Method for determining RNA secondary structure

Abstract

The present application relates to the field of biotechnology, and in particular to a method and a kit for determining the secondary structure of RNA. The method comprises a sample pretreatment step, an RNA extraction step, a library preparation step and a sequencing step. The library preparation step comprises a reverse transcription step, a first purification step, a second purification step, a linker ligation step, a third purification step, and an amplification and library construction step. The method of the present application has simplified operation steps, low requirements for the initial amount of RNA, and high enrichment efficiency.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a method and a kit for determining RNA secondary structure. BACKGROUND

[0002] Determination of RNA secondary structure is of great significance for understanding gene expression regulation, RNA function and disease mechanism. Traditional RNA structure determination methods, such as X-ray crystallography and nuclear magnetic resonance (NMR), require a large amount of high-purity RNA samples and are complex to operate, which limits their wide application in practice. In recent years, RNA structure determination methods based on chemical probes and high-throughput sequencing have gradually become mainstream. Among them, SHAPE-Seq, DMS-Seq and smartSHAPE technologies can efficiently analyze the secondary structure of RNA through chemical modification and sequencing analysis.

[0003] Related RNA structure detection technologies mainly fall into two categories: the first category is represented by DMS-seq and icSHAPE, which determines the RNA structure by detecting the termination signal generated by chemically modified nucleotides during reverse transcription, but they cannot detect the structure information of the 3' end of RNA; the second category is represented by DMS-MaPseq and SHAPE-MaP, which measures the mutation rate generated by chemically modified nucleotides during reverse transcription to overcome the loss of 3' end structure information. However, DMS-MaPseq can only provide partial nucleotide coverage, and the reagent used by SHAPE-MaP only has moderate cell membrane penetration ability, which limits its ability to detect intracellular RNA structure. In addition, smartSHAPE connects the two ends of the ssDNA through a cyclase in multiple steps, which is complicated and costly. These technologies all face the problems of complex operation or high requirement for starting amount.

[0004] Therefore, there is an urgent need for a RNA secondary structure determination method with simplified operation steps, low requirement for starting amount and high enrichment efficiency. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art.

[0006] To this end, embodiments of the first aspect of the present application provide a method for determining RNA secondary structure, comprising: a sample pretreatment step, the sample pretreatment step comprising treating the sample such that the single-stranded regions of the RNA molecules of the sample to be tested have a first modification; an RNA extraction step, the RNA extraction step comprising extracting the RNA having the first modification from the pretreated sample; a library preparation step, the library preparation step comprising: a reverse transcription step, the reverse transcription step comprising reverse transcription using the RNA molecules having the first modification as a template to obtain one or more DNA-RNA hybrid double-stranded molecules formed by one or more single-stranded cDNA molecules hybridized to the RNA molecules, wherein the one or more single-stranded cDNA molecules are separated by the first modification; a first purification step, the first purification step comprising specific cleavage at the single-stranded regions of the RNA molecules that are not hybridized to the one or more single-stranded cDNA molecules using a first enzyme to obtain one or more DNA-RNA hybrid double-stranded molecules; a second purification step, the second purification step comprising specific hydrolysis of the RNA strand in the one or more DNA-RNA hybrid double-stranded molecules using a second enzyme to obtain one or more DNA single-stranded molecules; a linker ligation step, the linker ligation step comprising adding a linker sequence to both the 5' end and the 3' end of the one or more DNA single-stranded molecules simultaneously using T4 DNA ligase; a third purification step, the third purification step comprising removing the rRNA corresponding DNA single-stranded molecules from the one or more DNA single-stranded molecules; an amplification and library construction step, the amplification and library construction step comprising amplifying the purified DNA single-stranded molecules and obtaining a sequencing library; and a sequencing step, the sequencing step comprising sequencing the sequencing library and analyzing the RNA secondary structure. The method has simplified operation steps, low requirements for the starting amount of RNA, and high enrichment efficiency.

[0007] In some embodiments, the first modification is at least one selected from the group consisting of NAI-N3, DMS, 1M7, NAI.

[0008] In some embodiments, further comprising: performing a click reaction after the RNA extraction step to connect a biotin modification to the first modification; and performing an enrichment step between the first purification step and the second purification step, wherein the enrichment step comprises using streptavidin magnetic beads to connect to the DNA-RNA hybrid double-stranded molecules having the biotin modification, thereby enriching the one or more DNA-RNA hybrid double-stranded molecules.

