WO2008138572A1 - Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique - Google Patents

Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique Download PDF

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Publication number
WO2008138572A1
WO2008138572A1 PCT/EP2008/003779 EP2008003779W WO2008138572A1 WO 2008138572 A1 WO2008138572 A1 WO 2008138572A1 EP 2008003779 W EP2008003779 W EP 2008003779W WO 2008138572 A1 WO2008138572 A1 WO 2008138572A1
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Prior art keywords
nucleic acid
methylation
reporter
reporter gene
expression
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PCT/EP2008/003779
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German (de)
English (en)
Inventor
Albert Jeltsch
Sergey Ragozine
Rachna Goyal
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Jacobs University Bremen Gmbh
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Priority to DE112008001210T priority Critical patent/DE112008001210A5/de
Publication of WO2008138572A1 publication Critical patent/WO2008138572A1/fr

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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
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the invention relates to the field of screening for and evaluation of inhibitors of eukaryotic nucleic acid methylation reactions, as well as useful reporter plasmids and methylation inhibitor standards.
  • Nucleic acid methylation, and particularly DNA methylation is described in eukaryotes, particularly in mammalian cells, and particularly in humans, by the DNA methyltransferases Dnmtl (NCBI accession numbers: P26358, AAH92517, AAI26228, NP_001370), Dnmt3a (NCBI accession numbers: AAH 18214 , AAH23612, Q9Y6K1 NP_715640, NP_783329, NP_783328, NP_072046, AAH51864, AAY14761, AAH43617, AAH32392, AAL57039, AAD33084) and Dnmt3b (NCBI accession numbers: CAB53071, CAB53069, CAB53070, Q9UBC3, NP_787046, NP_787045, NP_787044, NP_008823, ABC48951, AAL57040, AAF04015, AAD53063, AAD53062,
  • methyltransferases transfer a methyl group of coenzyme S-adenosyl-L-methionine ("AdoMet") to the nucleic acids to be methylated, in particular DNA ("target DNA”).
  • AdoMet coenzyme S-adenosyl-L-methionine
  • target DNA DNA
  • a change in DNA methylation pattern of genomic DNA is observed over the usual methylation pattern of genomic DNA.
  • Such aberrant DNA methylation is considered to be a trigger or promoter of the emergence and / or progression, especially of tumor diseases. Therefore, it has often been attempted to develop DNA methylation inhibitors in order to use them as drugs for preventing or treating aberrant DNA methylations.
  • DNA methylation inhibitors such as sinefungin are based on the principle to bind to the coenzyme binding site of the respective DNA methyltransferase and thus to prevent them from binding to the coenzyme AdoMet.
  • these DNA methylation inhibitors also bind to the coenzyme binding sites of other enzymes that require AdoMet as coenzyme. Due to this cross-reactivity, these DNA methylation inhibitors cause numerous side effects.
  • nucleotide analogs such as zebularin and decitabine are used to reduce DNA methylation. Once incorporated into DNA, the nucleotide analogs covalently bind the DNA methyltransferases to the DNA. This reduces the cellular supply of available DNA methyltransferases. Disadvantageously, this also leads to considerable DNA damage and to a very high DNA repair activity.
  • the object of the present invention was therefore to provide a method and, in particular, a method suitable for screening for determining the inhibiting effect of an inhibitor on a eukaryotic nucleic acid methylation reaction.
  • the object is achieved by a method for determining the inhibition effect of an inhibitor on a eukaryotic nucleic acid methylation reaction, comprising the steps:
  • step 4 comparing the expression of the reporter gene determined in step 3 with the expression of the reporter gene at a preselected level of inhibition to determine the inhibitory effect of the inhibitor to be tested on the nucleic acid methylation reaction.
  • the method according to the invention makes it possible in an advantageously simple manner to determine the inhibiting effect of a (suspected) inhibitor of a eukaryotic nucleic acid methylation reaction under standardized conditions.
  • the method is particularly useful as part of a screening method for finding a nucleic acid methylation inhibitor. It is particularly expedient to carry out at least step 3 in a high-throughput approach. Due to its standardisability and simplicity, the method according to the invention is particularly well suited for use in high-throughput approaches, more details below.
