Protein-DNA (Deoxyribose Nucleic Acid) interaction detection method aiming at whole genome open chromatin and independent of antibody

Abstract

The invention discloses a protein-DNA interaction detection method aiming at whole genome open chromatin and independent of an antibody. The method comprises the steps of cell nucleus preparation, transposase reaction, ampDAP library construction, protein incubation and DNA library construction. The cell nucleus preparation comprises cell activity detection and cell nucleus extraction; the transposase reaction comprises transposon and DNA (Deoxyribose Nucleic Acid) recovery; the construction of the ampDAP library comprises the steps of PCR amplification and product purification; the protein incubation comprises protein in-vitro expression and protein-library incubation; dNA library building comprises PCR (polymerase chain reaction) amplification and product purification. According to the present invention, the protein-DNA interaction detection is achieved, the experiment target can be achieved by only requiring the in-vitro expression of the interested protein during the process, and the one library can simultaneously meet the detection of a plurality of proteins, such that the new idea and the new method are provided for the protein-DNA interaction detection.

Description

Technical Field

[0001] The present invention relates to the field of database security, and particularly to a method for detecting protein-DNA interaction for the whole-genome open chromatin without relying on antibodies. Background Art

[0002] In many cellular life activities, such as DNA replication, mRNA transcription and modification, and virus infection, protein-DNA interaction is involved. Its types mainly include histones, transcription factors, DNA methyltransferases, and chromatin remodeling complexes, etc. Protein-DNA interaction is the key to gene transcriptional regulation and the prerequisite for initiating gene transcription. Precise analysis of it can reveal the mutual recognition mechanism and dynamic changes between the two, which is crucial for deeply understanding the gene regulation mechanism under physiological and pathological conditions.

[0003] Among the existing methods for studying protein-DNA interaction, CHIP-seq is a classic method for studying protein-DNA interaction. It can truly and completely reflect the target proteins bound to the DNA sequence. However, in this technology, some non-related proteins cross-link to form false positives during the formaldehyde fixation process, and some transcription factors with weak binding forces form false negatives during sonication due to insufficient formaldehyde cross-linking. Therefore, a large number of cells are required, and the steps are cumbersome and time-consuming. Subsequently, technologies such as CUT&RUN proposed in 2017 and CUT&Tag in 2019 further optimized the research methods for protein-DNA interaction. The CUT&RUN technology uses MNase to replace sonication in CHIP-seq, reducing the cell usage. CUT&Tag is further improved on the basis of CUT&RUN, using the adapter-ligated Tn5 transposase to replace MNase, simplifying the experimental operation while reducing the cell usage. Although compared with CHIP-seq, the new technologies solve the problems of large cell quantity and cumbersome steps, these technologies all have drawbacks. Their experimental processes are restricted by antibodies, with high requirements for antibodies. Only the binding information of one antibody can be detected at a time, the detection efficiency is low, the price is expensive, and the availability of suitable antibodies becomes the key to the success of the detection. Therefore, seeking new detection technologies or improving detection methods has become the key to breaking the limitations. Therefore, a method for detecting protein-DNA interaction for the whole-genome open chromatin without relying on antibodies is proposed. Summary of the Invention

[0004] The present invention provides a method for detecting protein-DNA interaction for the whole genome open chromatin without relying on antibodies, overcoming the problems of large cell volume, cumbersome steps, long time consumption, and most importantly, being restricted by antibodies in existing protein-DNA detection methods. By referring to the method of ATAC-seq, the present invention constructs an ampDAP library to obtain information about accessible chromatin in the whole genome, then uses an in vitro protein expression system to express the protein of interest, incubates the successfully expressed protein with the ampDAP library, and combines sequencing and other necessary technical means to achieve the detection of protein-DNA interaction. In this process, only the protein of interest needs to be expressed in vitro to achieve the experimental goal, and one library can simultaneously meet the detection of multiple proteins, providing new ideas and methods for the detection of protein-DNA interaction.

