Comprehensive and Quantitative Analysis Method for RNA-Protein Complex Interactions Using Synthetic RNA

Abstract

An object of the present invention is to provide a method for safely, rapidly, quantitatively, and comprehensively identifying a protein complex that binds to any RNA sequence. The present invention provides a comprehensive and quantitative RNA-protein complex interaction analysis method using synthetic RNA, which includes a method of designing a Flag-tagged synthetic RNA to be nested with respect to a target RNA in order to comprehensively identify binding proteins of all partial sequences from the 5'-end to the 3'-end of the target RNA.

Description

Technical Field

[0001] The present invention relates to a comprehensive and quantitative method for analyzing RNA-protein complex interactions using synthetic RNA, and an application method for drug discovery by screening for inhibitors of RNA-protein complex interactions using this method.

Background Art

[0002] As a method for identifying RNA-binding proteins, a method using an RNA probe has been performed. This method for identifying RNA-binding proteins using an RNA probe is known as RNA affinity Purification followed by Mass Spectrometry (Non-Patent Document 1). In the analysis of protein complexes by this RNA affinity method, it has been difficult to distinguish non-specific signals from significant signals, and it has also been difficult to identify the RNA partial sequences to which proteins bind (see Fig. 1A).

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a method for safely, rapidly, quantitatively, and comprehensively identifying protein complexes that bind to an arbitrary RNA sequence.

Means for Solving the Problems

[0005] The present invention provides a comprehensive and quantitative RNA-protein complex interaction analysis method using synthetic RNA, which includes a tiling design of synthetic RNA modified with a Flag-tag in an nested manner with respect to a target RNA in order to comprehensively identify the binding proteins of all partial sequences from the 5'-end to the 3'-end of the target RNA, and an RNA-protein complex interaction analysis method.

[0006] In the RNA-protein complex interaction analysis method of the present invention, proteins that bind to a plurality of RNA probes and a negative control containing only Flag without RNA are comprehensively identified by mass spectrometry, and by normalizing, relative quantification, and correcting experimental errors for each signal, a quantitative analysis of the relative binding strength of RNA-binding proteins is performed. It may be an RNA-protein complex interaction analysis method.

[0007] In the RNA-protein complex interaction analysis method of the present invention, by simultaneously determining the RNA-protein complex of a protein and RNA that interacts with a target RNA and its interaction sequence, it includes exploring the interaction protein complex with respect to the target RNA and specifying the interaction sequence. It may be an RNA-protein complex interaction analysis method.

[0008] Furthermore, the present invention is a comprehensive and quantitative RNA-protein complex interaction analysis method using synthetic RNA, (1) RNA probes fragmented at approximately 300 to 1000 nt intervals from the 5'-side of an arbitrary target RNA sequence are tiled designed so that 50% of the sequence length of each probe overlaps, and the RNA is prepared by in vitro transcription or chemical synthesis (see Figure 1B), (2) Performing a Flagging treatment on the prepared RNA probes, (3) Mixing the Flagged RNA probes with a cell lysate and allowing them to interact to form an RNA-protein complex. (4) Immunoprecipitate and purify the RNA-protein complex using an anti-Flag tag antibody. (5) Separate the peptide fragments of the protein in the purified RNA-protein complex by liquid chromatography, and identify the peptide sequence signals and the corresponding proteins by mass spectrometry (LC-MS / MS method). (6) Normalize the signals of the identified proteins for each RNA probe and each protein, standardize the relative protein binding strength between RNA probes for each protein for multiple experimental data, and calculate the z-score. And (7) Use the z-score to test and analyze whether the difference in protein binding strength between the negative control and each RNA probe is statistically significant. provided is a method for analyzing RNA-protein complex interactions, including the above steps.

[0009] Further, the present invention provides a method for screening an inhibitor of RNA-protein complex interaction, which uses the method for analyzing RNA-protein complex interaction of the present invention, adds a small molecule compound library for experiments, and screens for small molecule compounds that inhibit RNA-protein interaction.

