Method for distinguishing four terminal small RNAs through APB small RNA imprinting hybridization

Abstract

The invention belongs to the technical field of RNA modification detection, and particularly relates to a method for distinguishing four terminal small RNAs through APB small RNA imprinting hybridization. RNA to be detected is subjected to phosphatase and T4 polynucleotide kinase treatment, RNA purification and APB small RNA imprinting hybridization, the response condition of the RNA to phosphatase and T4 polynucleotide kinase is obtained by comparing whether the electrophoresis migration rate before and after treatment is changed or not, and then the terminal types of 2 ', 3' OH, 2 'OMe, 2', 3'cP and 3 'P before treatment are deduced. Compared with a traditional small RNA imprinting hybridization method, the method has the advantages that the migration rate difference of bands of different modified small RNAs during APB gel electrophoresis is larger, and the problems that the migration rate difference of different modified RNAs is small, and experimental results are often difficult to judge due to the influence of RNA purity, concentration and experimental conditions in a traditional method are solved; the method has a good application prospect in the fields of small RNA modification and functional research thereof.

Description

Technical Field

[0001] The present invention belongs to the technical field of RNA modification detection, and particularly relates to a method for accurately distinguishing four kinds of small terminal RNAs of 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P by adding APB to a urea denaturing gel. Background Art

[0002] Gene expression regulation is one of the basic scientific issues in the field of biological research. Gene silencing mediated by small molecule RNAs is an important mechanism of gene expression regulation. According to the source, generation method and action mode, small molecule RNAs are mainly divided into three categories: miRNA, siRNA, and piRNA. In addition to the above three types of classical small RNAs, small RNAs derived from structural RNAs such as tRNA, rRNA, snRNA, etc., and small RNAs derived from coding and long non-coding RNAs have gradually become research hotspots in the field.

[0003] Terminal modifications of small RNAs are crucial for maintaining their stability and exerting their functions. There are four main types as follows: 2',3'-OH (i.e., unmodified), 2'-OMe (2'-O-methylation, 2-position oxygen methylation modification), 2',3'-cP (2',3'-cyclic phosphate, 2-position and 3-position cyclic phosphate modification), and 3'-P (3'-phosphate, 3-position phosphate modification). Among them, the 2'-OMe modification plays an important role in maintaining the stability of plant miRNAs, animal piRNAs, and siRNAs bound to Drosophila AGO2. 2',3'-cP / 3'-P is the terminal modification type of the vast majority of 5'tsRNAs and 5'tRNA halves. Identifying the modification types of small RNAs is of great significance for the identification of catalytic enzymes and the study of their biological functions. Currently, the main method for identifying the modification types of small RNAs is LC-MS. However, how to obtain highly pure endogenous RNA from organisms is the main technical bottleneck restricting the application of LC-MS. Compared with the complex RNA sample pretreatment of LC-MS, it is relatively simple to identify the modification types by utilizing the differences in the electrophoresis speeds of RNAs with different termini. Multiple research teams have detected CIP / T4PNK-treated small RNAs by small RNA blot hybridization and judged the modification types of tsRNAs based on the differences in electrophoresis speeds (Gu, et al., 2021; Honda, et al., 2016; Mourelatos, et al., 2019). CIP treatment can convert 3'-P to 2',3'-OH, but cannot convert 2',3'-cP to 2',3'-OH. T4PNK treatment can convert both 3'-P and 2',3'-cP to 2',3'-OH. However, due to the fact that in traditional small RNA blot hybridization, the electrophoresis rates of small RNAs before and after CIP and T4PNK treatments only differ by 1-2 nucleotides, and are easily affected by their concentrations, purities, and the gel electrophoresis process. Therefore, it is sometimes difficult to accurately judge the terminal modification types of small RNAs based on the displacement.

[0004] Studies have shown that small RNAs with terminal ribose modifications (such as 3’P, 2',3'cP, 2’OMe) can be effectively distinguished from small RNAs with 2',3'OH termini in APB (acryloylaminophenylboronic acid) small RNA electrophoresis, and the displacement is relatively large. Considering that small RNAs with 3’P termini can respond to both CIP and T4PNK; small RNAs with 2’,3’-cP termini only respond to T4PNK; small RNAs with 2’OMe and 2',3'OH termini do not respond to either CIP or T4PNK. Therefore, according to the different responses of small RNAs with different terminal types to CIP and T4PNK, combined with APB small RNA blot hybridization, small RNAs with these four different terminal types (2',3'OH, 2’OMe, 2',3'cP, 3'P) can be effectively distinguished. Summary of the Invention

[0005] Aiming at the problem that small RNAs with 2',3'OH, 2’OMe, 2',3'cP, and 3'P termini are not significantly distinguishable in traditional small RNA blot hybridization, the purpose of the present invention is to provide a method for significantly distinguishing the above four terminal small RNAs, that is, through treatment with phosphatase and T4 polynucleotide kinase, and by means of APB small RNA blot hybridization, to solve the problem of poor discrimination effect of traditional methods.

