL of 1% formic acid aqueous solution is used to release the captured I-CMCT by vortexing for 3 minutes, and finally centrifuged at 13000 rpm for 3 minutes, and the supernatant is collected. After freeze-drying, the samples were dissolved in 50 μL water for subsequent LC-MS / MS analysis.

[0044] In step (4), the supernatant collected in step (3) is freeze-dried and dissolved in 50 μL of water for LC-MS / MS analysis. LC-MS / MS analysis is performed on a mass spectrometer, which needs to have an electrospray (ESI) source (turbo ion spray). The derivative products are separated on a C18 chromatographic column (100 mm×2.1 mm inner diameter, 2.0 μm) at 40°C. Mass spectrometry analysis is performed in positive ion mode. The mobile phase consists of 0.05% formic acid (solvent A) and acetonitrile (solvent B) with a flow rate of 0.3 mL / min. The analytes are separated using a 20 min gradient, 0-2 min, 5% B; 2-10 min, 5-70% B; 10-11 min, 70% B; 11-12 min, 70-5% B; 12-20 min, 5% B. Multiple reaction monitoring (MRM) mode is used for detection.

[0045] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but it should not be understood as limiting the present invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principle of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. Unless otherwise specified, the technical means used in the examples (including RNA extraction from tissue or cell samples, RNA enzymatic hydrolysis, and LC-MS / MS analysis) are conventional means well known to those skilled in the art.

[0046] Example 1: Inosine in total RNA in CMCT-derived 293T cells

[0047] The enzymatic digestion system of the RNA sample to be tested is: 20 ng RNA sample, 2 μL 10× buffer (500 mM Tris–HCl, 10 mM MgCl2, 100 mM NaCl, 10 mM ZnSO4, pH 7.0), 0.5 μL S1 nuclease (180 U / μL), 0.5 μL phosphodiesterase I (0.25 U / μL) and 0.25 μL alkaline phosphatase (30 U / μL), and water is added to make the total volume 20 μL. The mixture is incubated at 37°C for 4 hours. The conditions for the derivatization reaction of nucleosides are: CMCT (solvent is acetonitrile) and enzymatic digestion products are added to borate buffer (concentration of 50 mM, pH 9.0), the final concentration of borate buffer in the reaction system is 25 mM, and the final concentration of CMCT is 50 mM. After thorough mixing, the reaction is shaken at a constant temperature of 30°C for 2 hours at a speed of 850 rpm. The derivatized product was subjected to dispersed solid phase extraction using CeO2 according to step (3). The extracted sample was subjected to LC-MS / MS analysis according to step (4).

[0048] In this example, m / z (520.2→252.2) was identified as the ion pair of the derivative product (I-CMCT) ( Figure 3 A, B and Figure 4 ). CMCT was performed to derivatize inosine in total RNA of 293T cells, and the derivatization reaction of inosine in total RNA of 293T cells was investigated by LC-MS / MS ( Figure 3 C). In order to obtain the best derivatization efficiency, the CMCT derivatization conditions were further optimized, including reaction temperature, reaction time, and CMCT concentration. Regarding the reaction temperature, it can be seen that the reaction at 30°C provided the best reaction efficiency ( Figure 3 D). Moreover, the peak area of ​​I-CMCT reached a plateau at 120 minutes (2 hours) ( Figure 3 D). In addition, when the CMCT concentration was 50 mM, the derivatization efficiency was relatively good ( Figure 3 D). Therefore, the optimal CMCT derivatization reaction conditions were obtained as follows: 50 mM CMCT, 2 hours at 30°C. Based on the reduction in inosine before and after the reaction, it can be calculated that the derivatization efficiency of the reaction under the optimal reaction conditions is 93.4% ( Figure 5 The derivative reaction product exhibits good stability and will not degrade at least within 12 hours at room temperature ( Figure 6 ). This indicates that CMCT derivatization is suitable for LC-MS / MS analysis of inosine.

[0049] Example 2: CMCT derivatization combined with LC-MS / MS to quantitatively analyze inosine in single cells or trace amounts of RNA. The specific steps are as follows:

[0050] (1) Different amounts of inosine and a fixed amount of internal standard (adenosine) were mixed and reacted with CMCT under optimized conditions. Excess CMCT was removed by CeO2 dispersed solid phase extraction. LC-MS / MS analysis was performed and the relationship between the average peak area ratio of I-CMCT / adenosine (I-CMCT / A) and the average molar ratio of inosine / adenosine (I / A) was plotted to construct a calibration curve ( Figure 7 ) to quantify inosine levels in RNA.