[0010] Further, the present invention provides a method for screening an antiviral drug against an RNA virus, which selects an RNA virus sequence as a target RNA sequence, performs the method for analyzing RNA-protein complex interaction of the present invention to identify host interaction protein factors and interacting RNA sequences of the RNA virus at a resolution of several hundred bases, and adds a small molecule compound library for experiments to screen for antiviral prophylactic or therapeutic drugs that inhibit RNA-protein interaction.

[0011] Furthermore, the present invention provides a method for screening a prophylactic or therapeutic agent targeting a disease-specific mutant RNA sequence, which comprises performing the RNA-protein complex interaction analysis method of the present invention on an RNA sequence having a disease-specific mutation of a disease including cancer to identify a mutation-specific interacting protein factor and an interacting RNA sequence at a resolution of several hundred bases, and adding a small molecule compound library to perform experiments to screen for a disease-specific therapeutic agent that inhibits the RNA-protein interaction.

Advantages of the Invention

[0012] The present invention can provide a method for safely, rapidly, quantitatively, and comprehensively identifying a protein complex that binds to an arbitrary RNA sequence. In the method of the present invention, non-specific signals can be removed by relative quantification between tiled-designed RNA probes, and the relative binding strength can be precisely measured with statistical significance. In addition, since the full-length RNA sequence is not required for the experiment, a protein complex that binds to an RNA virus sequence that causes dangerous infectious diseases can be rapidly and safely detected in various biological species and tissues. Further, by subjecting the RNA probe to nucleic acid modification or sugar chain modification characteristic of a virus or the like, the actual viral RNA state can be artificially reproduced, and the interacting RNA-binding protein can be analyzed in detail. Thus, it is a technology applicable to drug discovery such as screening and designing compounds that inhibit RNA-protein interactions of known and novel RNA viruses and the like. This method can also be applied to the identification of a protein complex that specifically binds to a disease-specific RNA mutation site such as cancer as well as RNA viruses. Furthermore, it is a technology applicable to drug discovery such as screening and designing compounds that inhibit RNA-protein interactions. Therefore, it is expected to be an advanced technology that is very beneficial for next-generation drug discovery. It can also be applied as basic data when designing sequences in mRNA drug discovery and the like. Therefore, it is expected to be an advanced technology that is very beneficial for next-generation drug discovery.

Brief Description of the Drawings

[0013]

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Mode for Carrying Out the Invention

[0014] One of the present inventions is a comprehensive and quantitative method for analyzing RNA-protein complex interactions using synthetic RNA. The operating procedure of the RNA-protein interaction analysis method of the present invention will be described below.

[0015] 1. Preparation of RNA Probe Fragment an arbitrary target RNA sequence into fragments of approximately 300 to 1000 nt from the 5'-end, and prepare the RNA by in vitro transcription method or chemical synthesis method. At this time, each probe is designed with a tiling so that 50% of the sequence length overlaps. The synthesized RNA probe is subjected to Flag-tagging treatment.

[0016] 2. Generation and Purification of RNA-Protein Interaction Complex Form an RNA-protein complex by interacting the prepared RNA probe with a cell lysate. The cell lysate is prepared by lysing any cell with a reagent. The RNA-protein complex is immunoprecipitated and purified with an anti-Flag tag antibody.

[0017] 3. Identification of RNA-Binding Proteins by Mass Spectrometry The purified RNA-protein complex is further purified, and the RNA-binding protein is decomposed into peptide fragments. The decomposed peptide fragments are separated by liquid chromatography, and the peptide sequence signals and the corresponding proteins are identified by mass spectrometry (LC-MS / MS method).

[0018] 4. Relative Quantification of RNA-Binding Proteins The signals of the identified proteins are normalized for each RNA probe and each protein. For multiple experimental data, the relative binding strength of the protein between RNA probes is standardized for each protein, and the z-score is calculated. Using the z-score, it is tested whether the difference in the protein binding strength between the negative control and each RNA probe is statistically significant.

