N obtained by artificial evolution 6 -methyladenine modification-specific binding protein

Abstract

The present invention provides N obtained by artificial evolution 6 -methyladenine modification specific binding protein (e6mABP1, e6mABP2 and e6mABP3), having the amino acid sequence shown in SEQ ID NO.1, 2 or 3. The protein reduces the 6 ‑methyladenine binds to DNA and RNA, enhancing the binding of RNA N 6 ‑Methyladenine and DNA N 6 ‑methyladenine recognition and binding specificity. This protein can be used to enrich RNA / DNA N 6 ‑methylated adenine, develop affinity assays and RNA / DNA N 6 -methylated adenine sequencing technology, and the use of this specific binding to develop intracellular epigenetic intervention technology to achieve the improvement and treatment of epigenetic-related diseases such as tumors. The present invention further provides a method for preparing the protein and its application.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA / RNA detection containing methylated adenine, and in particular to the artificially evolved N 6 -Methyladenine-modified specific binding proteins (e6mABP1, e6mABP2 and e6mABP3), preparation methods and applications. Background Art

[0002] N 6 -methyladenine (6mA / m 6 A) is a functional epigenetic base modification found in DNA and RNA. Currently commercially available 6mA antibodies can non-specifically bind to short repeat sequences without modification. These non-specifically bound short repeat sequences account for 50%-99% of the data obtained by DNA immunoprecipitation enrichment and 6mA sequencing, resulting in widespread false positive peaks. This makes it difficult to accurately determine 6mA sites in DNA. Similarly, there is also the problem of how to identify mRNAs. 6 The difficulty in accurately measuring the A site. Summary of the Invention

[0003] In view of this, the main purpose of the present invention is to provide an artificially evolved N 6 -Methyladenine-modified specific binding proteins (e6mABP1, e6mABP2 and e6mABP3), preparation methods and applications thereof, in order to at least partially solve at least one of the above-mentioned technical problems.

[0004] To achieve the above object, the technical solution of the present invention is as follows:

[0005] As one aspect of the present invention, there is provided N 6 -Methyladenine modification specific binding protein, obtained through artificial evolution, having an amino acid sequence as shown in SEQ ID NO. 1, 2 or 3.

[0006] According to an embodiment of the present invention, the above protein reduces the 6 -The binding of methyladenine to DNA or RNA enhances the specificity of recognition and binding to methyladenine in DNA or RNA.

[0007] As another aspect of the present invention, a recombinant plasmid is provided, comprising a recombinant plasmid encoding the N 6 -Methyladenine modifications specifically bind to nucleic acids of proteins.

[0008] According to an embodiment of the present invention, the method for preparing the recombinant plasmid includes cloning a gene fragment encoding the protein having a sequence as shown in SEQ ID NO. 4, 5, or 6 into a linearized pMAL-C5X vector containing an MBP tag and a flag tag. However, the method for preparing the recombinant plasmid is not limited thereto.

[0009] According to an embodiment of the present invention, the recombinant plasmid is suitable for transformation into an expression strain, which includes but is not limited to Escherichia coli, etc., to express the target protein.

[0010] As another aspect of the present invention, a recombinant Escherichia coli is provided, which is prepared by introducing the recombinant plasmid described above into a host Escherichia coli, wherein the Dam gene of the host Escherichia coli is knocked out.

[0011] According to an embodiment of the present invention, the host Escherichia coli with the Dam gene knocked out has a low abundance 6mA background, so that the nucleic acid background content of Escherichia coli in the purified protein is low, which can meet the requirements of experiments such as specificity verification or practical applications.

[0012] As another aspect of the present invention, there is provided N as described above 6 The method for preparing a methyladenine-modified specific binding protein comprises the following steps: inoculating and culturing the recombinant Escherichia coli as described above, and inducing its expression; purifying the expression product of the recombinant Escherichia coli by prokaryotic means to obtain N 6 -Methyladenine modification-specific binding protein.

