EP1397688A2 - Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome - Google Patents

Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome

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Publication number
EP1397688A2
EP1397688A2 EP02754655A EP02754655A EP1397688A2 EP 1397688 A2 EP1397688 A2 EP 1397688A2 EP 02754655 A EP02754655 A EP 02754655A EP 02754655 A EP02754655 A EP 02754655A EP 1397688 A2 EP1397688 A2 EP 1397688A2
Authority
EP
European Patent Office
Prior art keywords
dna
protein
dna fragments
library
proteins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02754655A
Other languages
German (de)
English (en)
Inventor
Stefan Beyer
Ursula Bilitewski
Joop Van Den Heuvel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Original Assignee
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Helmholtz Zentrum fuer Infektionsforschung HZI GmbH filed Critical Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Publication of EP1397688A2 publication Critical patent/EP1397688A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures

Definitions

  • the present invention relates to a novel method for the parallel zn-vitro identification of DNA-protein changes effects, ie potential DNA binding sites for proteins of unknown function can be found in the genome of organisms.
  • the proteins to be examined are incubated with DNA fragments that map the entire genome and then the sequences to which a specific binding takes place are identified.
  • the DNA fragments are preferably used in immobilized form and the binding of the protein to be examined is detected.
  • Upstream regulatory elements in front of genes and operons that are bound by DNA-binding proteins such.
  • B. transcription factors can be identified, can be identified.
  • the clarification of regulatory networks in complex biological systems is simplified and accelerated.
  • this invention can be used in order to describe the function and networking of regulons and modulons in bacterial systems.
  • DNA-protein interactions can be detected either in vivo, in vitro or by a combination of both experimental systems. As a rule, the investigations must be limited to one or only a few interacting and previously identified DNA-protein binding partners. By linking a so-called in vivo footprint with in vitro DNA microarray analyzes, even complex DNA mixtures can be checked for interactions based on a known or suspected DNA binding protein.
  • In vivo analyzes determine the effect of a DNA binding protein in connection with a possible DNA binding site on the transcription rate of a gene located in ice downstream.
  • the mRNA formed is detected and possibly quantified by z.
  • the mRNA is radioactively marked during the in vivo transcription or the mRNA is subsequently hybridized with radioactive or fluorescence-labeled probes.
  • the transcription rate can be indirectly correlated with the amount of the protein translated by this mRNA or with the enzyme activity derived therefrom.
  • the DNA partner is usually radioactively marked.
  • Protein and DNA are incubated in solution under various conditions and then either paired with a nitrocellulose membrane (filter binding) or immunoprecipitated with an antibody (McCay assay).
  • McCay assay Another go The common method is the gel retardation assay, in which the electrophoretic running behavior of protein-bound DNA is compared with that of unbound DNA. Different incubation conditions such as salt concentration, pH value, competitor DNA etc. allow conclusions to be drawn about the specificity and binding constants between the protein and the DNA fragment.
  • Chromatin immunoprecipitation with subsequent DNA microarray hybridization is based on the covalent linkage of DNA with cellular proteins catalyzed by formaldehyde or glutaraldehyde in an intact cell system. Because of their proximity, DNA binding proteins are preferentially bound to the DNA.
  • the DNA from the cells treated in this way is extracted, fragmented (eg by scissors) and then immunoprecipitated using an antibody directed against the DNA binding protein to be examined.
  • the DNA After separation of the DNA binding protein, the DNA can be amplified, labeled and hybridized against DNA spotted on microarrays. Compared to a control, e.g. B. a deletion mutant relating to the protein to be examined, specifically bound DNA fragments can be identified in parallel.
  • the detection limit of this method is directly dependent on the presence or the amount of the regulatory protein to be examined, the intracellular concentration of which is generally very low.
  • Nucleic acids are polymers and therefore bind to suitable carrier materials such as e.g. Membranes.