[0019] The features of the RNA-protein interaction analysis method of the present invention are described below. The present invention is a method for safely, rapidly, quantitatively, and comprehensively identifying a protein complex that binds to an arbitrary RNA sequence and its RNA-binding sequence with a resolution of several hundred to several thousand nucleotides. By this method, the protein complex that binds to each partial sequence from the 5'-end to the 3'-end of the target RNA and its binding sequence can be comprehensively and quantitatively analyzed.

[0020] In the method for analyzing RNA-protein interactions of the present invention, by relatively quantifying the intensity of RNA-protein interactions between a negative control and a plurality of RNA probes designed by tiling, it is possible to remove non-specific signals, and the relative binding strength can be precisely measured with statistical significance. In addition, since the full-length RNA sequence is not required for the experiment, protein complexes that bind to RNA virus sequences that cause dangerous infectious diseases can be rapidly and safely detected in various species and tissues. By modifying the RNA probe with nucleic acid modifications or sugar chain modifications characteristic of viruses, etc., the actual viral RNA state can be artificially reproduced, and the interacting RNA-binding proteins can be analyzed in detail. Thereby, protein complexes and binding sequences that bind to known and emerging RNA viruses can be identified. This method can also be applied to the identification of protein complexes that specifically bind to disease-specific RNA mutation sites such as cancer as well as RNA viruses. Furthermore, it is a technology applicable to drug discovery such as screening and designing compounds that inhibit RNA-protein interactions. Therefore, it is expected to be an advanced technology that is very beneficial for next-generation drug discovery.

[0021] The features of the RNA-protein interaction analysis method of the present invention compared with the conventional method are described below. The method for identifying RNA-binding proteins using conventional RNA probes is a technique known as RNA affinity purification followed by mass spectrometry (RNA affinity Purification followed by Mass Spectrometry). However, the present invention has advantages in the following aspects. First, in the prior art, biotin is generally the type of antigen tag for RNA for performing immunoprecipitation (IP; immunoprecipitation). In the present invention, the Flag amino acid sequence with less noise is adopted. Next, in the RNA-protein interaction analysis by the RNA affinity method, which is a prior art technique, it was difficult to identify the RNA partial sequence to which the protein binds. Also, it was difficult to discriminate between non-specific binding signals and statistically significant binding signals. In particular, proteins with weak binding signals may not be subject to downstream additional analysis. This is because there is no appropriate method for correcting experimental errors in existing methods, and the results may vary greatly depending on the number of experimental runs. Therefore, it was difficult to perform the test for statistical significance itself. In the method of the present invention, first, by making the probe design method a tiling method that covers the entire target RNA in a nested manner, it is possible to comprehensively identify the proteins that bind to each partial sequence, and relative quantification of the binding proteins among the partial sequences of RNA becomes possible. Also, by standardizing the protein binding patterns among multiple RNA probes for each experiment, successful correction of experimental errors between multiple experimental data was achieved. As a result, removal of background noise and reproducible relative quantification of signals were realized. Thus, not only proteins with strong signal intensity but also proteins with weak signal intensity, which have hitherto sometimes been excluded from analysis, can be precisely and comprehensively analyzed using statistical test methods for specific binding proteins to the target RNA sequence. Therefore, binding proteins can be identified at the complex level. Furthermore, by changing the length of the RNA probe to be designed to an arbitrary length, it is possible to comprehensively determine the interacting RNA sequences of the binding protein complex at a resolution of an arbitrary length. Therefore, it is possible to comprehensively map the interacting proteins in the full length of the target RNA at a resolution of an arbitrary RNA length.There is no example similar to this mapping technology so far, and it is a novel invention.

[0022] In addition, since the pharmaceutical screening method based on this technology can be achieved only from the sequence information and base modification information of viral RNA, it is also effective for targets with little information such as emerging infectious diseases. As a methodology for the development of antiviral drugs, it is also very epoch-making and may dramatically improve the speed of antiviral drug development.