[0009] In some embodiments, the first enzyme is at least one selected from the group consisting of RNase I, RNase A, and RNase L, which are ribonucleases that specifically cleave single-stranded RNA, and the second enzyme is one selected from the group consisting of RNase H and RNase III, which are enzymes that specifically hydrolyze single strands of RNA in DNA-RNA hybrid double strands.

[0010] In some embodiments, the adapter sequence added to both ends is a double-ended adapter with a universal nucleotide single-stranded segment at one end, which is capable of hybridizing with a single-stranded DNA molecule.

[0011] In some embodiments, the adapter ligation step further includes second-strand synthesis of the one or more single-stranded DNA molecules to obtain one or more double-stranded DNA molecules.

[0012] In some embodiments, the third purification step uses an rRNA removal probe kit to remove the DNA single-stranded molecules corresponding to the rRNA.

[0013] In some embodiments, the sample includes live cells, primary cells, early mammalian embryos, post-infected cells, bacteria, fungi, cell lines, and viruses.

[0014] In some embodiments, it also includes:

[0015] A fourth purification step, comprising purification using a total RNA filtration kit between the RNA extraction step and the library preparation step, preferably the total RNA filtration kit being the ZYMO RNA Clean & Concentrator kit; and / or a fifth purification step, comprising purification using a total DNA filtration kit between the second purification step and the third purification step, preferably the total DNA filtration kit being the ZYMO DNA Clean & Concentrator kit.

[0016] An embodiment of the second aspect of the present invention provides a kit comprising: a first modification reagent, a first enzyme, a second enzyme, a T4 DNA ligase, an rRNA removal probe, and an adapter.

[0017] The advantages and technical effects brought about by the independent claims according to the embodiments of the present invention are as follows:

[0018] This protocol significantly improves RNA enrichment efficiency and reduces costs by optimizing the smartSHAPE method, while avoiding the loss of non-coding RNA due to Poly A-tail dependence, thus enhancing the integrity of transcriptome analysis. Furthermore, this protocol simplifies the adapter ligation step, reducing operation time and material consumption, and improving experimental efficiency. For low-starting-volume samples, this protocol optimizes the processing flow, improving assay sensitivity and accuracy, and requiring even lower starting-volume samples, making it widely applicable to studies of rare or valuable samples. These improvements make this protocol more efficient, economical, and adaptable in RNA structure determination. Attached Figure Description

[0019] Figure 1 This is a flowchart illustrating the adapter ligation step in the RNA secondary structure determination method according to an embodiment of the present invention.

[0020] Figure 2 This is a graph showing the gel electrophoresis results of the library obtained in the library preparation step of the RNA secondary structure determination method according to an embodiment of the present invention.

[0021] Figure 3 This is a graph showing the quality test results of the library obtained by the library preparation step of the RNA secondary structure determination method in this embodiment of the invention.

[0022] Figure 4 This is a comparison chart of the proportion of rRNA in the human genome between the library obtained by the library preparation step of the RNA secondary structure determination method in this embodiment of the invention and the human genome.

[0023] Figure 5 This is a graph showing the percentage of RNA types in the library obtained by the library preparation step of the RNA secondary structure determination method in this embodiment of the invention.

[0024] Figure 6 This is a distribution map of RNA species obtained using the RNA secondary structure determination method of this invention at a cell input level of 10.

[0025] Figure 7 This is a graph showing the library constructed using the RNA secondary structure determination method of this invention with an input of 20 fg RNA. Detailed Implementation

[0026] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0027] This invention is based on the inventor's discoveries and understanding of the following facts and problems:

[0028] The term "RNA secondary structure" refers to the specific secondary structures formed by base pairing of the primary sequence of an RNA molecule. Common RNA secondary structure domains include: RNA double helix (double-stranded RNA containing two complementary strands), hairpin loops, and protrusions. RNA secondary structure plays an important regulatory role in the life activities of bacteria, fungi, viruses, and mammals.

[0029] Determining RNA secondary structure is crucial for understanding gene expression regulation, RNA function, and disease mechanisms. Traditional RNA structure determination methods, such as X-ray crystallography and nuclear magnetic resonance (NMR), require large quantities of high-purity RNA samples and are complex to operate, limiting their widespread practical application. In recent years, RNA structure determination methods based on chemical probes and high-throughput sequencing have gradually become mainstream. Among them, technologies such as SHAPE-Seq, DMS-Seq, and smartSHAPE can efficiently resolve RNA secondary structures through chemical modification and sequencing analysis.

