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WO2021155712A1 - Application d'une substance permettant de détecter la mutation mecp2 pour détecter si la mutation mecp2 est une mutation pathogène et sélectionner un médicament - Google Patents

Application d'une substance permettant de détecter la mutation mecp2 pour détecter si la mutation mecp2 est une mutation pathogène et sélectionner un médicament Download PDF

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WO2021155712A1
WO2021155712A1 PCT/CN2020/135514 CN2020135514W WO2021155712A1 WO 2021155712 A1 WO2021155712 A1 WO 2021155712A1 CN 2020135514 W CN2020135514 W CN 2020135514W WO 2021155712 A1 WO2021155712 A1 WO 2021155712A1
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mecp2
mutation
dna
dna fragment
nucleosome
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Chinese (zh)
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李丕龙
李国红
王亮
胡明丽
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Tsinghua University
Institute of Biophysics of CAS
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Tsinghua University
Institute of Biophysics of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to the application of a substance for detecting a MeCP2 mutation in the field of biotechnology in detecting whether the MeCP2 mutation is a pathogenic mutation and screening drugs.
  • Phase transition as a property of matter has long been well known in the physical world and daily life. In recent years, scientists have gradually discovered that the mechanism of phase transition (or phase separation) is also widely present in biological cells, and in the time and space of the cell life cycle. Regulation and other aspects exercise important biological functions.
  • phase change liquid-liquid phase change
  • the multivalent valence refers to the number of binding regions contained in a macromolecule or its ligand that can interact with each other.
  • Methyl CpG binding protein 2 can bind methylated and unmethylated DNA, and has a stronger affinity for methylated DNA.
  • MeCP2 is an important transcriptional regulatory factor. Abnormal expression or mutations (including point mutations and duplication or deletion mutations) will cause changes in the expression levels of genes regulated by MeCP2, causing abnormal development of neurons, axons, and dendrites. Causes serious neurological diseases.
  • the technical problem to be solved by the present invention is how to detect whether the MeCP2 mutation is a pathogenic mutation, and how to screen drugs for treating and/or preventing diseases caused by the MeCP2 mutation.
  • the present invention first provides any of the following applications:
  • MeCP2 mutations are a MeCP2 pathogenic mutation
  • the MeCP2 mutation is a MeCP2 pathogenic mutation
  • the substance for detecting the MeCP2 mutation may include the MeCP2 mutant protein in which the MeCP2 mutation occurs and the following a1) or a2) or a3) or a4):
  • the nucleosome bead is a structure with a diameter of 11 nm formed by a DNA winding histone octamer.
  • the DNA fragment in a1) can be a11) or a12) or a13):
  • the nucleosome localization DNA of a11 may specifically be a "601" sequence.
  • the “601” sequence may be the 31st-177th position of the sequence 21 or the 24-170th position of the sequence 22.
  • the DNA fragment may be positions 16-192 of sequence 21, or positions 9-185 or 186-362 or positions 1956-2132 of sequence 22.
  • n is any natural number greater than or equal to 1. Specifically, n can be 4-12.
  • the DNA fragment may specifically be the DNA fragment shown in sequence 21 or sequence 22 in the sequence listing.
  • the DNA fragments can be methylated or unmethylated DNA fragments.
  • the nucleosome bead is formed by a DNA fragment shown in sequence 21 or sequence 22 and a histone octamer.
  • the substance mentioned in a3) can be a31) or a32):
  • the substance for detecting MeCP2 mutation may also include MeCP2 unmutated protein.
  • the substance for detecting the mutation of MeCP2 may further include a reporter group, and the reporter group is used to label MeCP2 or its mutant protein.
  • the reporter group can be a dye (such as Alexa Fluor TM 568 NHS Ester (Succinimidyl Ester)) or a fluorescent protein (such as mCherry).
  • a dye such as Alexa Fluor TM 568 NHS Ester (Succinimidyl Ester)
  • a fluorescent protein such as mCherry
  • the fluorescent protein can be connected to the MeCP2 mutein or MeCP2 unmutated protein directly or through a connecting peptide.
  • the substance for detecting the MeCP2 mutation may be composed of the MeCP2 mutein and the above a1), may also be composed of the MeCP2 mutein and the above a2), may also be composed of the MeCP2 mutein and the above a3), or may be composed of the MeCP2 mutein and the above a3).
  • the mutein is composed of the above a4), or may be composed of the MeCP2 mutein, the above a1) and the MeCP2 unmutated protein, or may be composed of the MeCP2 mutein, the above a2) and the MeCP2 unmutated protein, or may be composed of the MeCP2 mutant protein.
  • the group composition can also be composed of the MeCP2 mutein, the above a2), the MeCP2 unmutated protein and the reporter group, or the MeCP2 mutein, the above a3), the MeCP2 unmutated protein, and the reporter group.
  • the composition can also be composed of the MeCP2 mutein, the above a4), the MeCP2 unmutated protein and the reporter group.
  • the method for detecting whether the MeCP2 mutation is a pathogenic mutation using the substance for detecting the MeCP2 mutation includes: mixing the MeCP2 mutant protein with the MeCP2 mutation and the nucleosome beads to obtain the system to be tested; mixing; The MeCP2 unmutated protein is beaded with the nucleosome to obtain a control system; compare the phase transition of the test system and the control system to determine whether the MeCP2 mutation is a pathogenic mutation: as the phase change of the test system The change ability is less than the phase change ability of the control system, and the MeCP2 mutation is or candidate is a pathogenic mutation; if the phase change ability of the test system is not less than the phase change ability of the control system, the MeCP2 mutation is Or the candidate is a non-pathogenic mutation.