[0013] As another aspect of the present invention, there is provided N as described above 6 -Methyladenine modification specific binding protein is used in the preparation of 6 - Use of a reagent or kit for RNA or DNA enrichment analysis of methylated adenine.

[0014] As another aspect of the present invention, there is provided N as described above 6 -Methyladenine modification-specific binding protein in the preparation of N-methyladenine for RNA or DNA 6 -Application of reagents or kits for methylated adenine sequencing.

[0015] As one aspect of the present invention, there is provided N as described above 6 -Application of a methyladenine modification-specific binding protein in the preparation of drugs for intracellular epigenetic intervention to treat epigenetic diseases.

[0016] Based on the above technical solution, the artificial evolution of the present invention obtains N 6-Methyladenine-modified specific binding protein, preparation method and application thereof have at least one or part of the following beneficial effects:

[0017] The protein produced according to the amino acid sequence of the present invention has high stability and can selectively and specifically bind to N 6 -methyladenine modification, thereby 6 A's DNA / RNA specifically binds to help achieve 6mA / m 6 A enrichment analysis or site sequencing positioning, as well as the development of intracellular epigenetic intervention technology, can achieve the improvement and treatment of epigenetic-related diseases such as tumors.

[0018] Since the protein is a biosynthetic protein that does not rely on antibodies and can be purified using the E. coli prokaryotic system, the protein product of the present invention has a low economic cost and a short production time, and is an artificially evolved N protein with low price, low cost, safety, non-toxicity, strong stability and good specificity. 6 -Methyladenine modification-specific binding protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a chromatogram of the protein HIC hydrophobic purification of the present invention, wherein A is the MBP-Flag control group protein, B is the MBP-Flag-reader control group protein, C is the MBP-Flag-e6mABP1 experimental group protein, D is the MBP-Flag-e6mABP2 experimental group protein, and E is the MBP-Flag-e6mABP3 experimental group protein;

[0020] Figure 2 This is a chromatogram of the protein MBP affinity purification of the present invention, wherein A is the MBP-Flag control group protein, B is the MBP-Flag-reader control group protein, C is the MBP-Flag-e6mABP1 experimental group protein, D is the MBP-Flag-e6mABP2 experimental group protein, and E is the MBP-Flag-e6mABP3 experimental group protein;

[0021] Figure 3 It is the protein quantitative gel map of the present invention;

[0022] Figure 4 This is a graph showing protein activity verification using the 6mA-containing DNA probe of the present invention, wherein A represents the MBP-Flag-reader control group protein, B represents the MBP-Flag-e6mABP1 experimental group protein, C represents the MBP-Flag-e6mABP2 experimental group protein, and D represents the MBP-Flag-e6mABP3 experimental group protein;

[0023] Figure 5 This is a graph showing protein activity verification using the 5mC-containing DNA probe of the present invention, wherein A represents the MBP-Flag-reader control group protein, B represents the MBP-Flag-e6mABP1 experimental group protein, C represents the MBP-Flag-e6mABP2 experimental group protein, and D represents the MBP-Flag-e6mABP3 experimental group protein;

[0024] Figure 6 This is a graph showing protein activity verification using the 5hmC-containing DNA probe of the present invention, wherein A represents the MBP-Flag-reader control group protein, B represents the MBP-Flag-e6mABP1 experimental group protein, C represents the MBP-Flag-e6mABP2 experimental group protein, and D represents the MBP-Flag-e6mABP3 experimental group protein;

[0025] Figure 7 This is a graph showing protein activity verification using the 5fC-containing DNA probe of the present invention, wherein A represents the MBP-Flag-reader control group protein, B represents the MBP-Flag-e6mABP1 experimental group protein, C represents the MBP-Flag-e6mABP2 experimental group protein, and D represents the MBP-Flag-e6mABP3 experimental group protein;