  • Nucleic acids are always negatively charged due to the phosphate residues of the nucleotides. Accordingly, they can be transferred to positive carrier surfaces using purely electrostatic forces. tie surfaces. This is exploited when using supports that are functionalized with amino groups (eg polylysine-coated supports or supports modified with aminopropyltriethoxysilane (APTS)), or membranes with positively charged side groups. Both methods (pure adsorption or adsorption supported by electrostatic forces) are characterized by the fact that they are easy to carry out, since the simple incubation of the support with the nucleic acid is sufficient for coupling, and that they largely retain the functionality of the bound molecules , since they do not need any further manipulation of the biomolecules.
  • APTS aminopropyltriethoxysilane
  • nucleic acids have a phosphate group at their 5 'end. This can be activated (eg by carbodiimides and succinimides) so that chemical coupling to amino groups can take place.
  • modified nucleic acids in particular nucleic acids which have an amino group or a biotin residue at one end.
  • nucleic acids oligonucleotides
  • Such nucleic acids are commercially available and can be incorporated into the nucleic acid to be investigated via polymerase reactions.
  • Amino-functionalized nucleic acids bind quickly to aldehyde-modified supports (formation of Schiff bases), which are also commercially available as glass supports.
  • they can be coupled to epoxy-functionalized carriers (modification, for example by means of suitable silanizing reagents) or else to carriers with carboxyl groups (after activation with carbodiimide and succinimd).
  • the use of biotinylated nucleic acid allows stable coupling to avidin or streptavidin modified carriers.
  • the coupling via reactions of the amino groups with aldehyde groups or via biotin (strept) avidin takes place so quickly that they are also suitable for the production of arrays with spotters.
  • nucleic acids or proteins, for example avidin or streptavidin
  • suitable functional groups for example amino, aldehyde, epoxy, carboxyl groups.
  • such carriers are commercially available, in others these modifications must be carried out by yourself, which is usually done by reaction of the carrier with suitable silanizing reagents. No further requirements are placed on the carrier, so that planar glass carriers can be silanized just like glass beads or metallic carriers.
  • carrier surfaces are often described in order to suppress non-specific interactions of later added molecules with the carrier.
  • Such further modifications include the saturation of carrier surfaces with inert proteins, such as bovine serum albumin, but also the coating with polymers, such as dextrans, or polyacrylamide gel, which can also serve as the basis for the immobilization and thus increase the loading capacity of the surface.
  • the immobilization principles described above can also be used for proteins, since proteins have sufficient functional groups, in particular for covalent couplings, due to the side groups of the amino acids. Since the charge of proteins is not as uniform as that of nucleic acids, immobilization due to electrostatic interactions is not as important, but pure adsorption reactions, which also include hydrophobic interactions, often lead to stable protein layers.
  • the marking the binding properties of the protein are not changed.
  • the coupling of the fluorescent dye in the region of the binding site of the protein could prevent the binding, or the coupling of highly hydrophobic dyes can lead to additional non-specific hydrophobic interactions with a carrier surface.
  • markers radioisotopes, fluorescent dyes
  • detection via subsequent reactions of the markers is also known.
  • Enzymes whose reaction products are detected principle of enzyme immunoassays) or labels for which binding partners exist (eg biotin with streptavidin peroxidase and enzymatic reaction; digoxigenin with enzyme-labeled antibody and subsequent enzymatic reaction; His.) are also used as markers 6 with appropriate antibody).
  • markers are suitable for the detection of bound nucleic acids as well as for the detection of bound proteins.
  • the most elegant detection methods are based on the direct, label-free detection of an affinity reaction. They not only allow the detection of the binding of an unmodified binding partner, but also the quantification of this binding and the monitoring of the binding reaction in real time and thus the determination of kinetic constants, ie the association and dissociation constants. These methods are essentially based on the fact that the binding reaction to a partner immobilized on a support changes the mass coverage of this support or / and the thickness of the layers on this support. The change in the layer thickness can be tracked using interferometric, ie optical methods (RIFs principle).
  • arrays have mainly been used for the detection of DNA-DNA interactions (expression analysis or sequencing with gene chips). Due to the need to elucidate the functions of the proteins encoded by the genes, the development of protein arrays is becoming more important. This is primarily about the detection of proteins by specific antibodies or similar binding proteins (protein A / G; receptors), or of enzyme activities. DNA-protein interactions with DNA fragments immobilized in array format have not yet been described. However, there are publications on the detection of DNA-protein interactions.