[0023] Furthermore, by identifying protein interaction factors at disease-specific RNA mutation sites such as cancer and screening for inhibitors thereof, it can also be used as a screening method for disease therapeutic drugs such as novel anticancer drugs.

Example

[0024] The present invention will be further specifically described with the following examples, but it can be appropriately changed without departing from the gist of the present invention. Therefore, the scope of the present invention is not limitedly interpreted by the specific examples shown below.

[0025] 1. Design of RNA probe for target RNA LINE1 (long interspersed element 1), which is a major transposon retaining metastatic activity in the human genome, was selected as the target RNA, and a tiling RNA probe of about 1000 nt was designed for the target RNA of about 6000 nt in full length (upper part of Fig. 2A). In addition, for the first RNA probe region on the 5' side, tiling probes divided every about 300 nt were further designed (lower part of Fig. 2A).

[0026] 2. Preparation of RNA probe Based on the designed array, DNA serving as a template for RNA synthesis was chemically synthesized and purified. Using the synthesized DNA as a template, RNA was synthesized by in vitro transcription. Amplification of all RNA probe regions was performed by PCR using a primer with a T7 promoter sequence at the 5´ end, and RNA synthesis was carried out using the MEGAscript T7 kit (AMB13345, manufactured by Thermo Fisher Scientific) according to the attached protocol. A reaction was performed to covalently bond Flag-hydrazide to the 3´ end of the synthesized RNA using a known method (“ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway (Nucleic Acids Res. 2014 Sep;42(15):10037-49. doi: 10.1093 / nar / gku652.”) to label the 3´ end of the RNA with Flag. Specifically, 250 pmol of RNA was added to 60 μl of 0.1 M NaIO 4 and incubated at 0°C for 10 minutes to dialdehydeize the 3′ end of the RNA. Next, the RNA was precipitated with 2% LiClO 4 / acetone and washed with 1 ml of acetone. After removing the acetone, the RNA was dissolved in 10 μl of 0.1 M sodium acetate, pH 5.2, mixed with 30 mM hydrazide-Flag peptide, and subjected to a mixing reaction at room temperature for 30 minutes. The imine functional group of the RNA was reduced by adding 12 μl of 1 M NaCNBH 3 and performing a reduction reaction for 30 minutes. The RNA after the reaction was purified using the RNeasy Mini Kit (manufactured by Qiagen).

[0027] 3. Immunoprecipitation and Protein Identification Using a known method for purified Flag-labeled RNA (“ZFP36L1 and ZFP36L2 control LDLR mRNA stability via the ERK-RSK pathway (Nucleic Acids Res. 2014 Sep;42(15):10037-49.doi:10.1093 / nar / gku652.”), the binding protein was identified. Specifically, 10 pmol of purified Flag-labeled RNA was mixed with an anti-Flag antibody (M2, manufactured by Sigma Aldrich) and Protein G-magnetic beads (DB10004, manufactured by Thermo Fisher Scientific), and reacted at 4°C for 1 hour. Then, 1 mg of cell extract protein extracted from cultured K562 cells was added and the reaction was further carried out at 4°C for 1 hour. After washing away the unbound protein, the RNA and RNA-binding protein were eluted with Flag peptide. The sample obtained by elution was treated with RNaseA and then precipitated with TCA. The precipitate was washed with cold acetone and dissolved in a 7M guanidine solution. The dissolved protein sample was subjected to cysteine residue CAMylation by using TCEP and IAA, and then decomposed into peptides by lysyl endopeptidase (manufactured by Fujifilm Wako Pure Chemical Corporation) and trypsin (manufactured by Thermo Fisher Scientific). The decomposed peptides were analyzed by LC-MS / MS method. For the LC part, Easy-nLC 1200 manufactured by Thermo Fisher Scientific was used, and for the mass spectrometry part, Q-Exactive HF-X instrument manufactured by Thermo Fisher Scientific was used. Peptide separation was performed using a C18 reverse-phase column. The mobile phase of LC was 0.1% formic acid distilled water and 0.1% formic acid 80% acetonitrile (gradient of 80 minutes with a slope from 5 to 40%), and analysis was carried out at a constant flow rate of 300 nl / min. The eluted peptides were introduced directly into the mass spectrometer through a spray column, and data acquisition was performed using a data-dependent shotgun analysis method (scanning was performed between 380 and 1500 m / z, and MS / MS data acquisition for the TOP25 precursor ions obtained by MS was defined as one cycle).Regarding the obtained data, analysis was performed using Proteome Discoverer 2.2 (manufactured by Thermo Fisher Scientific) software to identify and quantify peptides. For peptide identification, an existing human protein database was used and performed under the condition of FDR < 1%.