  • the method for detecting whether the MeCP2 mutation is pathogenic using the substance for detecting the MeCP2 mutation includes: mixing the MeCP2 mutant protein with the MeCP2 mutation and the nucleosome beads to obtain the system to be tested; mixing the MeCP2 unmutated protein with The nucleosomes are beaded to obtain a control system; compare the phase change of the test system and the control system to determine whether the MeCP2 mutation is pathogenic: if the phase change ability of the test system is less than that of the control system If the phase change ability of the test system is not less than the phase change ability of the control system, the MeCP2 mutation does not cause disease or the candidate does not cause disease.
  • test system and the control system can both be an intracellular environment or a reaction buffer environment.
  • the reaction buffer can be composed of a solute and a solvent.
  • the solvent is water, and the solute and its content in the reaction buffer The concentrations in are 20mM HEPES and 100mM NaCl, pH 7.4.
  • phase change ability may be reflected in whether a phase change occurs, the proportion of the phase change phase in the field of view in the field of view, the clarity of the phase separation boundary, and/or the signal strength of the reporter group in the phase change phase.
  • the MeCP2 mutation may be a truncation mutation or a point mutation of MeCP2.
  • the truncation mutation of MeCP2 is an early termination at position 168, 255, 270 or 294 of MeCP2.
  • the point mutation of MeCP2 may be a mutation at positions 106, 111, 120, 133, 152, 157, 158, 225 or/and 306 of MeCP2.
  • the point mutation of MeCP2 may be R106W mutation, R111G mutation, Y120D mutation, R133C mutation, P152R mutation, F157I mutation, T158M mutation, P225R mutation or/and R306C mutation of MeCP2.
  • the disease caused by the MeCP2 mutation may be a dysplasia of the nervous system.
  • the disease caused by the MeCP2 mutation may be a dysplasia of the nervous system caused by a mutation of MeCP2, or a dysplasia of the nervous system containing a MeCP2 mutation.
  • the disease caused by the MeCP2 mutation may be Angel syndrome.
  • the present invention also provides a method for detecting or assisting in detecting whether the MeCP2 mutation is a pathogenic mutation, and the method is completed by using the substance for detecting the MeCP2 mutation.
  • the present invention also provides a method for screening or assisting in screening drugs for the treatment and/or prevention of diseases caused by the MeCP2 mutation.
  • the MeCP2 mutation is a MeCP2 pathogenic mutation; the method is completed by using the substance for detecting the MeCP2 mutation.
  • the present invention also provides a method for detecting or assisting in detecting whether the MeCP2 mutation is pathogenic, and the method is completed by using the substance for detecting the MeCP2 mutation.
  • the present invention also provides a method for diagnosing or assisting in diagnosing the diseases caused by the MeCP2 mutation, the disease caused by the MeCP2 mutation is a dysplasia of the nervous system, and the method is completed by using the substance for detecting the MeCP2 mutation.
  • the present invention also provides a product with any of the following functions, and the product is the substance for detecting MeCP2 mutation:
  • Figure 1 shows the phase transition analysis of MeCP2 and nucleosome beads.
  • the upper left picture in A is control 1, the upper right picture is control 2, and the lower three pictures are the experimental group; the upper left picture in B picture is control 1, the upper right picture is control 3, and the lower three pictures are control 4.
  • AI568 stands for Alexa Fluor TM 568 NHS Ester (Succinimidyl Ester).
  • Figure 2 shows the phase transition analysis of truncated MeCP2 and nucleosome beads.
  • A is a schematic diagram of the structure of truncated MeCP2.
  • B is the phase transition image analysis of different concentrations of full-length/truncated MeCP2 and 4 ⁇ nucleosome beads.
  • C is the quantitative analysis of the phase transition in Figure B.
  • Area Occupied represents the percentage of the area occupied by the phase transition phase or precipitation in the field of view, and No LLPS represents the percentage of the area occupied by the precipitation with fluorescent signal formed by the phase transition in the field of view, and the value is relatively small; LLPS represents the percentage of the area occupied by the phase change phase in the field of view.
  • R168 ⁇ , R255 ⁇ , R270 ⁇ , R294 ⁇ and MeCP2 represent R168 ⁇ -His, R255 ⁇ -His, R270 ⁇ -His, R294 ⁇ -His and MeCP2-His, respectively, and 4 ⁇ NA means 4 ⁇ non Methylated nucleosomes beaded.
  • Figure 3 shows the phase transition analysis of pathogenic point mutation MeCP2 and methylated/unmethylated nucleosome beads.
  • 12x Native Nucleosome Aray (DNA) means 12 x non-methylated nucleosome beads
  • 12x Nudeosome Aray (5me-DNA) means 12 x methylated nucleosome beads.
  • MeCP2 wt means MeCP2-His, MeCP2 R106W, MeCP2 R111G, MeCP2 Y120D, MeCP2 R133C, MeCP2 F157I, MeCP2 T158M, MeCP2 P225R, MeCP2 R306C respectively means MeCP2_R106W-His, MeCP2_R106W-His, MeCP2_R106W-His, MeCP2_R106W-His, MeCP2_R106W-His-HiCP1 His, MeCP2_T158M-His, MeCP2_P225R-His and MeCP2_R306C-His.
  • Figure 4 shows the effect of different point mutations of MeCP2 on intracellular phase transition.
  • A is a schematic diagram of the site distribution of pathogenic point mutation MeCP2 (top) and non-pathogenic point mutation MeCP2 (bottom).