  • the present invention enables a function assignment of proteins or genes whose biological function is hitherto unknown or only partially known.
  • this invention enables a comprehensive, parallel in vitro identification of DNA-protein interactions and is independent of the expression of the regulatory protein to be examined in the actual host. This makes it possible, unaffected by environmental conditions, to clarify global regulatory networks in organisms in order to test them for their suitability for use in modulating metabolic pathways. Subsequent "genetic engineering” allows the optimization of, for example, fermentation processes for the production of enzymes, chemicals and pharmaceutically usable substances.
  • the regulatory networks discovered on the basis of this invention can be used to identify new "drug targets" for the treatment of diseases in humans, animals and plants.
  • the object on which the invention is based is thus achieved by a method for identifying interactions between proteins and DNA fragments of a genome, in which at least one protein is incubated with at least one DNA fragment of a genome in vitro and then the protein and / or DNA - Identified sequences that have interacted.
  • the DNA fragments can map the genome of a procaryte or eukaryote, in particular a bacterium, a yeast or a fungus.
  • a procaryte or eukaryote in particular a bacterium, a yeast or a fungus.
  • DNA fragments which are present as a DNA vector library in particular as a DNA plasmid library.
  • the DNA vector library with Escherichia coli clones can be provided.
  • the DNA fragments can be arranged as a DNA library or as a DNA vector library in microtiter plates.
  • the DNA fragments can be arranged multiple times in duplicate.
  • the vector DNA of the library can be double-stranded and immobilized on a solid matrix.
  • the vector DNA can be provided in an orderly or systematic manner.
  • a solid, optionally functionalized support which is a glass support, a membrane or a gel, can be provided as the solid matrix.
  • a functionalization of the support can be provided for the method according to the invention, which can couple the immobilized DNA fragments, in particular can bind covalently or electrostatically.
  • the optionally functionalized support can be provided on a planar support element, in a column or in or on a capillary.
  • a protein which is derived from the genome shown can also be used in the method according to the invention.
  • the derived protein may have been heterologously expressed in a prokaryotic, in particular a bacterial, or in a eukaryoiitic expression system.
  • a prokaryotic in particular a bacterial
  • a eukaryoiitic expression system in particular a prokaryotic, in particular a bacterial, or in a eukaryoiitic expression system.
  • the derived protein may have been expressed with an artificial epitope tag, in particular a histidine hexapeptide as an epitope tag.
  • the incubation can be carried out simultaneously on all immobilized DNA fragments batchwise or in the flow.
  • the incubation can also be carried out sequentially, in particular when immobilized in a column or in or on a capillary.
  • the interacting partners, DNA fragment and protein can be covalently linked to one another, in particular using an aldehyde, preferably formaldehyde or glutaraldehyde.
  • the interacting protein can be detected by looking at the protein
  • the DNA fragments can have a size of 1.5 to 2.5 kB, 1.0 to 2.0 kB, 0.5 to 1.5 kB, 0.3 to 0.8 kB or 0.03 to 0.5 kB ,
  • the method according to the invention can be carried out to identify DNA-protein interactions in complex DNA fragment mixtures, in particular to identify DNA binding sites for proteins of known or unknown function.
  • the method according to the invention for identifying cis-arranged regulatory elements for the transcription of eukaryotic or prokaryotic genes can be carried out.
  • the method according to the invention can be carried out for the function assignment of genes and / or proteins which are related to gene regulation, in particular for the identification of transcription factors or of genes which are regulated by transcription factors.
  • the method according to the invention for elucidating regulatory networks in complex biological systems can be carried out, in particular for optimizing the cultivation of prokaryotes or eukaryotes on the basis of regulatory networks.
  • the method according to the invention for optimizing fermentation processes can be carried out with the help of prokaryotes or eukaryotes on the basis of regulatory networks.
  • the method according to the invention for identifying targets for the development of active substances or of methods for the treatment of diseases can be carried out.