[0028] 4. Mapping of interacting proteins to the target RNA An RNA-protein complex formed by interacting a tiling RNA probe of approximately 1000 nt with the target RNA and cell lysate was purified, and proteins binding to each probe were comprehensively identified using the LC-MS / MS method. To obtain relative binding strengths, normalization of the relative signal intensities between each probe and between proteins was performed. Mass spectrometry was repeated twice for each sample for each experiment, and independent experiments were performed three times to obtain a total of six sets of data. A heatmap showing the relative binding strengths indicated by z-score is shown with the proteins binding to each partial sequence on the vertical axis and the designed RNA probes (each with six sets of data) on the horizontal axis (Figure 2B). The six experimental data for each probe showed highly reproducible protein binding patterns respectively.

[0029] Next, the above was performed in the same manner using tiling probes divided into approximately 300 nt segments for the probe region at the 5' end. A heatmap showing the average values of the relative binding strengths of the proteins binding to each probe indicated by z-score is shown (Figure 3A). Mass spectrometry was performed twice for each probe and independent experiments were performed twice to obtain a total of four sets of data. At this time, the constituent proteins of each protein complex showed similar binding patterns to each other. As an example, the binding pattern of the constituent proteins of protein complex X is shown (Figure 3B). Protein complex X was shown to strongly bind to probe 4.

[0030] 5. Protein binding signal correction and statistical testing between experiments Examples of protein binding signal correction and statistical tests between experiments for Protein X identified as a target RNA-binding protein (in the case of results using tiling probes of ~1000 nt) are shown. The raw data of the relative protein binding strength of each RNA probe after normalization differed for each experimental run (upper panel of Fig. 4A). Therefore, the relative binding strength was standardized for each experimental run and converted to a z-score to correct for errors between experiments (middle panel of Fig. 4A). Also, to examine whether each RNA probe binds to Protein X statistically significantly compared to the negative control (Mock), statistical significance was tested using the experimental data from six runs (lower panel of Fig. 4A). As a result, it was found that Protein X binds significantly to probes 1, 3, and 4 in any of the test methods of student t-test, welch’s t-test, Mann-Whitney U test, Brunner-Munzel test, and permutation test (FDR < 0.05).

[0031] The above information processing and analysis were performed for all binding proteins. The number of proteins that bound statistically significantly to each RNA probe is shown in Fig. 4B (welch’s t-test, FDR < 0.05). The upper vertical bar graph shows the number of proteins that bind only to each probe, and the lower horizontal bar graph shows the total number of proteins that bind to each probe.

[0032] 6. Correlation coefficient (reproducibility) between experiments In the existing method, the experimental error between experiments was large, but the method of the present invention showed high reproducibility between experiments. For the example using 1000-nt tiling probes, the Pearson correlation coefficients of the protein binding patterns between each experiment and between RNA probes are shown in a heat map (Figure 5A). A high positive correlation is shown between different experiments within the same probe. Also, a tendency for a high positive correlation was confirmed even between adjacent probes with partially overlapping sequences. Further, a scatter plot of the z-scores of the binding proteins for each RNA probe in Experiment 1 and Experiment 3 is shown in Figure 5B. A high correlation between experiments was observed for all RNA probes. The r described in each of these graphs represents the Pearson correlation coefficient, and rs represents the Spearman correlation coefficient. Also, the distribution of the correlation coefficients between experiments of the RNA binding patterns for each protein is shown in a violin plot (left in Figure 5C: Pearson correlation coefficient, right: Spearman correlation coefficient). The vertical axis represents the correlation coefficient, and the horizontal axis represents the combination of experimental runs to be compared. A high correlation coefficient distribution was shown between any experiments. From the above results, it became clear that the method of the present invention is a highly practical method having high reproducibility between experiments.