[0006] The method provided by the present invention for significantly distinguishing small RNAs with 2',3'OH, 2’OMe, 2',3'cP, and 3'P termini includes the acquisition of the RNA to be detected; treatment with phosphatase and T4 polynucleotide kinase; RNA purification; APB small RNA blot hybridization; wherein:

[0007] The treatment with phosphatase and T4 polynucleotide kinase is to digest the RNA with phosphatase and T4 polynucleotide kinase, and the specific steps are as follows:

[0008] Step 1: Reaction of phosphatase and T4 polynucleotide kinase;

[0009] Step 2: Inactivation of phosphatase and T4 polynucleotide kinase;

[0010] The APB small RNA blot hybridization of the RNA is to separate small RNAs with different terminal types using an APB urea denaturing gel, and the specific steps are as follows:

[0011] Step 1: Preparation of APB urea denaturing gel;

[0012] Step 2: Electrophoresis of APB urea denaturing gel;

[0013] Step 3: APB small RNA blot hybridization.

[0014] Furthermore,

[0015] The method for distinguishing 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P terminal small RNAs can distinguish small RNAs with a length range of 10 - 50 nt.

[0016] When using phosphatase and T4 polynucleotide kinase to digest RNA, the phosphatase used is not limited to shrimp alkaline phosphatase (rSAP), but also includes other phosphatases with the same efficacy such as calf intestinal alkaline phosphatase (CIP), etc.; T4 polynucleotide kinase (T4PNK) or other treatments with the same efficacy can be used, such as HCl + phosphatase.

[0017] For the operation process of RNA purification, the purification method is not limited to phenol-chloroform purification of RNA, but also includes purification using kits, such as Zymo RNA Clean&Concentrator-5 (R1013) of Zymo RNA purification and concentration kit, etc.

[0018] During the operation process of distinguishing 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P terminal small RNAs by APB small RNA blot hybridization, the APB urea denaturing gel prepared in step 1 has an APB concentration of 0.05 - 2.5%, and the buffer in the denaturing gel is not limited to HEPES, but also includes buffers with the same efficacy such as TBE, TAE, MOPS, etc.; the electrophoresis buffer used for APB urea denaturing gel electrophoresis in step 2 and the buffer used for RNA loading are not limited to HEPES, but also include buffers with the same efficacy such as TBE, TAE, MOPS, etc.

[0019] Furthermore, the method for significantly distinguishing 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P terminal small RNAs has the following specific operation procedures:

[0020] (1) Obtaining the RNA to be detected: The RNA to be detected includes RNA extracted from organisms (by Trizol method or kit extraction), RNA purified by fragment selection (gel extraction method or magnetic bead purification), synthetic RNA from companies, etc.

[0021] (2) Phosphatase and T4 polynucleotide kinase reaction: Use phosphatase (such as rSAP, Quick CIP, etc.) and T4 polynucleotide kinase (T4PNK), configure the reaction system with reference to the instruction manual, and heat to inactivate the enzyme after the reaction ends.

[0022] (3) RNA purification: Phenol-chloroform purification method: Add an equal volume of phenol-chloroform to the reaction system to remove protein contaminants such as enzymes. Add glycogen and linear acrylamide as nucleic acid coprecipitants to the supernatant after centrifugation to promote the recovery of RNA in ethanol precipitation. Wash the RNA twice with 75% ethanol and dissolve the RNA with an appropriate amount of DEPC water. Kit purification method: Operate according to the kit instructions.

[0023] (4) APB small RNA blot hybridization: Perform electrophoresis using an APB urea denaturing gel (0.1 - 0.5% PBA), and use Turbo TM transfer system (Bio-Rad) for membrane transfer, cross-link, and bake at 80 °C to fix the RNA on the Zeta-Probe GT membrane. Incubate the membrane with the hybridization solution containing the probe overnight for 16 - 18 h, and use a chemiluminescent nucleic acid detection module kit for signal detection.