[0026] Figure 8 This is a graph showing protein activity verification using the 5caC-containing DNA probe of the present invention, wherein A represents the MBP-Flag-reader control group protein, B represents the MBP-Flag-e6mABP1 experimental group protein, C represents the MBP-Flag-e6mABP2 experimental group protein, and D represents the MBP-Flag-e6mABP3 experimental group protein;

[0027] Figure 9 The present invention is used m 6 A-containing RNA probes verify protein activity, where A is the MBP-Flag-reader control group protein, B is the MBP-Flag-e6mABP1 experimental group protein, C is the MBP-Flag-e6mABP2 experimental group protein, and D is the MBP-Flag-e6mABP3 experimental group protein. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments and the accompanying drawings. It should be noted that, unless otherwise defined, the technical terms used in the following examples have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. The experimental reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods described, unless otherwise specified, are all conventional methods.

[0029] In the process of realizing the present invention, it was found that by selecting RNA m 6 A reader YTHDF2 C-terminal YTH domain (YTHDF2C) protein was used as the initial template and error-prone PCR was used to create approximately 3×10 8 RNAm 6 A reader random mutation library, combined with yeast surface display technology and high-throughput flow screening, finally obtained artificial directed evolution of N 6 -Methyladenine super binding proteins (e6mABP1-3) can recognize 6mA / m in DNA and RNA with high specificity and affinity. 6 A base modification. On this basis, the artificial evolution of N 6 -Methyladenine modified specific binding protein, preparation method and application are beneficial for the detection of 6mA / m 6 A base modified DNA / RNA can selectively and specifically bind to it, which is beneficial to achieve the 6mA / m 6 A enrichment analysis or positioning sequencing will help develop intracellular epigenetic intervention technologies and achieve the improvement and treatment of epigenetic-related diseases such as tumors.

[0030] Example 1 Preparation of protein

[0031] e6mABP1, e6mABP2, and e6mABP3 (encoding gene fragments as set forth in SEQ ID NOs. 4, 5, or 6) were cloned into the pMAL-C5X-MBP-flag vector using homologous recombinases, generating recombinant plasmids (pMAL-C5X-MBP-Flag-e6mABP1, pMAL-C5X-MBP-Flag-e6mABP2, and pMAL-C5X-MBP-Flag-e6mABP3) with sequences as set forth in SEQ ID NOs. 7, 8, or 9. The resulting recombinant plasmids were transformed into expression strains, forming the MBP-Flag-e6mABP1, MBP-Flag-e6mABP2, and MBP-Flag-e6mABP3 experimental groups, respectively. Among them, the expression strain used was LAMBS (low adenine methylation background E. coli strain) with a low abundance 6mA background. For specific acquisition methods, please refer to Chen Z, Liu Y, Wang H. Conjoint expression and purification strategy for acquiring proteins with ultra-low DNA N-6-methyladenine backgrounds in Escherichia coli [J]. Biosci Rep, 2021, 41(3).

[0032] Similar to the experimental group, the difference is that the uncloned target protein sequence and the recombinant vector of the cloned reader protein were transformed into the expression strain as the MBP-Flag control group and the MBP-Flag-reader control group.

[0033] The proteins expressed by the recombinant strains in the experimental and control groups were purified by prokaryotic purification and passed through HIC hydrophobic column and MBP affinity column. The chromatograms are shown in Figure 2. Figure 1-2 As shown in the figure, it can be seen that the combination of HIC hydrophobic purification and MBP affinity purification can effectively purify the target protein. The background content of E. coli nucleic acid in the protein obtained by prokaryotic purification is low, which can meet the experimental requirements such as specificity verification and the requirements for practical applications. The purified protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the analysis results are shown in the figure. Figure 3 As shown, it can be seen that the target protein with higher purity can be obtained after purification.

[0034] The purified protein was stored in a buffer (pH 7.5, 50 mM K3PO4-K2HPO4, 150 mM potassium glutamate, 5 mM MgCl2, 2 mM ATP, 0.1 mM DTT, 0.1% PEG8000, 50% glycerol), which helps improve protein solubility and activity.