[0005] The present invention solves the above technical problems through the following technical solutions. The present invention includes nuclear preparation, transposase reaction, ampDAP library construction, protein incubation, and DNA library construction; the nuclear preparation includes cell viability detection and nuclear extraction; the transposase reaction includes transposition and DNA recovery; the ampDAP library construction includes PCR amplification and product purification; the protein incubation includes in vitro protein expression and protein-library incubation; the DNA library construction includes PCR amplification and product purification.

[0006] Further, in the nuclear preparation, for cell viability detection, cells are mixed with trypan blue and the cell viability is detected under a microscope; in nuclear extraction, an appropriate amount of cells is taken for nuclear extraction according to the results of viability detection for the next transposition reaction step.

[0007] Further, in the transposase reaction, for transposition, nuclease-free water, 5×TTBL, and TTE Mix are pipetted and mixed evenly to prepare a Tn5 transposase reaction solution, which is mixed with the nuclei extracted in the previous step and the transposition reaction is carried out in a water bath or a metal bath; in DNA recovery, after the transposition reaction is completed, the DNA is purified and recovered by column, and the amount of DNA is quality controlled.

[0008] Further, in the ampDAP library construction, for PCR amplification, an appropriate amount of DNA recovered after transposition, 5×TAB, TAE, and primer adapter are pipetted and mixed evenly, and the reaction solution is centrifuged to collect it at the bottom of the centrifuge tube, and the experiment is carried out in a PCR instrument; in product purification, the PCR amplification product is purified with 1×DNA Clean Beads and eluted with water, and the obtained product is used for the next protein-library incubation step.

[0009] Further, in the protein incubation and in vitro protein expression, the constructed protein expression vector with a streptavidin-binding peptide tag is mixed well with WEPRO TTmix, placed under the SUB-AMIX TT to form a bilayer for in vitro expression of the target protein; in the protein-library incubation, the in vitro expressed protein and the purified product from the previous step are co-incubated using streptavidin magnetic beads, and the obtained product is used for the next DNA library construction.

[0010] Further, in the DNA library construction, in the PCR amplification, an appropriate amount of the protein-library incubation product, primer adapter ligation buffer, DNA ligase, and the same primer adapter as in the previous round of amplification are pipetted and mixed well, and centrifuged to collect the reaction solution at the bottom of the centrifuge tube, and the experiment is carried out in a PCR instrument; in the product purification, the PCR amplification product is purified using DNA Clean Beads and eluted with water, and the obtained product is finally used for high-throughput sequencing.

[0011] The present invention has the following advantages compared with the prior art: This method for detecting protein-DNA interaction targeting open chromatin of the whole genome and not relying on antibodies, under the integration of the two technologies of ATAC-seq and DAP-seq, by obtaining information on chromatin open regions, uses an in vitro protein expression system to express the protein under study, replacing the antibody, co-incubates with the ampDAP library, and combines sequencing and other necessary technical means to find the DNA that interacts with the target protein; using less cells, simple process, good repeatability, and not requiring antibodies are the greatest advantages of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the overall flowchart of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0013] The following details the embodiments of the present invention. These embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0014] As Figure 1 shown, this embodiment provides a technical solution: A method for detecting protein-DNA interaction targeting open chromatin of the whole genome and not relying on antibodies, including five steps: nucleus preparation, transposase reaction, ampDAP library construction, protein incubation, and DNA library construction. The specific steps are as follows.

[0015] Figure 1 As shown, the nucleus preparation includes the following sub-steps: (1) Detect the cell viability of the cell sample. Take 105 cells, centrifuge at 500 g for 5 min, and remove the supernatant.

[0016] (2) Add 200 μl of PBS to step (1), centrifuge at 500 g for 5 min, and remove the supernatant.

[0017] (3) Add 200 μl of cell lysate to step (2), pipette to resuspend the cells, and place on ice for 10 min.

[0018] (4) Centrifuge at 500 g for 5 min and remove the supernatant.