  • the reporter nucleic acid can therefore be propagated in a eukaryotic and in particular a mammalian cell line without having to integrate into the genome.
  • the reporter nucleic acid is replicable independently of viral transcomplementing proteins in the cell line.
  • the reporter nucleic acid is then replicable in a variety of cell lines without the cell lines having to be previously transformed with viral genes needed for replication for trans-complementing factors.
  • the reporter nucleic acid according to the invention avoids a limitation of the method according to the invention to COS7 cells and the SV40 Large Transforming Antigen System. Accordingly, the method according to the invention can be carried out in a cell line which is unchanged from the reporter nucleic acid.
  • the method according to the invention is therefore particularly suitable for circumventing artifacts which can be caused by the expression of trans-complementing proteins.
  • the reporter nucleic acid can be stably transfected or otherwise expressably introduced into a target cell or cell line, preferably a eukaryotic and especially a mammalian cell line.
  • the reporter nucleic acid is expediently integrated into the genome, for example a chromosome, of the target cell or target cell line and replicated with the genome or chromosome.
  • cell lines reporter cell lines
  • This contributes to an improved reproducibility of the nucleic acid methylation analyzes and also facilitates the handling of the reporter cell lines.
  • Such episomally replicable reporter nucleic acids comprising a nuclear scaffold / matrix attachment region (S / MAR) Such a region is adapted for binding to the chromating scaffold of a eukaryotic cell nucleus, and preferably one Such reporter nucleic acids, but also reporter nucleic acids stably integrated into the genome of a target cell, advantageously allow the study of nucleic acid methylation inhibition in Mammalian cells and preferably in human cells.
  • a method according to the invention, which makes use of these reporter nucleic acids, is therefore set up particularly advantageously, suitable nucleic acid
  • Methylation inhibitors especially for the treatment of aberrant DNA methylation patterns in mammalian cells and especially in human cells.
  • the method according to the invention therefore particularly supports the development of medicaments for the prevention or treatment of tumor diseases.
  • the reporter nucleic acid may further comprise one or more genes for antibiotic resistance or other phenotypic trait (control feature) effective in prokaryotic and / or eukaryotic cells. This makes it possible to check the presence of the reporter nucleic acid in a pro- or eukaryotic cell independently of the expression of the reporter gene.
  • the reporter nucleic acid used in a method according to the invention additionally comprises a reporter gene expressed as a function of the degree of methylation of the reporter nucleic acid, preferably a gene for a fluorescent or luminescent protein, in particular green fluorescent protein (GFP, EMBL accession no .: gi
  • GFP green fluorescent protein
  • Enhanced GFP Enhanced Green Fluorescent Protein, EEFP, EMBL Accession No .: gi
  • Red Fluorescent Protein RFP, EMBL
  • luciferase EBL Accession No .: gi
  • the products of these reporter genes are either readily detectable by their fluorescence or they generate one In both cases, the signal associated with the product of the respective reporter gene (fluorescence or luminescence) is characteristic of the report so that false signals can be excluded by other cell components.
  • the inventive method thus allows a largely error-free determination of the degree of methylation of the reporter nucleic acid.
  • the reporter gene is preferably under the control of a methylation of the reporter nucleic acid dependent promoter which suppresses the expression of the reporter gene in the methylated state of reporter nucleic acid and allows in the demethylated state, preferably a cytomegalovirus immediate early promoter.
  • a reporter nucleic acid according to the invention preferably also contains an origin of replication effective in prokaryotes
  • the reporter nucleic acid according to the invention can therefore also be advantageously propagated in prokaryotic cells and harvested by conventional methods and processed for the transfection of eukaryotic cells.
  • the methylation reaction is carried out in the inventive method in a cell line.
  • the cell line is usually transformed with the reporter nucleic acid and subsequently amplified in the cell line, preferably by episomal replication, or by proliferation of cells with stably integrated reporter nucleic acid.