[0030] RNA structure detection techniques are mainly divided into two categories: the first category, represented by DMS-seq and icSHAPE, determines RNA structure by detecting the termination signal generated during reverse transcription of chemically modified nucleotides, but they cannot detect the structural information at the 3' end of RNA. The second category, represented by DMS-MaPseq and SHAPE-MaP, overcomes the loss of 3' end structural information by measuring the mutation rate at the positions of chemically modified nucleotides during reverse transcription. However, DMS-MaPseq only provides partial nucleotide coverage, and the reagents used in SHAPE-MaP have only moderate cell membrane penetration, limiting their ability to detect the structure of intracellular RNA. Furthermore, while SHAPE-Seq and DMS-Seq methods can analyze the secondary structure of RNA through chemical modification and sequencing, they are complex to operate, require high starting amounts, and are poorly adaptable to low starting sample amounts. Reducing the amount of RNA input at the start of the reaction leads to significant RNA loss, severely impacting library yield and limiting their application in precious or limited samples.

[0031] Related RNA structure detection techniques include the smartSHAPE method, which utilizes the ability of NAI-N3 to selectively modify the 2'-hydroxyl group of RNA within cells. This allows for the detection of RNA structures across the entire transcriptome in vivo, and also enables full-length structural analysis of small RNAs, significantly improving the accuracy and precision of in vivo RNA structure detection. However, the smartSHAPE method uses mRNA capture beads to enrich mRNA. While this reduces the proportion of rRNA and tRNA in the target library, it is prone to losing non-mRNA sequences within the cell. Specifically, in the smartSHAPE method, mRNA capture beads with Poly T probes specifically bind to the Poly A tail of mRNA through complementary AT base pairing, thereby enriching mRNA. Although this method reduces the proportion of rRNA and tRNA in the library, it is prone to losing other types of RNA without Poly A. Alternatively, in improved methods, smartSHAPE uses riboerase to enrich the desired rRNA, but this is done at the initial stage of the reaction, and the technology does not realize that this can actually lead to a drastic reduction in sample size by more than ten times, easily causing sample loss. In addition, the smartSHAPE method involves multiple steps to ligate adapters to both ends of ssDNA using cyclase or T4 ligase. That is, it involves ligating a single-end adapter sequence, interspersed with multiple steps, before ligating another adapter sequence. This makes the process cumbersome and costly.

[0032] In this regard, an embodiment of the first aspect of the present invention provides a method for determining the secondary structure of RNA, comprising:

[0033] The sample pretreatment step includes processing the sample to give a first modification to the single-stranded regions of the RNA molecules in the sample to be tested; an RNA extraction step includes extracting the RNA with the first modification from the pretreated sample; a library preparation step includes: a reverse transcription step, which includes reverse transcription using the RNA molecules with the first modification as templates to obtain a DNA-RNA hybrid double strand formed by hybridization of one or more single-stranded cDNA molecules with the RNA molecules, wherein the one or more single-stranded cDNA molecules are separated by the first modification; a first purification step, which includes using a first enzyme to specifically cleave the single-stranded regions of the RNA molecules that have not hybridized with the one or more single-stranded cDNA molecules to obtain one or more DNA-RNA hybrid double strands and optional free RNA single-stranded molecules; a second purification step, which includes using a second enzyme to specifically hydrolyze the RNA strand and free RNA single-stranded molecules in the one or more DNA-RNA hybrid double strands to obtain one or more DNA single-stranded molecules; and a adapter ligation step, which includes using T4... DNA ligase simultaneously adds adapter sequences to both the 5' and 3' ends of the one or more single-stranded DNA molecules; a third purification step, which includes removing the DNA single-stranded molecule corresponding to the rRNA from the one or more single-stranded DNA molecules; an amplification and library preparation step, which includes amplifying the purified DNA single-stranded molecules to obtain a sequencing library; and a sequencing step, which includes sequencing the sequencing library and analyzing the RNA secondary structure. This method has simplified operation steps, low requirements for the starting amount of RNA, and high enrichment efficiency.

[0034] In some embodiments, the first modification is selected from at least one of the group consisting of NAI-N3, DMS, 1M7, and NAI. In some embodiments, the first modification is NAI-N3. In some specific embodiments, the sample pretreatment step includes treating the cell sample to be tested such that the single-stranded regions of the RNA molecules in the sample to be tested have NAI-N3 modification. In other words, the NAI-N3 modification is applied to non-intra-stranded complementary single-stranded regions such as RNA secondary structure loops and protrusions.