[0019] Figure 1 As shown, the transposase reaction includes the following sub-steps: (1) The nuclei prepared in the nucleus preparation step are mixed well by pipetting with the transposase reaction solution prepared with RNase-free H2O, 5×TTBL, and TTE Mix V50, and incubated at 37 °C for 45 min.

[0020] (2) Column purification step: Add 26 μl of RNase-free H2O to the product of step (1) to recover the DNA.

[0021] (3) Measure the DNA concentration by Qubit.

[0022] Figure 1 As shown, the construction of the ampDAP library includes the following sub-steps: (1) The product of the transposase reaction step, 5×TAB, Primer, TAE, and RNase-free H2O are mixed well and then subjected to experiments in a PCR instrument under the conditions of 72 °C for 3 min, 98 °C for 30 s, 98 °C for 15 s, 60 °C for 30 s, 72 °C for 1 min, 72 °C for 5 min, and 4 °C hold (maintained at 4 °C). Among them, 8 - 12 cycles are carried out under the conditions of 98 °C for 15 s, 60 °C for 30 s, and 72 °C for 1 min, thereby obtaining the ampDAP library.

[0023] (2) The product of step (1) is purified with 1×DNA Clean beads and eluted with 32 μl of RNase-free H2O, and the obtained product is used for the protein incubation step.

[0024] Figure 1 As shown, the protein incubation includes the following sub-steps: (1) Mix 2.5 μl of the protein expression vector and 2.5 μl of WEPRO TTmix well (the mixture), set up a bilayer with 50 μl of SUB-AMIX TT, keep the mixture to form the lower layer, and incubate at 26 °C for 2 - 24 h.

[0025] (2) Take 100 μl of streptavidin magnetic beads and remove the supernatant.

[0026] (3) Resuspend the magnetic beads with 100 ul of PBST buffer and wash twice.

[0027] (4) After the last wash, resuspend the magnetic beads with 50 ul of PBST buffer, add 50 ul of the incubation product from step (1), and mix well (if less than 50 ul, make up with RNase-free H2O). Incubate with rotation at room temperature for 15 - 30 min.

[0028] (5) Remove the supernatant, resuspend the magnetic beads with 80 ul of PBST buffer to wash the protein, and repeat the wash twice.

[0029] (6) Wash the magnetic beads with 40 ul of PBST buffer, add 30 - 100 ng of the ampDAP library, and make up the volume to 80 ul.

[0030] (7) Incubate with rotation at room temperature for 1 h.

[0031] (8) After incubation, remove the supernatant, resuspend the magnetic beads with 85 ul of PBST buffer, and repeat the wash four times. Transfer the magnetic beads and buffer to a new tube during the last wash.

[0032] (9) Use MPC to remove the supernatant, resuspend the magnetic beads with 22 ul of RNase-free H2O, treat at 98 °C for 10 min, incubate on ice for 5 min, and take the supernatant for the DNA library construction part.

[0033] Figure 1 As shown, the DNA library construction includes the following sub-steps: (1) For 20 ul of the product from the previous step, 5 ul of Primer mix, and 25 ul of VAHTS HiFi Amplification Mix, perform experiments in a PCR instrument under the conditions of 95 °C for 3 min, 98 °C for 20 s, 60 °C for 15 s, 72 °C for 30 s, 72 °C for 5 min, and 4 °C hold. Among them, perform 3 - 22 cycles under the conditions of 98 °C for 20 s, 60 °C for 15 s, and 72 °C for 30 s to obtain the library for sequencing.

[0034] (2) Purify and sort the product from step (1) with 0.5× / 0.3× DNA Clean beads, and elute the DNA with 32 ul of RNase-free H2O for high-throughput sequencing.