[0024] The beneficial effects of the present invention are as follows:

[0025] The present invention applies APB small RNA blot hybridization to the study of distinguishing small RNAs with four terminal types: 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P. Compared with the traditional small RNA blot hybridization method, the displacement of the small RNA electrophoresis bands before and after treatment with phosphatase and T4 polynucleotide kinase is greater, and it can more accurately judge the terminal type of small RNAs, solving the problem that the migration rate difference of different modified RNAs in the traditional method is small and the experimental results are often difficult to determine due to the influence of RNA purity, concentration, and experimental conditions. It has good application prospects in the field of small RNA modification and its function research. Brief Description of the Drawings

[0026] Figure 1 Shows the responses of small RNAs with four different terminal types: 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P to rSAP and T4PNK.

[0027] Figure 2 Schematic diagram of the method for APB small RNA blot hybridization to distinguish small RNAs with 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P terminals.

[0028] Figure 3 Result diagram of separating 2',3'-OH terminal small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization.

[0029] Figure 4 Result diagram of separating 2'-OMe terminal small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization.

[0030] Figure 5 Results of separating 2',3'-cP-ended small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization

[0031] Figure 6 Results of separating 3'-P-ended small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization Specific implementation manners

[0032] The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0033] Unless otherwise specified, the following experiments are all experimental operations well-known to those skilled in the art, and the reagent consumables used can be purchased from regular reagent companies. The Arabidopsis thaliana materials used in the following examples are wild-type materials of the Col-0 ecotype.

[0034] The method of the present invention will be specifically described below:

[0035] Step 1: Obtaining the RNA to be detected

[0036] In this example, the small RNAs with 2',3'OH, 2’OMe, and 3'P at the ends were all synthesized by a reagent company, and the small RNA with 2',3'cP at the end was a self-made RNA referring to (Lai, et al., 2023). The preparation of the small RNA with 2',3'cP at the end is described as follows: First, the small RNA with 3'P at the end synthesized by the company was treated with Mth RNA ligase to convert the 3'P end to a 2',3'cP end (300 pmol of 3'P-end small RNA, 6 μl of 5'DNA adenylation reaction buffer (10×), 6 μl of 1 mM ATP, 300 pmol of Mth RNA ligase, supplemented with DEPC H2O to 60 μl, reacted at 65°C for 3 h, and inactivated the enzyme at 85°C for 5 min). Then, rSAP was used to convert the remaining 3'P-end RNA to 2',3'OH-end RNA (RNA after Mth RNA ligase treatment, 3 μl of CutSmart buffer (10×), 3 μl of rSAP, supplemented with DEPC H2O to 60 μl, reacted at 37°C for 3 h, and inactivated the enzyme at 65°C for 20 min). APB small RNA electrophoresis was used to distinguish between 2',3'OH-end and 2',3'cP-end RNA, and the 2',3'cP-end RNA was recovered by gel cutting (add 3 volumes of solubilization buffer (0.3 M NaCl, 5 mM EDTA) to the ground gel particles, rotate overnight at 4°C, add 1 μl of glycogen and 1 ml of absolute ethanol to precipitate RNA to each 400 μl of gel supernatant filtered through 0.45 μm, wash the RNA precipitate twice with 75% ethanol, and then dissolve it in an appropriate amount of DEPC water).

[0037] Step 2: rSAP and T4PNK reactions

[0038] The rSAP and T4PNK reactions were carried out according to the following systems respectively. T4PNK reaction system (50 μl): 42 μl of RNA (if insufficient, make up with DEPC water), 5 μl of T4PNK buffer (10×), 1 μl of RNase inhibitor (40 U / μl), 2 μl of T4PNK (10 U / μl), reaction conditions: 37°C, 3 h, incubate at 65°C for 20 min after the reaction to inactivate; rSAP reaction system (50 μl): 42 μl of RNA (if insufficient, make up with DEPC water), 5 μl of CutSmart buffer (10×), 1 μl of RNase inhibitor (40 U / μl), 2 μl of rSAP (5 U / μl), reaction conditions: 37°C, 3 h, incubate at 80°C for 2 min after the reaction to inactivate.