  • the reporter nucleic acid is optionally methylated under the action of methyltransferases of the cell line. It is particularly preferable to transform the cell line with a methylated reporter nucleic acid.
  • the replication products are also methylated; a reporter nucleic acid integrated into the genome is methylated during the replication of the genome.
  • the effect of a nucleic acid methylation inhibitor can then be demonstrated by the fact that the replication products of the reporter nucleic acid are no longer or no longer completely methylated.
  • the effect of a methylation inhibitor can be detected by the detection of the reporter gene product. This detection is possible in a very simple and reproducible manner, particularly in the case of fluorescent or luminescent reporter gene products, so that a corresponding method according to the invention can be carried out in a high-throughput assay.
  • the methylation mixture in particular the cell line transformed with the reporter nucleic acid, preferably further comprises, therefore optionally in addition to a gene for a control feature as described above, a control nucleic acid comprising a control reporter gene which can be expressed independently of the degree of methylation of the control nucleic acid.
  • a control nucleic acid comprising a control reporter gene which can be expressed independently of the degree of methylation of the control nucleic acid.
  • the cells of the cell line transformed with the report nucleic acid and preferably also with the control nucleic acid are examined for the expression of the reporter gene and preferably also of the control reporter gene. It is expedient to submit the transformed cells of the cell line in individual microtiter vessels, to multiply therein and thereby to pressurize the inhibitor to be examined.
  • the application may be effected in any desired manner, for example by adding the inhibitor or by inducing the expression of an inhibitor gene.
  • the cells of the cell line are propagated after or with continued exposure to the inhibitor, wherein the reporter nucleic acid and, if present, the control nucleic acid are also increased.
  • the report nucleic acid is methylated if the inhibitor has no or only a slight effect in the particular concentration present, or if it has no or less methylated if the inhibitor has a nucleic acid methylation-inhibiting activity in the particular concentration present.
  • the expression of the reporter gene and optionally the control reporter gene is determined, preferably by determining the fluorescence intensity on the Wavelength of the gene product of the reporter gene and optionally of the control reporter gene.
  • the inhibitory effect of the inhibitor to be examined on the nucleic acid methylation reaction can be simple and reproducible Be determined manner.
  • step 4 the expression of the reporter gene in the presence of the inhibitor to be tested is compared with an expression of the reporter gene in the presence of a preselected concentration of a standard methylation inhibitor selected from the group consisting of 5'-azacytidine and a peptide of up to 17 amino acids in length, containing an amino acid sequence according to Figure 7, wherein "x" is phosphorylated serine, and particularly preferably the amino acid sequence EKIY (I or M) SKI (V or L) (V or I ) E, where the letters are individual amino acids according to IUPAC
  • Methylation inhibitor is used as a standard for several rounds of the inventive method or for several inhibitors to be examined. Particularly preferred is a standard methylation inhibitor consisting of a peptide of up to 17 amino acids comprising the amino acid sequence E K I Y I phosphorylated S K I V V E. This peptide has proven to be a particularly good nucleic acid methylation inhibitor.
  • the reporter nucleic acid should be episomally replicable in plasmid form in eukaryotic cells, especially in human cell lines.
  • the reporter gene selected was a gene for green fluorescent protein (enhanced green fluorescent protein, eGFP, EMBL accession number: see above).
  • the vector also contains a gene for neomycin phosphotransferase as an antibiotic resistance gene active in prokaryotes and eukaryotes (kanamycin and neomycin resistance), as well as a pUC "origin of replication" as a bacterial replication initiation area.
  • FIG. 1 shows a map of the reporter nucleic acid.
  • the reporter nucleic acid was isolated with a methyltransferase from Spiroplasma sp. (M.Sssl, New England Biolabs). Methyltransferase methylates all cytosine residues in double-stranded cytosine guanine dinucleotides. Methylation was carried out in vitro according to the manufacturer's instructions.