  • B is the imaging analysis of cells transfected with pathogenic point mutation MeCP2 (left column) and non-pathogenic point mutation MeCP2 (right column).
  • Figure 5 shows the statistical analysis of the partition coefficient of mCherry fusion protein in the phase transition phase and the normal phase of the transfected cell line.
  • the distribution coefficient shown in the figure is the ratio of the intensity of the mCherry fluorescence signal in the DAPI dark-stained area and the DAPI non-stained area of the transfected cell line.
  • MeCP2, R106W, R111G, Y120D, R133C, P152R, F157I, K144R, P176R, T197M, A201V, R250H, R294P respectively represent the transfection of pmCherry-C1-MeCP2, pmCherry-C1-MeCP2_R106W, pmCherry-C1-Mepm2_R111G, Cherry-C1 -MeCP2_Y120D, pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_F157I, pmCherry-C1-MeCP2_K144R, pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_T201CV1-MeCP2Cherry-C1 , PmCherry-C1-MeCP2_R294P cells.
  • positions 1-6 and 1461-1466 of sequence 2 are the recognition sequences of NcoI and XhoI, respectively.
  • the DNA molecule coding sequence shown in positions 3-1484 of sequence 2 is MeCP2-His shown in sequence 1
  • sequence 1 Positions 1-486 are the amino acid sequence of MeCP2
  • positions 489-494 of Sequence 1 are the amino acid sequence of His-tag.
  • positions 1-6 and 504-509 of sequence 4 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-527 of sequence 4 are the R168 ⁇ -His sequence shown in DNA molecule coding sequence 3.
  • Position 1-167 of sequence 3 is position 1-167 of MeCP2, and position 170-175 of sequence 3 is the amino acid sequence of His-tag.
  • positions 1-6 and 765-770 of sequence 6 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-788 of sequence 6 are the R255 ⁇ -His sequence shown in the coding sequence 5 of the DNA molecule,
  • the 1-254th position of sequence 5 is the 1-254th position of MeCP2, and the 257-262th position of sequence 5 is the amino acid sequence of His-tag.
  • the recombinant vector pET-28a(+)-R270 ⁇ -His contains the DNA fragment shown in sequence 8, It can express the protein shown in sequence 7 (the His protein tag is fused at positions 1-269 of MeCP2, denoted as R270 ⁇ -His).
  • positions 1-6 and 810-815 of sequence 8 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-833 of sequence 8 are the R270 ⁇ -His sequence shown in the coding sequence 7 of the DNA molecule, Position 1-269 of Sequence 7 is position 1-269 of MeCP2, and position 272-277 of Sequence 7 is the amino acid sequence of His-tag.
  • positions 1-6 and 882-887 of sequence 10 are the recognition sequences of NcoI and XhoI, respectively, and positions 3-905 of sequence 10 are the R294 ⁇ -His sequence shown in the coding sequence 9 of the DNA molecule, Position 1-293 of sequence 9 is position 1-293 of MeCP2, and position 296-301 of sequence 9 is the amino acid sequence of His-tag.
  • the 318th position of the DNA fragment shown in sequence 2 was replaced by a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 1).
  • the sequence of the protein encoded by this DNA fragment is the 106th position of sequence 1
  • the amino acid sequence obtained by replacing the amino acid residue R with the tryptophan residue W, and the protein is designated as MeCP2_R106W-His.
  • the 333rd position of the DNA fragment shown in sequence 2 was replaced with G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 2).
  • the sequence of the protein encoded by this DNA fragment is that the 111th position of sequence 1 is replaced by fine
  • the amino acid sequence obtained by replacing the amino acid residue R with the glycine residue G, and the protein is designated as MeCP2_R111G-His.
  • the 360th position of the DNA fragment shown in sequence 2 was replaced by T with G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 3).
  • the sequence of the protein encoded by this DNA fragment is the 120th position of sequence 1 from The amino acid sequence obtained by replacing the amino acid residue Y with the aspartic acid residue D, and the protein is designated as MeCP2_Y120D-His.
  • the 399th position of the DNA fragment described in sequence 2 was replaced by a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 4).
  • the sequence of the protein encoded by this DNA fragment was changed from the 133rd position of sequence 1 to fine
  • the amino acid sequence obtained by replacing the amino acid residue R with the cysteine residue C, and the protein is designated as MeCP2_R133C-His.
  • the 471th position of the DNA fragment shown in sequence 2 was replaced with A from T to obtain the point mutation MeCP2 DNA fragment (denoted as DNA fragment 5).
  • the amino acid sequence obtained by replacing the alanine residue F with the isoleucine residue I, the protein is designated as MeCP2_F157I-His.
  • the 475th position of the DNA fragment shown in sequence 2 was replaced with T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 6).
  • the sequence of the protein encoded by this DNA fragment is that the 158th position of The amino acid sequence obtained by replacing the amino acid residue T with the methionine residue M, and the protein is designated as MeCP2_T158M-His.
  • the 676th position of the DNA fragment shown in sequence 2 was replaced by C to G, and the DNA fragment of point mutation MeCP2 (denoted as DNA fragment 7) was obtained.
  • the amino acid sequence obtained by replacing the amino acid residue P with the arginine residue R, and the protein is designated as MeCP2_P225R-His.
  • the 918th position of the DNA fragment shown in sequence 2 was replaced with a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 8).
  • the sequence of the protein encoded by this DNA fragment is that the 306th position of sequence 1 is replaced by fine
  • the amino acid sequence obtained by replacing the amino acid residue R with the cysteine residue C, and the protein is designated as MeCP2_R306C-His.