[0039] Step 3: Phenol-chloroform purification of RNA

[0040] Add 50 μl of DEPC water to the above 50-μl reaction system. After mixing, add 100 μl of phenol-chloroform solution (phenol:chloroform = 1:1). After thorough mixing, let it stand at room temperature for 5 min, then centrifuge at 13,000 rpm at 4 °C for 15 min. Transfer the supernatant to a new centrifuge tube. Add 200 μl of DEPC water, 1 μl of glycogen, 1 μl of linear acrylamide, and 30 μl of 3 M NaAc to the RNA supernatant. After mixing, add 1 ml of absolute ethanol and place it at -80 °C for at least 2 h.

[0041] Step 4. APB small RNA blot hybridization

[0042] The procedure for APB small RNA blot hybridization is as follows: First, prepare an APB urea denaturing gel (16% Acr-Bis, 42% Urea, 1×HEPES, 0.2% PBA). After all the APB powder is dissolved, add APS and TEMED. The electrophoresis buffer used is 1×HEPES (20 mM HEPES, 5 mM NaAC, 1 mM EDTA, pH 8.2), and the 2×RNA Loading Buffer is HEPES loading buffer (add 1 ml of 20×HEPES, 1 ml of DEPC water to 8 ml of formamide, add an appropriate amount of bromophenol blue and xylene cyanol, mix thoroughly and dispense, store in the dark); Stop electrophoresis when the dimethyl blue dye is 2 cm from the bottom of the glass plate, and transfer the membrane according to the program of "25 V - 1.3 A - 7 min" (transfer twice in total); Place the membrane face up in the UV cross-linker, select an energy value of 1200 (×100 μJ / cM 2 ), and cross-link twice; Place the membrane between two filter papers and heat at 80 °C for 20 min; Add the biotin-labeled probe (10 μM) to the hybridization solution at a ratio of 1:1000, and hybridize the membrane in a hybridization oven (42 °C) for 16 - 18 h; Use a chemiluminescent nucleic acid detection module kit for signal detection, and the detection steps refer to the instruction manual.

[0043] Result analysis

[0044] Figure 1 It is about the responses of small RNAs with four different terminal types of 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P to rSAP and T4PNK. A - B shows that the terminal types of small RNAs with 2',3'-OH (A) and 2'-OMe (B) termini remain unchanged after treatment with rSAP and T4PNK; C. The small RNA with 2',3'-cP terminal modification responds to T4PNK but not to rSAP; D. The small RNA with 3'-P terminal modification responds to both rSAP and T4PNK.

[0045] Figure 2Flow chart of the method for differentiating 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P terminal small RNAs by APB small RNA blot hybridization. Treat the RNA to be detected with rSAP and T4PNK, purify the RNA after the reaction with phenol-chloroform, and then perform APB small RNA blot hybridization. Judge the terminal type according to its response to rSAP and T4PNK.

[0046] Figures 3 to 6 The lengths of the four kinds of terminal small RNAs of 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P used are all 35 nt. After the four kinds of terminal small RNAs are treated with rSAP and T4PNK respectively (NT is the untreated group), they are detected by a urea denaturing gel without APB (A, traditional method) and a urea denaturing gel containing 0.2% concentration of APB (B, the method of the present invention). The urea denaturing gel used in Figure A does not contain APB, and the marker used is single-stranded DNA labeled with 3'-FAM (with lengths of 28, 35, and 50 nt). The urea denaturing gel used in Figure B contains 0.2% concentration of APB, and the markers used are single-stranded DNA labeled with 3'-FAM (with lengths of 28, 35, and 50 nt, simulating "modified ribose at the end") and single-stranded RNA labeled with 5'-FAM (with a length of 35 nt, simulating "unmodified ribose at the end").

[0047] Figure 3 Results diagram of separating 2',3'-OH terminal small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization. The results show that: in the traditional method (A), the band positions of the 2',3'-OH modified small RNAs are the same before and after treatment with rSAP and T4PNK; in the method of the present invention (B), although the band positions of the 2',3'-OH modified small RNAs are the same before and after treatment with rSAP and T4PNK, the electrophoresis bands all shift upward to the marker of "unmodified ribose at the end".

[0048] Figure 4 Results diagram of separating 2'-OMe terminal small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization. The results show that: in the traditional method (A), the band positions of the 2'-OMe modified small RNAs are the same before and after treatment with rSAP and T4PNK; in the method of the present invention (B), although the band positions of the 2'-OMe modified small RNAs are the same before and after treatment with rSAP and T4PNK, the electrophoresis bands are all consistent with the marker of "modified ribose at the end". In the traditional method, the band distances between the 2',3'-OH terminal and 2'-OMe terminal small RNAs are relatively close ( Figure 3 A, Figure 4 A), while in the method of the present invention, the distances between the two are relatively far and are easy to distinguish ( Figure 3 B, Figure 4 B).