  • Another embodiment of the invention relates to a microtiter plate with a DNA library or DNA vector library arranged therein for carrying out the method according to the invention.
  • Another embodiment of the invention relates to a solid matrix according to the invention with DNA fragments immobilized on the matrix.
  • Another embodiment of the invention relates to a planar support element according to the invention with a solid matrix according to the invention applied thereon.
  • the plasmid DNA of the library is immobilized in an orderly manner as a double strand 'on a solid matrix.
  • a treated / coated glass surface, a membrane or a gel of different materials can be used as the matrix.
  • the treatment or coating of the glass surface can be carried out in order to permit the coupling of the DNA (covalent or electrostatic), to suppress unspecific bonds (see 5.), or to enable detection (coating with, for example, a gold layer for detection via surface plasmon resonance).
  • Other supports than glass supports can also be used if other detection methods than optical are selected.
  • only the “insert DNA” of the recombinant plasmids can be immobilized in a directional manner. B. done using the PCR.
  • the matrix can be on a planar support, but also in a column or on the wall of a capillary.
  • the protein to be examined is selected by computer-aided analysis of DNA and protein sequences that are already available, e.g. B. from fully or partially sequenced genomes.
  • a comparison of the derived amino acid sequences of ORFs with protein databases can provide first indications of DNA binding proteins.
  • the assignment of the derived proteins to so-called COGs (clusters of orthologous groups) or the presence of special structural motifs (eg “helix-turn-helix” motifs) can make it possible to predict a potential DNA-binding activity.
  • the gene / gene product selected under 3) is heterologously expressed in a bacterial or eukaryotic expression system with or without an artificial “epitope tag (eg histidine hexapeptide) and then purified in its native state.
  • an artificial “epitope tag eg histidine hexapeptide
  • the protein present in buffer solution is incubated with the double-stranded DNA immobilized on a matrix. Non-specific interactions of the protein with the matrix or the DNA are suppressed by suitable surface treatments or washing steps, or made visible by competition experiments.
  • the incubation can be carried out simultaneously on all immobilized DNA samples in batch or in flow, or sequentially (preferably when the DNA is immobilized in columns or capillaries and corresponding flow systems).
  • the interacting partners are covalently linked to one another using crosslinkers (for example formaldehyde or glutaraldehyde).
  • crosslinkers for example formaldehyde or glutaraldehyde.
  • the detection of the regulator protein is then carried out by means of immunological methods using (enzyme, fluorescence or radioactive) labeled antibodies which are directed against the regulator protein or the "epitope tag" present on the regulator protein. Detection methods are used which allow a label-free measurement (eg surface plasmon resonance; "resonant mirror”;interferometer; piezocrystals), the attachment of the protein can be followed directly without the need for antibodies.
  • the method described above identifies protein-DNA binding partners with the aid of a heterologously expressed protein and a DNA library.
  • the ordered plasmid library allows the clear assignment to genomic DNA regions of the organism to be examined.
  • the sequencing of the "inserts" of the plasmids filtered out makes it possible to identify the genes which are in the immediate vicinity of the protein binding region. In the case of fully sequenced genomes, DNA sequences which influence the regulation of genes and operons can be identified in this way.
  • the biochemical and biological characterization of the DNA-protein interactions can then be carried out using various "state-of-the-art" methods (for example, by sequence comparison, marker gene analysis in combination with deletion studies, DNAse protection analysis, reporter gene analysis, etc.).
  • LexA modulates the expression of many regulons of the SOS system.
  • TyrA controls 8 operons bifunctionally as a repressor and activator.
  • FnrR is involved in the regulation of more than 30 transcription units.
  • LrpR affects the transcription of 8 operons.
  • PhoPQ is involved in the regulation of at least 7 operons.
  • DNA chips a) Nature Genet. 21 (1999) Suppl. B) K. Niikura, K. Nagata, Y. Okahata, Quantitative detection of protein binding onto DNA by using a quartz-crystal microbalance, Chem. Lett. 1996, 863-4 c) D. Guschin, G. Yershov, A. Zaslavsky, A. Gemmell, V. Shick, D. Proudnikov, P. Arenkov, A. Mirzabekov, Manual Manufacturing of oligonucleotides, DNA, and protein microchips, Anal. Biochem. 250, 1997, 203-11