  • pmCherry-C1-MeCP2 can express the protein shown in sequence 11 (mCherry fusion MeCP2, denoted as mCherry-MeCP2).
  • positions 1-6 of sequence 12 are the recognition sequence of BspEI
  • positions 1465-1470 are the recognition sequence of HindIII
  • positions 7-1464 are the coding gene sequence of MeCP2
  • positions 1-236 of sequence 11 are mCherry's Amino acid sequence
  • the 239-724th position of sequence 11 is the amino acid sequence of MeCP2.
  • the 316th position of the MeCP2 coding sequence in the sequence 12 was replaced with a T to obtain a DNA fragment of the point mutation MeCP2 (denoted as DNA fragment 9).
  • Artificially synthesized DNA fragment 9 and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 9, to obtain the recombinant vector pmCherry-C1-MeCP2_R106W, pmCherry-C1-MeCP2_R106W
  • the 331st position of the MeCP2 coding sequence in sequence 12 was replaced by A to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 10).
  • Artificially synthesized DNA fragment 10 and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 10 to obtain the recombinant vector pmCherry-C1-MeCP2_R111G, pmCherry-C1-MeCP2_R111G
  • the fusion protein obtained by replacing the arginine residue R with the glycine residue G at position 111 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is denoted as mCherry-MeCP2_R111G.
  • the 358th position of the MeCP2 coding sequence in sequence 12 was replaced by T to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 11).
  • pmCherry-C1-MeCP2_Y120D It can express the fusion protein obtained by replacing the tyrosine residue Y with the aspartic acid residue D at position 120 of MeCP2 in the mCherry-MeCP2 of step 4, and this protein is denoted as mCherry-MeCP2_Y120D.
  • the 397th position of the MeCP2 coding sequence in sequence 12 was replaced with a T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 12).
  • Artificially synthesized DNA fragment 12 and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 12 to obtain the recombinant vector pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_R133C
  • the fusion protein obtained by replacing the arginine residue R with the cysteine residue C at position 133 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_R133C.
  • the 455th position of the MeCP2 coding sequence in sequence 12 was replaced by C to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 13).
  • Artificially synthesize DNA fragment 13 replace the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 13, to obtain the recombinant vector pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_P152R
  • the fusion protein obtained by replacing the proline residue P with the arginine residue R at position 152 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_P152R.
  • the 469th position of the MeCP2 coding sequence in sequence 12 was replaced with A from T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 14).
  • the fusion protein obtained by replacing the phenylalanine residue F with the isoleucine residue I at position 157 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and the protein is designated as mCherry-MeCP2_F157I.
  • the 431st position of the MeCP2 coding sequence in sequence 12 was replaced by A to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 15).
  • the fusion protein obtained by replacing the lysine residue K with the arginine residue R at position 144 of MeCP2 in pmCherry-C1-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_K144R.
  • the 527th position of the MeCP2 coding sequence in Sequence 12 was replaced by C to G to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 16).
  • Artificially synthesized DNA fragment 16 and replaced the DNA fragment between the BspEI and HindIII recognition sequences of pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) with DNA fragment 16, to obtain the recombinant vector pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_P176R
  • the fusion protein obtained by replacing the proline residue P with the arginine residue R at position 176 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_P176R.
  • the 590th position of the MeCP2 coding sequence in sequence 12 was replaced with T to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 17).
  • pmCherry-C1-MeCP2_T197M It can express the fusion protein obtained by replacing the threonine residue T with the methionine residue M at position 197 of MeCP2 in the mCherry-MeCP2 of step 4, and this protein is denoted as mCherry-MeCP2_T197M.
  • the 602th position of the MeCP2 coding sequence in the sequence 12 was replaced with a T to obtain a DNA fragment of the point mutation MeCP2 (denoted as DNA fragment 18).
  • the fusion protein obtained by replacing the alanine residue A with the valine residue V at position 201 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_A201V.
  • the 749th position of the MeCP2 coding sequence in sequence 12 was replaced by G to A, and a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 19) was obtained.
  • the 881th position of the MeCP2 coding sequence in sequence 12 was replaced by G to C to obtain a DNA fragment of point mutation MeCP2 (denoted as DNA fragment 20).
  • the DNA fragment 20 was artificially synthesized, and the DNA fragment between the BspEI and HindIII recognition sequences of the pmCherry-C1 vector (including the recognition sequences of BspEI and HindIII) was replaced with DNA fragment 20 to obtain the recombinant vector pmCherry-C1-MeCP2_R294P, pmCherry-C1-MeCP2_R294P
  • the fusion protein obtained by replacing the arginine residue R with the proline residue P at position 294 of MeCP2 in the mCherry-MeCP2 of step 4 can be expressed, and this protein is designated as mCherry-MeCP2_R294P.
  • the DNA molecule shown in sequence 14 in the sequence listing was artificially synthesized, and the DNA fragment between the NdeI and BamHI recognition sequences of the pET-3a vector (Invitrogen) (including the recognition sequences of NdeI and BamHI) was replaced with the sequence shown in sequence 14 in the sequence listing DNA molecules, the recombinant vector pET-3a-H2A is obtained, and pET-3a-H2A can express the H2A protein shown in sequence 13.
  • positions 1-6 and 399-404 of sequence 14 are the recognition sequences of NdeI and BamHI, respectively, positions 3-396 of sequence 14 are the H2A sequence of Xenopus laevis, and sequence 13 is the H2A sequence Amino acid sequence.