  • Methylation was used: 10 ⁇ g reporter nucleic acid, 1 ⁇ buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl 2 , 1 mM dithiothreitol, pH 7.9 at 25 ° C.), 160 ⁇ M S-adenosylmethionine (Sigma), 20 U M.Sssl (NEB, 5 ⁇ l) in a total volume of 50 ⁇ l. The mixture was incubated for one hour at 37 0 C. The methylation was stopped by 20 minutes heating at 65 0 C. The methylated reporter nucleic acid was nigtditerei- by column purification.
  • methylation of reporter nucleic acid was checked by restriction digestion with Hpall and subsequent gel electrophoresis. This restriction nuclease cleaves only unmethylated CCGG nucleic acid segments. As a control, the methylated reporter nucleic acid was also digested with Mspl, a methylation insensitive isoschizomer of Hpall. The completeness of the methylation could be confirmed.
  • Fig. 2 shows an electrophoresis gel of reporter nucleic acid digested with Hpall.
  • the methylated reporter nucleic acid and an unmodified, red fluorecent protein (EMBL accession number: see above) encoding plasmid (control nucleic acid) were transfected with the transfection reagent FuGene-6 (Roche) in HEK293 cells.
  • the HEK 293 cells were first in Dulbecco's Modified Eagle medium (DMEM), supplemented with 5% fetal bovine serum (FBS), 100 U / ml penicillin / streptomycin and 2 mM glutamine in a humid incubator at 5% CO 2 and 37 0 C incubated. The cells were in the 25th-35th Passage. The day before transfection, cells were seeded in 6-well plates.
  • DNA reporter nucleic acid and control nucleic acid
  • DNA reporter nucleic acid and control nucleic acid
  • the transfected cells were examined for red and green fluorescence with a Carl Zeiss LSM501 meta-microscope. Green and red fluorescence were plotted against each other. The highest fluorescence value of non-transfected cells was used as limit for a detected fluorescence.
  • Example 4 Methylation of the reporter nucleic acid leads to significant transcriptional repression of the reporter gene Hek293 cells were transfected with methylated and non-methylated reporter nucleic acid and simultaneously with control nucleic acid as described in Example 3. Fig. 3 shows the transfection results.
  • Example 5 Inhibition of endogenous DNA methylation leads to the recovery of expression of the reporter gene of methylated reporter nucleic acids
  • Hek293 cells were transfected with methylated reporter nucleic acid and unmethylated control nucleic acid as described in Example 3.
  • the cells were treated with either the demethylating reagent 5'-azacytidine (Sigma) or buffer control. This time was taken as the "time 0 h" all expression studies.
  • the reagent was added freshly for 7 days daily at a final concentration of 2 ⁇ M, while in the control experiment only buffer and solvent of the reagent were added. The experiments were repeated twice.
  • FIG. 4 shows the development of the expression of the reporter gene of reporter nucleic acid and demethylating and under non-demethylation cultivation conditions.
  • an increase in the green fluorescence and thus an increase in the expression of the reporter gene of the reporter nucleic acid could be detected (FIG. 4C).
  • the increase in reporter gene expression was continuously observed until the 7th day of the experiment, then the experiments were discontinued.
  • After culturing for 7 days under demethylating conditions about 75% of the expression of the reporter gene was observed, as is achieved when using non-methylated reporter nucleic acid (maximum achievable expression).
  • expression of the reporter gene of the methylated reporter nucleic acid was only 5% of the maximum achievable expression.
  • FIG. 5 shows the expression of the reporter gene of the reporter nucleic acid and of the control nucleic acid.
  • HEK293 cells cultured under non-demethylating conditions showed virtually no expression of the reporter gene eGFP (Fig. 5A) in accordance with the findings of Example 4.
  • HEK293 cells cultured under demethylating conditions showed strong expression of the reporter nucleic acid reporter gene (Figure 5B).
  • HEK293 cells were transfected with methylated reporter nucleic acid and non-methylated control nucleic acid as described in Example 3, and after transfecting into 96-well microtiter plates.
  • the cell cultures were measured for green and red fluorescence with a Safire XFLU0R4 (Tecan) fluorimeter.
  • the cell cultures were treated as described in Example 5 5'-azacytidine (Sigma) for preparing demethylierender cultivation conditions.
  • Figure 7 shows schematically the flow of the approach.
  • the mixture was repeated using, instead of the demethylating reagent 5'-azacytidine, a peptide having the amino acid sequence EKIYISKIWE with phosphorylated serine residue.
  • a significantly stronger eGFP expression after 5 days under demethylating cultivation conditions could be detected in comparison to non-demethylating cultivation conditions.