[0035] In some embodiments, the RNA extraction step includes extracting NAI-N3 modified RNA from the pretreated sample, wherein the RNA is total RNA including mRNA and non-coding RNAs such as rRNA, tRNA, miRNA, lncRNA, and snRNA.

[0036] In some embodiments, the library preparation steps include a reverse transcription step, a first purification step, a second purification step, a adapter ligation step, a third purification step, and an amplification and library construction step.

[0037] In some embodiments, the reverse transcription step includes reverse transcription using the RNA molecule with the first modification as a template to obtain a DNA-RNA hybrid double strand formed by hybridization of one or more single-stranded cDNA molecules with the RNA molecule, wherein the one or more single-stranded cDNA molecules are separated by the first modification. Specifically, since the NAI-N3 modification is located at the position of non-intra-strand complementary single-stranded regions such as loops and protrusions in the RNA secondary structure, when reverse transcription is performed using the RNA molecule as a template, cDNA molecules (here, single-stranded DNA obtained by reverse transcription) are obtained from the 5' to the 3' end, and reverse transcription terminates when the extension reaches the NAI-N3 modification. During reverse transcription, multiple reverse transcription primers may bind to one RNA molecule, resulting in the transcription of multiple cDNA molecules. Therefore, when the DNA-RNA hybrid double strand is formed, the multiple single-stranded cDNA molecules are presented in a form separated by the first modification.

[0038] In some embodiments, the first purification step includes using a first enzyme to specifically cleave the RNA molecule at a single-stranded region that has not hybridized with the one or more single-stranded cDNA molecules to obtain one or more DNA-RNA hybrid double strands and optionally free single-stranded RNA molecules. In some embodiments, the first enzyme is at least one selected from the group consisting of RNase I, RNase A, and RNase L, which are ribonucleases that specifically cleave single-stranded RNA. In some embodiments, the first enzyme is RNase I. RNase I can specifically cleave single-stranded RNA but cannot cleave RNA-cDNA hybrid strands. Therefore, RNase I specifically cleaves the RNA molecule at a single-stranded region that has not hybridized with the one or more single-stranded cDNA molecules (i.e., adjacent to NAI-N3 modification), thereby cleaving one or more DNA-RNA hybrid double strands and optionally free single-stranded RNA molecules into separate fragments.

[0039] In some embodiments, the second purification step includes using a second enzyme to specifically hydrolyze the RNA strand in the one or more DNA-RNA hybrid duplexes to obtain one or more DNA single-stranded molecules. In some embodiments, the second enzyme is selected from the group consisting of RNase H enzymes and RNase III enzymes, which specifically hydrolyze the RNA single strand in DNA-RNA hybrid duplexes. In some embodiments, the second enzyme is RNase H enzyme. RNase H enzyme specifically hydrolyzes the RNA single strand in DNA-RNA hybrid duplexes, thus allowing the extraction of one or more DNA single-stranded molecules from the DNA-RNA hybrid duplexes. By using RNase H enzyme, the potential damage to DNA or incomplete hydrolysis of RNA caused by the HCl / NaOH method is avoided, simplifying the cumbersome operation steps that require multiple additions of reagents to adjust the pH, reducing substrate loss, and thus lowering the required initial sample volume.

[0040] In some embodiments, a click reaction is performed after the RNA extraction step to link the biotin modification to the first modification. In some embodiments, this modification is NAI-N3. Thus, an enrichment step can be performed between the first and second purification steps, i.e., using streptavidin magnetic beads to link the biotin-modified DNA-RNA hybrid duplex to enrich the one or more DNA-RNA hybrid duplexes. The DNA-RNA hybrid duplexes are then further enzymatically digested.