  • the DNA molecule shown in sequence 16 in the sequence listing was artificially synthesized, and the DNA fragment between the XbaI and BamHI recognition sequences of the pET-3a vector (including the recognition sequences of XbaI and BamHI) was replaced with the DNA molecule shown in sequence 16 in the sequence listing,
  • the recombinant vector pET-3a-H2B is obtained, and pET-3a-H2B can express the H2B protein shown in sequence 15.
  • positions 1-6 and 423-428 of sequence 16 are the recognition sequences of XbaI and BamHI, respectively, positions 40-408 of sequence 16 are the H2B sequence of Xenopus laevis, and sequence 15 is the amino acid sequence of H2B.
  • the DNA molecule shown in sequence 18 in the sequence listing was artificially synthesized, and the DNA fragment between the XbaI and BlpI recognition sequences of the pET-3a vector (including the recognition sequences of XbaI and BlpI) was replaced with the DNA molecule shown in sequence 18 in the sequence listing,
  • the recombinant vector pET-3a-H3 is obtained, and pET-3a-H3 can express the H3 protein shown in sequence 17.
  • positions 1-6 and 503-509 of sequence 18 are the recognition sequences of XbaI and BlpI, respectively, positions 42-452 are the H3 sequence of Xenopus laevis, and sequence 17 is the amino acid sequence of H3.
  • the DNA molecule shown in sequence 20 in the sequence listing was artificially synthesized, and the DNA fragment between the recognition sequences of NdeI and BlpI of the pET-3a vector (including the recognition sequences of NdeI and BlpI) was replaced with the DNA molecule shown in sequence 20 in the sequence listing.
  • the recombinant vector pET-3a-H4 is obtained, and pET-3a-H4 can express the H4 protein shown in sequence 19.
  • positions 1-6 and 368-374 of sequence 20 are the recognition sequences of NdeI and BlpI, respectively, positions 4-315 of sequence 20 are the H4 sequence of Xenopus laevis, and sequence 19 is the amino acid sequence of H4.
  • the DNA molecule shown in sequence 21 in the sequence listing was artificially synthesized, and the DNA fragment between the PstI and SalI recognition sequences of the EZ-T vector (Invitrogen) (including the recognition sequences of PstI and SalI) was replaced with the sequence shown in sequence 21 in the sequence listing DNA molecules to obtain the recombinant vector EZ-T-177-4.
  • positions 1-6 and 801-806 of sequence 21 are the recognition sequences of PstI and SalI, respectively, positions 7-12 and 727-732 are the recognition sequences of EcoRV, respectively, and positions 16-192 of sequence 21 are Position, position 193-369, position 370-546 and position 547-723 are 4 177bp DNA sequences respectively.
  • each 177bp sequence contains a 147bp "601" sequence, which can bind to histone octamer with high affinity.
  • the interval between the bodies is 30 bp.
  • the "601" sequence is also called the nucleosome positioning DNA sequence, which is a region where nucleosomes are more easily formed than other sequences.
  • the DNA molecule shown in sequence 22 in the sequence listing was artificially synthesized, and the DNA fragment between the PstI and ClaI recognition sequences of the pWM530 vector (Invitrogen) (including the recognition sequence of PstI and ClaI) was replaced with the DNA molecule shown in sequence 22 in the sequence listing , The recombinant vector pWM530-177-12 was obtained.
  • positions 1-6 and 2133-2138 of sequence 22 are the recognition sequences of PstI and ClaI, respectively
  • positions 6-11 and 2130-2135 are the recognition sequences of EcoRV, respectively
  • positions 9-185 of sequence 22 are No. 186-362, No. 363-539, No. 540-716, No. 717-893, No. 894-1070, No. 1071-1247, No. 1248-1424, No. 1425-1601
  • the 1602-1778th, 1779-1955th, and 1956-2132th positions are 12 177bp DNA sequences, respectively.
  • each 177bp sequence contains a 147bp "601" sequence, which can bind to histone octamer with high affinity.
  • the interval between the bodies is 30 bp.
  • MeCP2 binds to the import and export ends of the linker DNA and has no sequence specificity.
  • step 2) After step 2) is completed, centrifuge the obtained culture solution, discard the supernatant, resuspend it in two 50ml tubes with 100ml PBS and centrifuge (4°C, 4000rpm, 30min), discard the supernatant.
  • step 3) After step 3) is completed, the obtained bacterial pellet is resuspended in a beaker with 80 mL of lysis buffer, and lysozyme (sigma, catalog number 10837059001) is added, with a final concentration of 0.2 mg/mL) placed at 4°C for 10 minutes.
  • lysozyme Sigma, catalog number 10837059001
  • step 4 After step 4 is completed, ultrasonically crush the obtained product, and then perform high-speed centrifugation on the crushed product (4° C., 18000 RPM, centrifugation for 30 minutes), and collect the supernatant.
  • step 6) After step 5) is completed, incubate the supernatant with 2mL His-beads at 4°C for 3h, transfer to the bio-rad empty column tube and collect the flow-through fluid.
  • step 6) After step 6) is completed, wash the beads with 100ml wash buffer.
  • step 7) After step 7) is completed, the protein is eluted with elution buffers of different imidazole concentrations, and the eluate is collected.
  • step 8) SDS-PAGE gel electrophoresis is used to detect the purification result in the eluate, and the eluate containing the target protein obtained in step 8 is purified by Hitrap SP HP column.
  • step 9) After step 9) is completed, use low-salt buffer A and high-salt buffer B to elute the protein bound to the Hitrap SP HP column, collect the eluate, and use SDS-PAGE gel electrophoresis again to detect the purification results. The results showed that high purity target protein was obtained.