[0049] Figure 5 The figure shows the results of separating 2',3'cP-terminated small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization. The results show that: in the traditional method (A), the band positions of 2',3'cP-modified small RNAs are the same before and after treatment with rSAP and T4PNK. In the method of the present invention (B), after treatment with rSAP, the band positions of 2',3'cP-modified small RNAs remain unchanged, but after treatment with T4PNK, they migrate from the position of "modified terminal ribose" before treatment to the position of "unmodified terminal ribose".

[0050] Figure 6 The figure shows the results of separating 3'P-terminated small RNAs by traditional small RNA blot hybridization and APB small RNA blot hybridization. The results show that: in the traditional method (A), the band positions of 3'P-terminated small RNAs are the same before and after treatment with rSAP and T4PNK. In the method of the present invention (B), after treatment with rSAP and T4PNK, the 3'P-terminated small RNAs both migrate from the position of "modified terminal ribose" before treatment to the position of "unmodified terminal ribose".

[0051] The above are all preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, various changes and modifications can be made to the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for distinguishing four types of small RNAs with different ends by APB small RNA blot hybridization. The four end types of small RNAs are 2',3'-OH, 2'-OMe, 2',3'-cP, and 3'-P, and it is characterized in that It includes the acquisition of RNA to be detected, treatment with phosphatase and T4 polynucleotide kinase, RNA purification, and APB small RNA blot hybridization; among which: The treatment with phosphatase and T4 polynucleotide kinase is to digest RNA with phosphatase and T4 polynucleotide kinase. The specific steps are as follows: Step 1: Phosphatase and T4 polynucleotide kinase reaction; Step 2: Inactivation of phosphatase and T4 polynucleotide kinase; The APB small RNA blot hybridization of RNA is to separate small RNAs of different end types using an APB urea denaturing gel. The specific steps are as follows: Step 1: Preparation of APB urea denaturing gel; Step 2: Electrophoresis of APB urea denaturing gel; Step 3: APB small RNA blot hybridization.

2. The method for differentiating four types of terminal small RNAs according to claim 1, wherein The length range of the distinguished small RNAs is 10 - 50 nt.

3. The method for distinguishing four types of terminal small RNAs according to claim 1, wherein In the specific operation process of digesting RNA with phosphatase and T4 polynucleotide kinase, the phosphatase used is shrimp alkaline phosphatase or calf intestinal alkaline phosphatase, and T4 polynucleotide kinase or HCl + phosphatase is used.

4. The method for differentiating four types of terminal small RNAs according to claim 1, characterized in that, The method for RNA purification is phenol-chloroform purification or kit purification.

5. The method for distinguishing four types of terminal small RNAs according to claim 1, characterized in that, During the APB small RNA blot hybridization process, for the APB urea denaturing gel prepared in Step 1, the APB concentration is 0.05 - 2.5%, and the buffer in the denaturing gel is HEPES, TBE, TAE, or MOPS; for the electrophoresis solution used in Step 2 for APB urea denaturing gel electrophoresis and the buffer used for RNA loading, they are HEPES, TBE, TAE, or MOPS.

6. The method for distinguishing four types of terminal small RNAs according to any one of claims 1-5, characterized in that, The specific operation is as follows: (1) Acquisition of RNA to be detected: The RNA to be detected includes RNA extracted from organisms, RNA purified by fragment selection, and synthetic RNA; (2) Phosphatase and T4 polynucleotide kinase reaction: Use phosphatase and T4 polynucleotide kinase to configure a reaction system, and heat to inactivate the enzyme after the reaction ends; (3) RNA purification: Phenol-chloroform purification method: Add phenol-chloroform with the same volume as the reaction system to remove protein contamination. Add glycogen and linear acrylamide as nucleic acid coprecipitants to the supernatant after centrifugation to promote the recovery of RNA in ethanol precipitation. After washing the RNA precipitate with 75% ethanol, dissolve the RNA with an appropriate amount of DEPC water; or use kit purification; (4) APB small RNA blot hybridization: Using APB urea denaturing gel electrophoresis, using Turbo TM transfer system for membrane transfer, cross-linking, baking at 80 °C to fix RNA on the Zeta-Probe GT membrane, incubating the membrane with the hybridization solution containing the probe overnight for 16 - 18 h, and using a chemiluminescent nucleic acid detection module kit for signal detection.

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