[0035] Figure 1As shown, through the combined actions of nuclear preparation, transposase reaction, ampDAP library construction, protein incubation, and DNA library construction in the present invention, open chromatin information is obtained through the Tn5 transposition reaction. The obtained library is co-incubated with the protein expressed in vitro, and combined with sequencing and other necessary technical means, the goal of exploring protein-DNA interactions in open chromatin regions is achieved, breaking through the limitations of antibodies and providing strong technical support for the detection of protein-DNA interactions. Existing methods for detecting protein-DNA interactions, such as CHIP-seq, require a large amount of cells, have high requirements for antibodies, are cumbersome to operate, and have a long experimental period, and are for genome-wide interaction detection; in addition, CUT&RUN and CUT&Tag have requirements for antibodies similar to CHIP, and CUT&Tag is not recommended for chromatin-related protein analysis. The present invention combines DAP-seq on the basis of ATAC to simultaneously analyze and screen specific regulatory factors of interest, which is simpler in operation, higher in accuracy, better in repeatability, and does not require antibodies compared with the prior art, overcoming the disadvantages such as detection obstruction due to antibody limitations in existing detection technologies.

[0036] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0037] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0038] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill 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 detecting protein-DNA interactions in whole genome open chromatin and independent of antibodies, characterized in that: The following steps are involved: Step 1: Cell nucleus preparation; Step 2: Transposase reaction; Step 3: ampDAP library construction; Step 4: protein incubation; Step 5: DNA library construction.

2. A method for detecting protein-DNA interactions targeting whole genome open chromatin and independent of antibodies according to claim 1, characterized in that: The cell nucleus preparation includes cell activity detection and cell nucleus extraction; The transposase reaction includes transposition and DNA recovery; ampDAP library construction includes PCR amplification and product purification; Protein incubation includes protein in vitro expression and protein-library incubation; DNA library construction includes PCR amplification and product purification.

3. The method for detecting protein-DNA interactions targeting whole genome open chromatin and not relying on antibodies according to claim 2, characterized in that: In the cell activity detection, cells are mixed with trypan blue and the cell activity is detected under a microscope; In the cell nucleus extraction, an appropriate amount of cells are taken for cell nucleus extraction according to the activity detection results for the next transposition reaction step.

4. The method for detecting protein-DNA interactions targeting whole genome open chromatin and not relying on antibodies according to claim 2, characterized in that: During the transposase reaction, nuclease-free water, 5×TTBL, and TTEMix were mixed by pipetting to prepare Tn5 transposase reaction solution, which was then mixed with the cell nuclei extracted in the previous step and subjected to transposition reaction in a water bath or metal bath; In DNA recovery, after the transposition reaction is completed, the DNA is purified and recovered by column, and the amount of DNA is quality controlled.

5. The method for detecting protein-DNA interactions targeting whole genome open chromatin and not relying on antibodies according to claim 2, characterized in that: In the PCR amplification, an appropriate amount of DNA recovered after transposition, 5×TAB, TAE, and primer adapters are pipetted and mixed evenly, and the reaction solution is collected to the bottom of the centrifuge tube by centrifugation, and the experiment is carried out in a PCR instrument; During product purification, the PCR amplification product was purified with 1× DNACleanBeads, eluted with water, and the obtained product was used in the next protein-library incubation step.

6. The method for detecting protein-DNA interactions targeting whole genome open chromatin and not relying on antibodies according to claim 2, characterized in that: In the in vitro expression of the protein, the constructed protein expression vector with a streptavidin-binding peptide tag is mixed with WEPROTTmix and placed in the lower layer of SUB-AMIXTT to form a bilayer for in vitro expression of the target protein; In the protein-library incubation, the in vitro expressed protein is incubated with the purified product in the previous step using streptavidin magnetic beads, and the obtained product is used for the next step of DNA library construction.

7. The method for detecting protein-DNA interactions targeting whole genome open chromatin and not relying on antibodies according to claim 2, characterized in that: In the PCR amplification, an appropriate amount of protein-library incubation product, primer-adapter connection buffer, DNA ligase, and the same primer-adapter as the previous round of amplification are pipetted and mixed, and the reaction solution is collected to the bottom of the centrifuge tube by centrifugation, and the experiment is carried out in a PCR instrument; During product purification, the PCR amplification product was purified using DNACleanBeads and eluted with water. The resulting product was ultimately used for high-throughput sequencing.

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