  • Dialysis the eluate containing the target protein obtained in step 10) (the dialysate is a reaction buffer). After the dialysis is completed, freeze the protein sample at -80°C for later use.
  • the obtained protein samples are MeCP2-His solution, R168 ⁇ -His solution, R255 ⁇ -His solution, R270 ⁇ -His solution, R294 ⁇ -His solution, MeCP2_R106W-His solution, MeCP2_R111G-His solution, MeCP2_Y120D-His solution, MeCP2_R133C-His solution, MeCP2_F157I-His solution, MeCP2_T158M-His Solution, MeCP2_P225R-His solution and MeCP2_R306C-His solution.
  • Analysis buffer 20mM Tris, 500mM NaCl, 20mM imidazole, 1mM PMSF, pH 7.4, the balance is water.
  • Wash buffer 20mM Tris, 300mM NaCl, 20mM imidazole, pH 7.4, the balance is water.
  • Elution buffer 20mM Tris, 300mM NaCl, 100mM/300mM/500mM/1M imidazole, pH 7.4, the balance is water.
  • Buffer A 20mM Tris, 300mM NaCl, pH 7.4, the balance is water.
  • Buffer B 20mM Tris, 1M NaCl, pH 7.4, the balance is water.
  • reaction buffer 20mM HEPES, 100mM NaCl, pH 7.4, the balance is water.
  • the recombinant vectors pET-3a-H2A, pET-3a-H2B, pET-3a-H3 and pET-3a-H4 obtained in step 1 were respectively introduced into E. coli BL21(DE3) Plys (Tiangen Biochemical Technology (Beijing) Co., Ltd.), Obtain recombinant strains BL21(DE3)Plys-pET-3a-H2A, BL21(DE3)Plys-pET-3a-H2B, BL21(DE3)Plys-pET-3a-H3 and BL21(DE3)Plys-pET-3a-H4 .
  • step 1) transfer the obtained bacterial solution to 750mL liquid LB medium at a volume ratio of 1:50 and cultivate until the OD600 reaches 0.5-0.6, then add IPTG to a final concentration of 0.5mM, and incubate at 37°C for 3hrs. Protein.
  • step 2) centrifuge the obtained bacterial solution and collect the precipitate.
  • 100ml 1 ⁇ wash buffer for every 6 bottles of bacteria 50mM Tris, 100mM NaCl, 1mM EDTA, 5mM B-ME, PH8.0, the balance is water
  • the bacteria were resuspended and sonicated (5s/5s, 400W, 2-3 rounds of sonication, 99 times per round), then the sonicated product was ultracentrifuged (23000g for 20min), the supernatant was discarded, and the precipitate was collected.
  • step 3 After step 3), resuspend the obtained precipitate in 100ml 1 ⁇ wash buffer containing 1%(v/v) TritonX-100 and then ultrasonically break it again (5s/5s, 400W, two rounds of ultrasound, 99 per round Times), then ultracentrifuge the sonicated product (20000g for 10min), discard the supernatant, and collect the precipitate.
  • step 4) once (or more times).
  • step 6) Resuspend the pellet obtained in step 5) with 100ml 1 ⁇ wash buffer and centrifuge (centrifuge at 20000g for 10min), discard the supernatant, and collect the pellet.
  • step 6) Repeat step 6) once (or multiple times).
  • step 7 Use 30ml unfolding buffer (20mM Tris(PH8.0), 7M Guanidine hydrochloride, 5mM B-ME) to resuspend the precipitate obtained in step 7 (stir and dissolve at room temperature for 1hr), ultracentrifuge (23000g for 20min), collect the supernatant, Obtain a protein solution.
  • step 9 high purity H2A solution, H2B solution, H3 solution and H4 solution containing the target protein are obtained respectively.
  • H2A solution, H2B solution, H3 solution and H4 solution into the dialysis bag.
  • the amount of H2A, H2B, H3 and H4 are 4mg each, and then put in the dialysate—refolding buffer (2M NaCl, 10mM Tris, 1mM EDTA, pH 8.0, The remainder is water). After stirring for 12 hours at 4°C, the dialysate is changed again and dialyzed at 4°C for 24 hours.
  • step 1 centrifuge the obtained protein sample (4°C, 21000g for 5 min), collect the supernatant and concentrate to 500 ⁇ l, purify the histone octamer by gel filtration chromatography, the purification steps are as follows:
  • Balance the superdex 200 column balance the superdex 200 column with refolding buffer (2M Nacl, 10mM Tris, 1mM EDTA, pH 8.0);
  • Loading Load 500 ⁇ l of sample into the column through the loading loop;
  • Separate octamers use refolding buffer to send the sample to the column for separation, collect different effluents, and identify histone octamers by SDS-PAGE.
  • the two recombinant vectors are introduced into E. coli DH5 ⁇ (Tiangen Biochemical Technology (Beijing) Co., Ltd. In ), two recombinant bacteria are obtained, and then the two recombinant bacteria are operated separately according to the following steps:
  • the recombinant bacteria were inoculated in 50ml of liquid LB medium and cultured overnight at 37°C to obtain a culture solution.
  • the culture solution obtained in step 1 was transferred to 800ml liquid LB medium containing 100ug/ml ampicillin, and after culturing at 37°C for 4-5 hours, the culture temperature was increased to 42°C and the culture was continued for about 12-13 hours to obtain a culture solution.