[0041] In some embodiments, the adapter ligation step includes simultaneously adding adapter sequences to both the 5' and 3' ends of the one or more single-stranded DNA molecules using T4 DNA ligase. In some specific embodiments, the adapter sequences added to both ends are double-stranded headers containing a universal nucleotide single-stranded segment at one end, which can hybridize with the single-stranded DNA molecule. In some specific embodiments, the adapter sequences have nucleotide sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. In some embodiments, the one or more single-stranded DNA molecules are used to synthesize a second strand based on the adapter sequence to obtain one or more double-stranded DNA molecules. When cyclases process the fixed sequence ligation reaction, the steps include single-strand ligation, biotin enrichment, second-strand synthesis, end repair, DSA ligation, and PCR. The method of this application embodiment only requires one step of double-end ligation, which simplifies the steps and reduces product loss. Furthermore, the ligation efficiency of the template-based T4 DNA ligase is higher than that of the template-free circliase, thereby improving reaction efficiency. Therefore, the ligation method of simultaneously adding adapter sequences to both the 5' and 3' ends of one or more single-stranded DNA molecules using T4 DNA ligase reduces the number of steps and improves ligation efficiency.

[0042] In some embodiments, the third purification step includes removing the DNA single-stranded molecule corresponding to the rRNA from the one or more DNA single-stranded molecules. In some embodiments, the third purification step uses an rRNA removal probe kit to remove the DNA single-stranded molecule corresponding to the rRNA. In some specific embodiments, the rRNA removal probe may be a BIO-RAD SEQuoia RiboDepletion kit, Reagent Box A, cat.#12013675. In this embodiment of the invention, rRNA removal is performed after obtaining intact DNA single-stranded molecules. That is, after obtaining total RNA and performing multiple steps such as click chemistry, reverse transcription, enzyme digestion, enrichment, re-enzyme digestion, and purification, the rRNA removal step is performed. This effectively avoids the sharp decrease in sample volume caused by immediately performing rRNA removal after total RNA extraction in low-starting-volume samples, further improving the adaptability to low-starting-volume samples. In other words, setting the rRNA removal step after cDNA generation can increase the amount of substrate in the early experimental processes, reduce substrate loss, and even avoid substrate loss altogether. This makes the requirement for a lower initial sample volume lower.

[0043] In some embodiments, mitochondrial RNA removal probes and / or tRNA removal probes may be used as needed.

[0044] In some embodiments, the amplification and library preparation steps include amplifying purified single-stranded DNA molecules to obtain a sequencing library. The sequencing step includes sequencing the sequencing library and analyzing the RNA secondary structure.

[0045] In some embodiments, the RNA secondary structure determination method of the present invention further includes a fourth purification step, which comprises purification using a total RNA filtration kit between the RNA extraction step and the library preparation step. In some embodiments, the total RNA filtration kit is the ZYMO RNA Clean & Concentrator kit.

[0046] In some embodiments, the RNA secondary structure determination method of this invention further includes a fifth purification step, which is performed between the second and third purification steps using a total DNA filtration kit. In some embodiments, the total DNA filtration kit is the ZYMO DNA Clean & Concentrator kit.

[0047] In some embodiments, the reverse transcription step includes reverse transcription using the RNA molecule with the first modification as a template to obtain one or more single-stranded cDNA molecules; and subjecting the one or more single-stranded cDNA molecules and the RNA molecule to a renaturation step to hybridize and form the DNA-RNA hybrid double strand.

[0048] In some embodiments, the sample includes live cells, primary cells, early mammalian embryos, post-infected cells, bacteria, fungi, cell lines, and viruses.

[0049] In some embodiments, the sample has 10-10^6 cells. In other words, the method described in this embodiment requires a very low initial sample quantity, as low as 10-10^6 cells or 20 fg RNA.

[0050] In some embodiments, the RNA secondary structure determination method proposed in this invention first performs a sample pretreatment step, which includes treating the cell sample to be tested to induce NAI-N3 modification in the single-stranded regions of the RNA molecules in the sample. Then, an RNA extraction step is performed, which includes extracting NAI-N3 modified RNA from the pretreated sample, wherein the RNA is total RNA including rRNA, mRNA, tRNA, and non-coding RNAs such as miRNA, lncRNA, and snRNA. Finally, a library preparation step is performed, which includes reverse transcription, a first purification step, a second purification step, adapter ligation, a third purification step, and amplification and library construction steps. The reverse transcription step includes reverse transcription using the NAI-N3 modified RNA molecule as a template to obtain a DNA-RNA hybrid double strand formed by hybridization of one or more single-stranded cDNA molecules with the RNA molecule, wherein the one or more single-stranded cDNA molecules are separated by the NAI-N3 modification; the first purification step includes using RNase I enzyme to specifically cleave the single-stranded regions of the RNA molecule that have not hybridized with the one or more single-stranded cDNA molecules to obtain one or more DNA-RNA hybrid double strands and optional free RNA single-stranded molecules; the second purification step includes using RNase H enzyme to specifically hydrolyze the RNA strand and free RNA single-stranded molecules in the one or more DNA-RNA hybrid double strands to obtain one or more DNA single-stranded molecules; the adapter ligation step includes using T4 DNA ligase to simultaneously add adapter sequences to the 5' and 3' ends of the one or more DNA single-stranded molecules in one step; the third purification step includes using an rRNA depletion probe to remove the rRNA-corresponding DNA single-stranded molecules from the one or more DNA single-stranded molecules; the amplification and library construction step includes amplifying the purified DNA single-stranded molecules and obtaining a sequencing library. This sequencing library is used for subsequent sequencing and analysis.