[0035] Example 2: Using gel shift analysis to visually characterize and identify proteins specifically binding to 6mA-containing DNA

[0036] (1) Design of DNA probe

[0037] To verify the affinity and selectivity of the protein binding to 6mA-containing DNA probes and probes containing other DNA modifications 5mC, 5hmC, 5fC, and 5caC, the following DNA probes were designed, each 30 nt in length and labeled with Cy5 fluorescently at the 5' end.

[0038] Table 1 Probe sequences

[0039]

[0040] (2) Interaction between protein and DNA probe

[0041] The DNA probe (10 nM) was incubated with the three experimental e6mABP proteins (0 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM) from Example 1, the MBP-Flag-reader control group (0 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM), and the MBP-Flag control group protein (400 nM) in a buffer (20 mM HEPES, 250 mM NaCl, 10% glycerol, pH 7.4) at 4°C for 30 minutes. After the reaction, the samples were separated by 12% native PAGE in 0.5× Tris-borate-EDTA (TBE) (45 mM Tris, 45 mM Boric acid, 1 mM EDTA) at 200 V for 30 minutes in the absence of light. The results are shown in Figure 2. Figures 4 to 8 As shown in the figure, the experimental group binds to the 6mA-modified DNA band at 50-400 nM, while the DNA-modified bands of the other probes are less prominent. In particular, at 100-200 nM, e6mABP1 to 3 show better specificity than the 6mA reader protein.

[0042] Example 3 Using gel shift analysis to visually characterize and identify protein pairs 6Specific binding to A-containing RNA

[0043] (1) Design of RNA probes

[0044] To verify the protein and m 6 To improve the affinity and selectivity of A-containing RNA probe binding, the following probes were designed, each 30 nt in length and labeled with Cy5 fluorescent marker at the 5' end.

[0045] Probe sequence

[0046]

[0047] (2) Interaction between protein and RNA probe

[0048] The RNA probe (10 nM) was incubated with the three experimental groups of e6mABP protein (0 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM) from Example 1, the MBP-Flag-reader control group (0 nM, 10 nM, 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM), and the MBP-Flag control group protein (400 nM) in a buffer (20 mM HEPES, 250 mM NaCl, 10% glycerol, pH 7.4) at 4°C for 30 minutes. After the reaction, the samples were separated by 12% native PAGE in 0.5× Tris-borate-EDTA (TBE) (45 mM Tris, 45 mM Boric acid, 1 mM EDTA) solution at 200 V for 30 minutes in the absence of light. The results are shown in Figure 2. Figure 9 As shown in the figure, it can be seen that the experimental group has a band bound to 6mA modified DNA at 50-400 nM, especially at 100 nM, e6mABP3 showed a higher 6 A better specificity for reading proteins.

[0049] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A N 6 -Methyladenine modification specific binding protein, obtained through artificial evolution, the sequence of the protein is shown in SEQ ID NO.

2.

2. A recombinant plasmid comprising a recombinant plasmid encoding the N 6 -Methyladenine modifications specifically bind to nucleic acids of proteins.

3. A recombinant Escherichia coli prepared by introducing the recombinant plasmid according to claim 2 into a host Escherichia coli, wherein the Dam gene of the host Escherichia coli is knocked out.

4. A N as claimed in claim 1 6 - A method for preparing a methyladenine-modified specific binding protein, comprising the following steps: Inoculating and culturing the recombinant Escherichia coli according to claim 3 and inducing its expression; The expression product of the recombinant Escherichia coli was purified by prokaryotic purification to obtain the N 6 -Methyladenine modification-specific binding protein.

5. An N as claimed in claim 1 6 -Methyladenine modification specific binding protein is used in the preparation of 6 - Use of a reagent or kit for RNA or DNA enrichment analysis of methylated adenine.

6. An N as claimed in claim 1 6 -Methyladenine modification-specific binding protein in the preparation of N-methyladenine for RNA or DNA 6 -Application of reagents or kits for methylated adenine sequencing.

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