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Abstract

L'invention concerne un procédé pour identifier des interactions entre des protéines et des fragments d'ADN d'un génome, consistant à incuber in vitro au moins une protéine avec au moins un fragment d'ADN d'un génome, puis à identifier les séquences de protéine et/ou d'ADN qui sont entrées en interaction.
EP02754655A 2001-06-12 2002-06-11 Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome Withdrawn EP1397688A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10128321 2001-06-12
DE10128321A DE10128321A1 (de) 2001-06-12 2001-06-12 Verfahren zur Identifizierung von Wechselwirkungen zwischen Proteinen und DNA-Fragmenten eines Genoms
PCT/EP2002/006406 WO2002101393A2 (fr) 2001-06-12 2002-06-11 Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome

Publications (1)

Publication Number Publication Date
EP1397688A2 true EP1397688A2 (fr) 2004-03-17

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Application Number Title Priority Date Filing Date
EP02754655A Withdrawn EP1397688A2 (fr) 2001-06-12 2002-06-11 Procede pour identifier des interactions entre des proteines et des fragments d'adn d'un genome

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US (1) US20040209267A1 (fr)
EP (1) EP1397688A2 (fr)
AU (1) AU2002320845A1 (fr)
DE (1) DE10128321A1 (fr)
WO (1) WO2002101393A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070154906A1 (en) * 2005-10-05 2007-07-05 Spirogen Ltd. Methods to identify therapeutic candidates
US20080248958A1 (en) * 2007-04-05 2008-10-09 Hollenbach Andrew D System for pulling out regulatory elements in vitro

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5700637A (en) * 1988-05-03 1997-12-23 Isis Innovation Limited Apparatus and method for analyzing polynucleotide sequences and method of generating oligonucleotide arrays
US5556752A (en) * 1994-10-24 1996-09-17 Affymetrix, Inc. Surface-bound, unimolecular, double-stranded DNA
US6669906B1 (en) * 1997-04-22 2003-12-30 Thomas Schalkhammer Reinforced cluster optical sensors
WO1999000520A1 (fr) * 1997-06-30 1999-01-07 The Government Of The United States Of America, Reresented By The Secretary Of The Department Of Health And Human Services Le clonage spectral - une nouvelle approche technique du clonage et caracterisation de toutes les aberrations chromosomiques dans des echantillons cancereux
US20020025552A1 (en) * 1998-01-06 2002-02-28 Institut Pasteur Screening interactor molecules with whole genome oligonucleotide or polynucleotide arrays
WO2000053813A1 (fr) * 1999-03-11 2000-09-14 Massachusetts Institute Of Technology Bibliotheques pangenomiques
WO2002086095A2 (fr) * 2001-04-23 2002-10-31 Chou Michael F Methodes permettant de decouvrir un reseau de facteurs de transcription

Non-Patent Citations (1)

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Title
See references of WO02101393A2 *

Also Published As

Publication number Publication date
AU2002320845A1 (en) 2002-12-23
WO2002101393A3 (fr) 2003-11-06
US20040209267A1 (en) 2004-10-21
DE10128321A1 (de) 2003-01-02
WO2002101393A2 (fr) 2002-12-19

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