[0051] A second aspect of the present invention provides a kit comprising: a first modification reagent, a first enzyme, a second enzyme, a T4 DNA ligase, an rRNA removal probe, and an adapter.

[0052] In some embodiments, it also includes RNA extraction reagents, reverse transcription reagents, biotin modification reagents, and streptavidin magnetic beads.

[0053] In some embodiments, the first modifying reagent is a NAI-N3 modifying reagent, the first enzyme is RNase I enzyme, and the second enzyme is RNase H enzyme.

[0054] The following embodiments are used to further illustrate the advantages and features of this method, and are not intended to limit the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the field or according to the product instructions.

[0055] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0056] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0057] Example 1

[0058] 1.1 Sample Pretreatment

[0059] Lung cancer cell lines (such as NCI-H2030 and NCI-H1793, purchased from Nanjing Kebai, catalog numbers CBP60068 and CBP60117 respectively) were resuspended in 100 mM NAI-N31xPBS solution (purchased from Ponopharm, catalog number E2S31132) and treated for 5 min.

[0060] 1.2 RNA extraction

[0061] (1) RNA extraction: Total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen, catalog number 15596018CN) according to the instructions and dissolved in 50 μL RNase-free water (purchased from TransGen Biotech, catalog number GI101-03). The RNA concentration was measured and recorded using Nanodrop.

[0062] (2) Click reaction: Click reaction was carried out with DBCO-biotin (purchased from Guangzhou Weihua Biotechnology, catalog number WH031N09) and RiboLockRNase Inhibitor (purchased from Thermo Scientific, catalog number EO0832). The specific reaction system is shown in Table 1 below.

[0063] Table 1 Click Reaction System

[0064]

[0065] (3) RNA purification: RNA was purified using the ZYMO RNA Clean & Concentrator kit (purchased from ZYMO research, catalog number 411008), and finally eluted with 10 μL TE buffer to collect 9.5 μL of product.

[0066] 1.3 Library Preparation

[0067] (1) Renaturation: Mix the RNA purification product from the previous step according to the system in Table 2 below (the components in Table 2 and Table 3 are from SuperScript III Reverse Transcriptase, purchased from Invitrogen, catalog number 2740346). After mixing, place the sample in a PCR instrument and treat at 85℃ for 3 minutes, then slowly cool to 4℃ (0.1℃ / s).

[0068] Table 2 Refolded Systems

[0069]

[0070] (2) Reverse transcription:

[0071] Add the reagents from Table 3 below to the PCR tube from the previous step and mix well. Place the sample in the PCR instrument and perform reverse transcription. The specific reaction program is as follows: 4℃ 2 min → 15℃ 3 min → 25℃ 10 min → 42℃ 45 min → 50℃ 25 min → 4℃. Hold.

[0072] Table 3 Reverse Transcription Reaction System

[0073]

[0074] (3) First purification: Mix the PCR product from the previous step with the reagent in Table 4 (Thermo Fisher, catalog number EN0602), place it in a PCR tube, and incubate at 37 ℃ for 30 min.

[0075] Table 4 Purification System

[0076]

[0077] (4) Enrichment and second purification: Take 10 μL of Streptavidin C1 magnetic beads (purchased from Invitrogen; catalog number: 2901190) and enrich the product of the previous step according to the instructions. Add the enriched product to the enzyme digestion system shown in Table 5 below for elution. 37℃, 1000 rpm, 20 minutes, mix occasionally.

[0078] Table 5 Purification System

[0079]

[0080] Add 30 μL of the RNase H reaction system shown in Table 5 to digest the purified product of C1 magnetic beads.

[0081] (6) Purification: The enzyme digestion products were purified using ZYMO DNA Clean & Concentrator (purchased from ZYMO research, catalog number D4014) to obtain the target DNA single-stranded molecules.