  • step 2 carefully add 210ml of ice-cold S3 (3M potassium acetate, adjust the pH to 5.2 with acetic acid, the balance is water) to the obtained liquid, and shake the centrifuge tube in one direction so that the liquid phase does not separate.
  • the impurities are in the shape of egg flowers, put on ice for 10 minutes.
  • step 3 centrifuge the obtained liquid at 4°C at 4000 rpm for 30 minutes, carefully aspirate the supernatant, filter through 4 layers of gauze to remove suspended impurities, and transfer to a 4L beaker to obtain solution 1.
  • step 1 centrifuge the obtained liquid (15000g 15min), and recover the nucleic acid precipitate with the supernatant.
  • step 2 rinse the sediment on the wall and bottom of the centrifuge tube with 70% (v/v) ethanol aqueous solution, centrifuge (15000g 15min, 4°C), discard the supernatant, and make the remaining ethanol evaporate clean (no alcohol smell and precipitation) It is damp and opaque) to obtain nucleic acid precipitation.
  • TE10/50 10mM Tris, 50mM EDTA, pH8.0, the balance is water
  • RNase final concentration 100ug/ml
  • step 1 After step 1 is completed, add 1/5 volume of 4M NaCl aqueous solution and 2/5 volume of 40% (mass ratio) PEG 6000 aqueous solution to the obtained product, mix well, incubate at 37°C for 5 min, and place on ice for 30 min.
  • step 2 centrifuge the obtained liquid at 4°C (20000g ⁇ 15min), discard the supernatant, wash once with 70% (v/v) ethanol aqueous solution, and dissolve the obtained precipitate in 50ml TE10/0.1(10mM Tris, 0.1 mM EDTA, pH 8.0, the balance is water) to obtain solution 2.
  • step 2 add 1/10 volume of 3M NaAc (PH5.2) and 2.5 times volume of absolute ethanol to the water phase, and place it at -20°C for at least 1 hour.
  • step 4 The 177-12 DNA obtained in step 4 is subjected to methylation reaction (using NEB methyltransferase M.SssI to complete), the system is as follows
  • NEBuffer2(10x), S-adenosylmethionine and Methyltransferase are all NEB products.
  • the resulting system was incubated at 37°C for 8h.
  • step 1 1) Take 3 ⁇ l of the reaction mixture in step 1, add 7 ⁇ l of water, add 10 ⁇ l of 0.2M NaOH aqueous solution after mixing, then incubate at 95°C for 10min;
  • step 1) After step 1) is completed, add 20 ⁇ l 1M ammonium acetate to the system to neutralize the reaction;
  • step 2) After step 2) is completed, take 0.1 ⁇ l and 0.3 ⁇ l of the neutralized samples respectively and drop them on the N+ membrane (GE Healthcare) to dry;
  • step 3) place the membrane at 80°C and incubate for 30 minutes;
  • step 4) After step 4) is completed, seal the N+ membrane with 5% (mass ratio) milk at room temperature, and wash 3 times with TBST for 1 hour;
  • step 6) After step 5) is completed, bind the membrane with an anti-5mC primary antibody (active motif), and carry out the binding at 4°C for 8 hours; wash with TBST three times;
  • step 6) After step 6) is completed, place the membrane in a goat anti-mouse secondary antibody (Zhongshan Jinqiao) and incubate at room temperature for 1 hour;
  • step 8) After step 7) is completed, add a luminescent substrate (invitrogen) to the film for 3 minutes at room temperature for development.
  • a luminescent substrate invitrogen
  • Step 3 Purify the 177-12 DNA that has undergone methylation modification.
  • the purification method is the same as Step 4 and Step 7 to obtain the 177-12 methylation modified DNA solution.
  • the 177-4 DNA and 177-12 DNA obtained in step four, the 177-12 methylated modified DNA obtained in step five and the histone octamer obtained in step three were used to assemble nucleosome beading.
  • the assembly system is as follows:
  • 1 ⁇ TE 10mM Tris, 1mM EDTA, pH 8.0, and the balance is water.
  • the samples were mixed according to the above assembly system and put into the refolding buffer for gradient dialysis.
  • the initial buffer was 450ml refolding buffer (the solution with NaCl concentration of 2M obtained by adding NaCl to 1 ⁇ TE), and the 1050ml 1 ⁇ TE pump was used by the peristaltic pump.
  • the refolding buffer gradually reduce the salt ion concentration, and after at least 16 hours of dialysis, the salt ion concentration of the assembled system is reduced to 0.6M NaCl. Then it is further dialyzed into a low-salt reaction buffer to obtain nucleosome beads.
  • nucleosome beads obtained by 177-4DNA, 177-12DNA, and 177-12 methylation modified DNA were recorded as 4 ⁇ unmethylated nucleosome beads, 12 ⁇ unmethylated nucleosome beads, and 12 ⁇ Methylated nucleosomes beaded.
  • MeCP2-His obtained in step 2 at a molar ratio of 1:1 with the dye Alexa Fluor TM 568 NHS Ester (Succinimidyl Ester) (ThermoFisher, catalog number A20003), mix and incubate (placed on a rotating shaker, room temperature for 1h) to achieve Fluorescent labeling of proteins. Then use reaction buffer to perform gel filtration chromatography on the above sample (the method is the same as step 3) to remove the remaining fluorescent dye to obtain dye-labeled MeCP2-His, which is frozen at -80°C for use.
  • Alexa Fluor TM 568 NHS Ester Succinimidyl Ester
  • the obtained dye-labeled MeCP2-His and unlabeled MeCP2-His are mixed to obtain a protein mixture, so that the molar concentration of the labeled protein accounts for 5%.