[0051] (2) The total RNA was enzymatically hydrolyzed into single nucleosides, reacted with CMCT under optimized conditions, and the excess CMCT was removed by CeO2 dispersed solid phase extraction. LC-MS / MS analysis was performed ( Figure 3 C); investigate the lowest amount of total RNA that can be detected.

[0052] (3) The single cells obtained by serial dilution were ruptured at 90°C for 10 minutes, enzymatically hydrolyzed into single nucleosides in the same centrifuge tube, reacted with CMCT under optimized conditions, and the excess CMCT was removed by CeO2 dispersive solid phase extraction. LC-MS / MS analysis ( Fig. 9 A).

[0053] (4) Calculate the level of inosine based on the quantitative curve in (1) and the peak area in LC-MS / MS.

[0054] The quantitative equation drawn is y = 0.5034x + 0.0551, R 2 is 0.9903( Figure 7 ). A clear peak of inosine-derived product (I-CMCT) can be observed in total RNA as low as 13 pg, with a signal-to-noise ratio of 26 ( Figure 8 Its retention time is consistent with the retention time of I-CMCT (5.1 minutes) verified by the spike experiment ( Figure 8 ). Using the constructed calibration curve, the quantitative results showed that the inosine level of 13pg RNA was 0.22% (I / A), which is comparable to the result obtained using a larger amount (300pg) of RNA (0.28%, I / A). Using this method, inosine can be detected in a small number of cells or even a single cell ( Fig. 9 B). The content of inosine in a single cell was 0.26% (I / A), the content of inosine in two cells was 0.27% (I / A), and the content of inosine in four cells was 0.22% (I / A), and the data were consistent.

[0055] Example 3: Quantification and comparison of inosine levels in different tissues of sleep deprived mice and normal mice

[0056] The method of the present invention was applied to detect the level of inosine in the RNA of the heart, liver and spleen of sleep-deprived mice and normal mice. The experimental method was the same as in Example 2, except that RNA extraction was performed on the tissues. The ratios of different peak areas were substituted into the quantitative curve. The results showed that the level of inosine in all the tested tissues (heart, liver or spleen) of the sleep-deprived mice was significantly increased compared with that of the control mice ( Fig. 9 C), indicating that inosine is associated with sleep deprivation and may be a potential indicator of sleep disorders (heart: p = 0.0033, liver: p = 0.0093, spleen: p = 0.0074).

Claims

1. A method for highly sensitive quantitative detection of inosine in RNA, characterized in that: The steps include: (1) Enzymatically digesting the cell sample or RNA sample to be tested into single nucleosides; (2) Derivatizing the digested nucleosides using CMCT; the derivatization reaction conditions are: 50 mM CMCT, 30° C. for 2 hours; the CMCT is N -Cyclohexyl- N '- β -(4-Methylmorpholine)ethylcarbodiimide p-toluenesulfonate; (3) using CeO2 to perform dispersed solid phase extraction on the derivatized product; the dispersed solid phase extraction is adsorption under weak alkaline conditions and elution under weak acidic conditions; (4) The extracted samples were subjected to LC-MS / MS analysis, and the ion pairs detected in the multiple reaction monitoring mode were: m / z :520.2→252.

2.

2. The method for highly sensitive quantitative detection of inosine in RNA according to claim 1, characterized in that: In step (1), the enzymatic digestion of the cell sample is to first heat the cells at high temperature for lysis and then enzymatically digest them using at least one of nuclease and phosphodiesterase and alkaline phosphatase; the high temperature heating lysis condition is incubation at 90°C to 95°C for 5 to 10 minutes.

3. The method for highly sensitive quantitative detection of inosine in RNA according to claim 1, characterized in that: In step (1), the enzyme used for enzymatic digestion of the RNA sample is at least one of nuclease and phosphodiesterase and alkaline phosphatase.

4. The method for highly sensitive quantitative detection of inosine in RNA according to claim 2 or 3, characterized in that: The enzymatic digestion conditions are incubation at 37° C. for 3 to 6 hours.

5. The method for highly sensitive quantitative detection of inosine in RNA according to claim 1, characterized in that: In step (2), the derivatization reaction is carried out in a borate buffer solution with a CMCT concentration of 50 mM and a pH of 9.0.

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