[0082] (7) Connector connection:

[0083] Configure the phosphorylation reaction system as shown in Table 6 below. Phosphorylate DNA single-stranded molecules at 37°C for 15 minutes, denature them at 95°C for 5 minutes, and then immediately place them on ice for 2 minutes.

[0084] Table 6 Formulations for Phosphorylation and Denaturation Reaction Solutions

[0085]

[0086] Then, as shown in Table 7 below, add the ssDNA double-end adapter ligation reaction system, which includes the adapter sequence and T4 DNA ligase (NEB M0204S). The adapter sequence was purchased from Sangon Biotech.

[0087] Table 7. Formulation of Reaction Solution for Double-Ended Connectors

[0088]

[0089] Table 8

[0090]

[0091] Table 9

[0092]

[0093] (8) Third purification:

[0094] The adapter-containing DNA single-stranded molecules were purified and recovered using the ZYMO DNA Clean & Concentrator kit, and finally eluted with 7 μL of TE buffer. The recovered DNA single-stranded molecules were mixed with an rRNA depletion probe, and the rRNA removal reaction system was prepared as shown in Table 10 below (BIO-RAD SEQuoia RiboDepletion kit, Reagent Box A, cat.#12013675), and slowly renatured according to the following procedure: 98 0 C 1 min→62 0 C 10 min→72 0C 1 min→20 0 C1 is maintained. 50 μL of C1 beads is placed on a magnetic rack for 2 minutes, and the supernatant is discarded. 30 μL of the above renaturation system is added and mixed with the C1 beads. The C1 beads adsorb rRNA from the system. After purification, 27 μL of the supernatant system and its DNA single-stranded molecules are recovered.

[0095] Table 10 rRNA removal reaction system

[0096]

[0097] 1.4 Amplification, Library Construction, and Sequencing

[0098] (1) Amplification: Mix the recovered product with the qPCR system shown in Table 11 below and place it in a qPCR instrument to amplify the target product.

[0099] Table 11 qPC System

[0100]

[0101] Table 12

[0102]

[0103] (2) Recovery: After removing large fragments by adding 0.4x DNA clean beads, the product was finally recovered with 0.9x DNA clean beads, eluted with 20 μL TE buffer, and 20 μL of supernatant was collected. The concentration was determined and labeled using the Qubit™ HS dsDNA Quantitative Kit (Thermofisher Q32851) after purification.

[0104] (3) Library construction: The product was constructed according to the library construction procedure of BGI sequencer, using MGIEasy circularization module PN1000005260 and DNBSEQ DNB preparation kit REF10000016115. Then, the product was sequenced by MGISEQ-2000 sequencing platform according to PE150 protocol.

[0105] (4) RNA secondary structure determination was performed according to the smartSHAPE analysis pipeline.

[0106] 1.5 Quality Inspection

[0107] (1) The library was detected by gel electrophoresis using a nucleic acid non-deformable polyacrylamide gel preparation kit (Solepro, Lot. No. 240001001).

[0108] (2) The length and distribution of the library were detected using an Agilent 2100 bioanalyzer, and the quality was tested using a High Sensitivity DNA kit (purchased from Agilent; catalog number: 5067-1504).

[0109] (3) The proportion of rRNA in the RNA library of lung cancer cell lines was statistically analyzed (rRNA accounts for about 83% of the total RNA in cells).

[0110] (4) The types of RNA amplification products from four lung cancer cell lines were statistically analyzed.

[0111] Figure 2 This is a graph showing the gel electrophoresis results of the library obtained in the library preparation step of the RNA secondary structure determination method according to an embodiment of the present invention. Figure 3 This is a graph showing the quality test results of the library obtained by the library preparation step of the RNA secondary structure determination method in this embodiment of the invention.

[0112] Depend on Figure 2 and Figure 3 It can be seen that the target library is relatively concentrated, and the size of the library is exactly concentrated within the sequencing range of the sequencer.

[0113] Figure 4 This is a comparison chart of the proportion of rRNA in the human genome between the library obtained by the library preparation step of the RNA secondary structure determination method in this embodiment of the invention and the human genome. Figure 5 This is a graph showing the percentage of RNA types in the library obtained by the library preparation step of the RNA secondary structure determination method in this embodiment of the invention.