  • the concentration of methylated nucleosome beads in the reaction system was 112.5 nM, and the concentration of the total protein in the protein mixture in the reaction system was 10 ⁇ M.
  • control system set up the following control system:
  • Control 1 reaction system only add the above protein mixture, and use the reaction buffer to dilute to a total protein concentration of 20 ⁇ M.
  • Control 2 reaction system only add 4 x unmethylated nucleosome beads, and use reaction buffer to dilute to a concentration of 225 nM.
  • Control 3 reaction system only add the 177-4 DNA obtained in step 4, and use the reaction buffer to dilute to a concentration of 37.5 nM.
  • Control 4 reaction system only add the 177-4DNA obtained in step 4 and the above-mentioned protein mixture, the concentration of 177-4DNA and MeCP2-His total protein are 37.5nM and 10 ⁇ M, respectively.
  • reaction systems obtained were allowed to stand overnight at 4°C, and imaged and analyzed by a laser confocal scanning microscope.
  • the obtained five dye-labeled proteins are respectively mixed with the respective unlabeled proteins to obtain a protein mixture, so that the molar concentration of the labeled protein is 5%.
  • the five protein mixtures obtained were mixed with the 4 ⁇ unmethylated nucleosome beads (4 ⁇ NA) obtained in step 6 according to the final concentration shown in Figure 2 to obtain different reaction systems, 4 ⁇ unmethylated nucleosome
  • the concentration of body beads in the reaction system was set to 14.06, 28.13, 56.25, 112.5 nM, and the concentration of total protein in the protein mixture in the reaction system was set to 1.25, 2.5, 5, and 10 ⁇ M, respectively.
  • the obtained reaction systems were allowed to stand overnight at 4°C, and imaged and analyzed by a laser confocal scanning microscope.
  • the wild-type MeCP2-His obtained in step two and the MeCP2-His of each point mutation were labeled according to the operation process of step eight to obtain dye-labeled MeCP2-His, dye-labeled MeCP2_R106W-His, dye-labeled MeCP2_R111G-His, Dye-labeled MeCP2_Y120D-His, dye-labeled MeCP2_R133C-His, dye-labeled MeCP2_F157I-His, dye-labeled MeCP2_T158M-His, dye-labeled MeCP2_P225R-His and dye-labeled MeCP2_R306C-His, frozen at -80°C for later use.
  • the obtained nine dye-labeled proteins are respectively mixed with the respective unlabeled proteins to obtain a protein mixture, so that the molar concentration of the labeled protein is all 5%.
  • the concentration settings of nucleosome beads and 12 ⁇ methylated nucleosome beads in the reaction system are 4.6875, 9.375, 18.75, 37.5nM, and the concentration of total protein in the protein mixture in the reaction system except MeCP2_R111G-His Set to 1.25, 2.5, 5, 10 ⁇ M, and the concentration of MeCP2_R111G-His to 0.63, 1.25, 2.5, 5 ⁇ M.
  • the resulting reaction system was allowed to stand overnight at 4°C, and imaged and analyzed by a laser confocal scanning microscope.
  • the obtained DAPI stained cells were analyzed by laser confocal scanning microscopy.
  • the partition coefficient is the ratio of the mCherry fluorescence signal intensity in the DAPI darkly stained area of the transfected cell line and the DAPI non-stained area.
  • pmCherry-C1-MeCP2_R106W pmCherry-C1-MeCP2_R111G, pmCherry-C1-MeCP2_Y120D, pmCherry-C1-MeCP2_R133C, pmCherry-C1-MeCP2_P152R, pmCherry-C1-MeCP2_K-C144I, pmCherry-C1-MeCP2_F157I
  • the distribution coefficients of pmCherry-C1-MeCP2_P176R, pmCherry-C1-MeCP2_T197M, pmCherry-C1-MeCP2_A201V, pmCherry-C1-MeCP2_R250H, pmCherry-C1-MeCP2_R294P are 2.2877 ⁇ 1.733792, 1.620161 ⁇ 1.110083, 1.065981 ⁇ 0.177234, 1.0659

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Abstract

Application d'une substance permettant de détecter la mutation mecp2 pour détecter si la mutation mecp2 est une mutation pathogène et sélectionner un médicament. On constate que MeCP2 peut se lier aux billes de nucléosomes pour générer un changement de phase, et qu'à la fois une mutation de troncature pathogène de MeCP2 et une mutation ponctuelle provoquent un changement de phase anormal entre MeCP2 et les billes de nucléosomes ; un résultat expérimental intracellulaire montre que seule une mutation ponctuelle pathogène peut provoquer le changement de phase anormal, mais qu'une mutation ponctuelle non pathogène n'a aucune influence évidente sur le changement de phase, et il est en outre prouvé que le changement de phase anormal est étroitement lié à une maladie provoquée par la mutation de MeCP2. Par conséquent, la réversion du changement de phase anormal est susceptible de constituer une nouvelle conception et une nouvelle orientation pour le traitement d'une maladie connexe, une substance régulatrice capable d'inverser le changement de phase anormal peut être obtenue au moyen d'un criblage à haut débit et être appliquée au développement d'un médicament ciblé potentiel, et la stratégie peut également servir de référence pour le traitement d'autres maladies liées au changement de phase anormal.
PCT/CN2020/135514 2020-02-03 2020-12-11 Application d'une substance permettant de détecter la mutation mecp2 pour détecter si la mutation mecp2 est une mutation pathogène et sélectionner un médicament Ceased WO2021155712A1 (fr)

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