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Abstract

L'invention concerne le domaine du criblage pour l'étude des réactions de méthylation d'acide nucléique et pour l'évaluation d'inhibiteurs de réactions de méthylation d'acide nucléique eucaryotes, ainsi que des normes de plasmides rapporteurs et d'inhibiteurs de méthylation utilisés pour cette étude et cette évaluation.
PCT/EP2008/003779 2007-05-09 2008-05-09 Procédé et réactifs pour l'étude des réactions de méthylation d'acide nucléique WO2008138572A1 (fr)

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DE112008001210T DE112008001210A5 (de) 2007-05-09 2008-05-09 Verfahren und Reagenzien zur Untersuchung von Nukleinsäure-Methylierungsreaktonen

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DE200710022274 DE102007022274A1 (de) 2007-05-09 2007-05-09 Verfahren und Reagenzien zur Untersuchung von Nukleinsäure-Methylierungsreaktionen

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2281045B1 (fr) * 2008-05-27 2012-08-01 Koninklijke Philips Electronics N.V. Administration et surveillance de traitement utilisant une protéine de fusion pour gène rapporteur d'intérêt et imagerie optique

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0999280A2 (fr) * 1998-10-17 2000-05-10 MultiGene Biotech GmbH Vecteur réplicatif épisomique, sa production et utilisation
WO2002044408A2 (fr) * 2000-12-01 2002-06-06 Arrow Therapeutics Limited Methode d'identification d'inhibiteurs d'enzymes
EP1384787A1 (fr) * 2002-07-25 2004-01-28 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procédé de criblage pour l'identification et la charactérisation d'inhibiteurs d'ADN méthyltransférase
WO2005082144A1 (fr) * 2004-02-25 2005-09-09 Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Office Of Technology Transfer Composes inhibiteurs de methylation
WO2007039920A1 (fr) * 2005-10-05 2007-04-12 Universita' Degli Studi Di Milano-Bicocca Méthode pour le transfert de vecteurs épisomiques dans des cellules animales
WO2007046803A1 (fr) * 2005-10-18 2007-04-26 University Of South Florida Dosage de promoteurs méthylés actifs en transcription

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0999280A2 (fr) * 1998-10-17 2000-05-10 MultiGene Biotech GmbH Vecteur réplicatif épisomique, sa production et utilisation
WO2002044408A2 (fr) * 2000-12-01 2002-06-06 Arrow Therapeutics Limited Methode d'identification d'inhibiteurs d'enzymes
EP1384787A1 (fr) * 2002-07-25 2004-01-28 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Procédé de criblage pour l'identification et la charactérisation d'inhibiteurs d'ADN méthyltransférase
WO2005082144A1 (fr) * 2004-02-25 2005-09-09 Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Office Of Technology Transfer Composes inhibiteurs de methylation
WO2007039920A1 (fr) * 2005-10-05 2007-04-12 Universita' Degli Studi Di Milano-Bicocca Méthode pour le transfert de vecteurs épisomiques dans des cellules animales
WO2007046803A1 (fr) * 2005-10-18 2007-04-26 University Of South Florida Dosage de promoteurs méthylés actifs en transcription

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GOYAL R. ET AL.: "Phosphorylation of serine-515 activates the mammalian maintenance methyltransferase Dnmt1", EPIGENETICS, vol. 2, no. 3, July 2007 (2007-07-01), pages 155 - 160, XP009105943, ISSN: 1559-2294(print) 1559-2308(ele *
JENKE A.C.W. ET AL.: "Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 101, no. 31, 3 August 2004 (2004-08-03), pages 11322 - 11327, XP002497602, ISSN: 0027-8424 *
PAPAPETROU E.P. ET AL.: "Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element", GENE THERAPY, vol. 13, no. 1, January 2006 (2006-01-01), pages 40 - 51, XP002497601, ISSN: 0969-7128 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2281045B1 (fr) * 2008-05-27 2012-08-01 Koninklijke Philips Electronics N.V. Administration et surveillance de traitement utilisant une protéine de fusion pour gène rapporteur d'intérêt et imagerie optique
US9528113B2 (en) 2008-05-27 2016-12-27 Koninklijke Philips N.V. Therapy delivery and monitoring using a gene of interest-reporter fusion protein and optical imaging

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