[0114] Depend on Figure 4 and Figure 5 It can be seen that this method can significantly reduce the proportion of rRNA in the RNA library and effectively capture non-coding RNAs without poly A tails (such as lncRNA, snRNA, miRNA, etc.).

[0115] Example 2

[0116] Following the method in Example 1, RNA secondary structure was determined for samples with an initial sample size of 10 cells or 20 fg RNA (cell / sample source was the same as in Example 1).

[0117] The results are as follows Figure 6 and Figure 7 As shown, this demonstrates that the method in the embodiments of this application has high sensitivity and low requirements for the initial sample amount.

[0118] In summary, this protocol significantly improves RNA enrichment efficiency and reduces costs by optimizing the smartSHAPE method, while avoiding the loss of non-coding RNA due to Poly A-tail dependence, thus enhancing the integrity of transcriptome analysis. Furthermore, this protocol simplifies the adapter ligation step, reducing operation time and material consumption, and improving experimental efficiency. For low starting sample volumes, this protocol optimizes the processing flow and improves assay sensitivity (e.g., ...). Figure 6 As shown, this method can still detect the structures of multiple RNAs within cells with a starting sample size of 10 cells, requiring a lower initial sample size, making it widely applicable to the study of precious or rare samples. These improvements make this protocol more efficient, economical, and adaptable in RNA structure determination.

[0119] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0120] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0121] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for determining the secondary structure of RNA, characterized in that, The steps performed sequentially include: sample pretreatment, total RNA extraction, library preparation, and sequencing. The sample pretreatment step includes processing the sample to give the single-stranded regions of the RNA molecules in the sample to be tested a first modification; The total RNA extraction step includes extracting RNA with a first modification from a pretreated sample, and performing a click reaction after the total RNA extraction step to link the biotin modification to the first modification. The library preparation steps include the following steps performed in sequence: The reverse transcription step includes reverse transcription using an RNA molecule with a first modification as a template to obtain a DNA-RNA hybrid double strand formed by hybridization of one or more single-stranded cDNA molecules with the RNA molecule, wherein the one or more single-stranded cDNA molecules are separated by the first modification. The first purification step includes using a first enzyme to specifically cleave the single-stranded region of the RNA molecule that has not hybridized with the one or more single-stranded cDNA molecules to obtain one or more DNA-RNA hybrid double strands and optionally free RNA single-stranded molecules. The enrichment step includes using streptavidin magnetic beads to link to the DNA-RNA hybrid double strands having the biotin modification, thereby enriching the one or more DNA-RNA hybrid double strands; The second purification step includes using a second enzyme to specifically hydrolyze the RNA strand and free single-stranded RNA molecules in the one or more DNA-RNA hybrid double strands to obtain one or more DNA single-stranded molecules; The adapter ligation step involves using T4 DNA ligase to simultaneously add adapters to both the 5' and 3' ends of one or more single-stranded DNA molecules. The third purification step includes removing the DNA single-stranded molecule corresponding to the rRNA from the one or more DNA single-stranded molecules; and Amplification and library construction steps are used to obtain sequencing libraries.

2. The method for determining RNA secondary structure according to claim 1, characterized in that, The first modification is selected from at least one of the group consisting of NAI-N3, DMS, 1M7, and NAI.

3. The method for determining RNA secondary structure according to claim 1, characterized in that, The first enzyme is at least one selected from the group consisting of RNase I, RNase A, and RNase L, which are ribonucleases that specifically cleave single-stranded RNA. The second enzyme is RNase H, an enzyme that specifically hydrolyzes the RNA single strand in a DNA-RNA hybrid double strand.

4. The method for determining RNA secondary structure according to claim 1, characterized in that, The connector is a double-linked connector with a universal nucleotide single-stranded segment at one end, which can hybridize with the DNA single-stranded molecule.

5. The method for determining RNA secondary structure according to claim 1, characterized in that, The third purification step uses an rRNA removal probe kit to remove the DNA single-stranded molecules corresponding to the rRNA.

6. The method for determining RNA secondary structure according to claim 1, characterized in that, The samples are selected from live cells, primary cells, bacteria, fungi, cell lines, viruses, and infected cells. The method described is not a disease diagnostic method.

7. The method for determining RNA secondary structure according to claim 1, characterized in that, Also includes: The fourth purification step, which includes purification using a total RNA filtration kit between the click reaction and library preparation steps; and / or The fifth purification step, which is performed between the second and third purification steps, uses a total DNA filtration kit for purification.

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