Claims

1. A comprehensive and quantitative RNA-protein complex interaction analysis method using synthetic RNA, comprising: (1) preparing a plurality of fragmented RNA probes of an arbitrary target RNA sequence, designed such that each RNA probe overlaps with respect to the target RNA, by in vitro transcription or chemical synthesis; (2) mixing the RNA probes with a cell lysate and allowing them to interact to form an RNA probe-protein complex; (3) purifying the RNA probe-protein complex; (4) separating peptide fragments of the protein in the purified RNA probe-protein complex by liquid chromatography and identifying the peptide sequence signals and corresponding proteins by mass spectrometry (LC-MS / MS); and (5) normalizing the signals of the identified proteins for each RNA probe and each protein, and quantitatively calculating the relative protein binding strength between RNA probes for each protein with respect to the experimental data. An RNA-protein complex interaction analysis method characterized by including the above steps.

2. The comprehensive and quantitative RNA-protein complex interaction analysis method using synthetic RNA probes, characterized by including nested tiling design such that each RNA probe overlaps with respect to the target RNA in order to comprehensively identify the binding proteins of all partial sequences from the 5' end to the 3' end of the target RNA, the RNA-protein complex interaction analysis method according to Claim 1.

3. The RNA-protein complex interaction analysis method according to Claim 1, characterized by performing Flag-tagging on the prepared RNA probes and performing immunoprecipitation purification on the RNA probe-protein complex using an anti-Flag tag antibody.

4. The RNA-protein complex interaction analysis method according to Claim 1, characterized by comprehensively identifying the proteins binding to a plurality of RNA probes and a negative control without an RNA probe by mass spectrometry, and testing and analyzing whether the difference in protein binding strength between the negative control and each RNA probe is statistically significant. **Claim 5**: For each of a plurality of experimental data, standardize the relative protein binding strength between RNA probes for each protein, calculate the z-score, and perform relative quantification and inter-experiment error correction, thereby performing quantitative analysis of the relative binding strength of RNA-binding proteins. The RNA-protein complex interaction analysis method according to claim 1, characterized by including this. **Claim 6**: By simultaneously determining an RNA probe-protein complex of a protein that interacts with a target RNA and its interaction sequence, searching for an interaction protein complex for the target RNA and identifying the interaction sequence. The RNA-protein complex interaction analysis method according to claim 1, characterized by including this. **Claim 7** Using the RNA-protein complex interaction analysis method according to any one of claims 1 to 6, adding a small molecule compound library and conducting experiments to screen for small molecule compounds that inhibit RNA-protein interaction. A screening method for an RNA-protein complex interaction inhibitor, characterized by this. **Claim 8** Select an RNA virus sequence as the target RNA sequence, perform the RNA-protein complex interaction analysis method according to any one of claims 1 to 6 to identify the host interaction protein factors and interaction RNA sequences of the RNA virus at a resolution of several hundred bases, and add a small molecule compound library and conduct experiments to screen for antiviral prophylactic or therapeutic drugs that inhibit RNA-protein interaction. A screening method for an antiviral drug against an RNA virus, characterized by this. **Claim 9** Performing the RNA-protein complex interaction analysis method according to any one of claims 1 to 6 on an RNA sequence having a disease-specific mutation of a disease including cancer, identifying mutation-specific interaction protein factors and interaction RNA sequences at a resolution of several hundred bases, and adding a small molecule compound library and conducting experiments to screen for disease-specific therapeutic drugs that inhibit RNA-protein interaction. A screening method for a prophylactic or therapeutic drug targeting a disease-specific mutant RNA sequence.

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