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WO1999036430A1 - Oligoligands a pouvoir de liaison - Google Patents

Oligoligands a pouvoir de liaison Download PDF

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Publication number
WO1999036430A1
WO1999036430A1 PCT/EP1999/000120 EP9900120W WO9936430A1 WO 1999036430 A1 WO1999036430 A1 WO 1999036430A1 EP 9900120 W EP9900120 W EP 9900120W WO 9936430 A1 WO9936430 A1 WO 9936430A1
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WIPO (PCT)
Prior art keywords
ligands
oligo
building blocks
mono
oligoligands
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PCT/EP1999/000120
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German (de)
English (en)
Inventor
Alexander Gasch
Douglas Friday
Kornelia Berghof
Lutz MÜLLER-KUHRT
Original Assignee
BioteCon Gesellschaft für Biotechnologische Entwicklung und Consulting mbH
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Application filed by BioteCon Gesellschaft für Biotechnologische Entwicklung und Consulting mbH filed Critical BioteCon Gesellschaft für Biotechnologische Entwicklung und Consulting mbH
Priority to DE19980033T priority Critical patent/DE19980033D2/de
Priority to AU26163/99A priority patent/AU2616399A/en
Publication of WO1999036430A1 publication Critical patent/WO1999036430A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/047—Simultaneous synthesis of different peptide species; Peptide libraries
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • 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
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the invention relates to a system which consists of molecular mono ligands which are produced by in vitro selection against small biological molecules (building blocks). These mono ligands are subsequently linked together to create combinatorial libraries of oligo ligands.
  • Such libraries can be used for the identification of affinity ligands for the purification of macromolecules and for the identification of novel, therapeutically or diagnostically usable substances.
  • the advantage of such libraries is that the macromolecule does not have to be present in pure form in order to produce or identify suitable oligo ligands with the desired affinity or activity. That's why it is possible to obtain ligands for almost any macromolecular target substance, even if this is unknown.
  • Building blocks or building block molecules are the individual molecular units (monosaccharides, amino acids, nucleotides, fatty acids) as well as those units in modified form (e.g. glycosylated, phosphorylated, acetylated, sulfonylated or methylated) from which larger biological molecules (hereinafter: macromolecules) are formed, for example Carbohydrates, proteins, glycoproteins, DNA / RNA / nucleic acids, fats. Building blocks can also be short oligomeric molecules, e.g. Dimers or trimers etc.
  • Monomers are individual building block molecules.
  • the products are oligomers when two (dimer), three (trimer) or more building block molecules are linked to one another by class-specific bonds (e.g. peptide bond with amino acids).
  • Oligomers are also molecules in which building block molecules of different classes are linked to one another (e.g. glycosylated peptide).
  • In vitro selection is a process by which molecules with certain properties can be isolated relatively quickly from a complex mixture. To do this, it must be possible to separate molecules with a desired property from other molecules without the desired property. Some methods for the in vitro selection of molecules with special properties are described.
  • a parent library is a mixture of ligands isolated by in vitro selection against a single building block molecule.
  • a parent library can consist of several ligands that show the desired activity, or in extreme cases a single ligand with high affinity or activity.
  • a macromolecule is a larger molecule that is derived from the above There are building blocks and usually have a biological function (e.g. enzyme, receptor, genetic information carrier, structural component, energy source).
  • An oligonucleotide can be characterized as being 10 to 250 and preferably 20 to 120 nucleotides in length.
  • an oligonucleotide according to the invention can be characterized in that it is single-stranded or has a complementary strand.
  • an oligonucleotide according to the invention can be characterized in that it is present i) as DNA or ii) as RNA or iii) as PNA, the oligonucleotide molecule optionally in one for analytical detection methods, in particular based on
  • Hybridization and / or amplification modified or labeled in a manner known per se.
  • an oligonucleotide according to the invention can be characterized in that it is a modified or labeled or additionally labeled nucleic acid molecule which, in a manner known per se for analytical detection methods, contains one or more radioactive groups, colored groups, fluorescent groups, groups for immobilization on solid Phase, groups for uptake in cells, groups for an indirect or direct reaction, in particular for an enzymatic reaction, preferably with the aid of antibodies, Antigens, enzymes and / or substances with affinity for enzymes or enzyme complexes, and / or other known modifying or modified groups of nucleic acid-like structure.
  • Aptamers are oligonucleotides selected in vitro which (due to their secondary structure) have a desired activity.
  • a mono ligand is a ligand that consists of a single molecule of a parent library and has affinity for a building block molecule.
  • a monoaptamer is a mono ligand that consists of an oligonucleotide molecule. Two or more mono ligands can be linked together by various methods to produce an oligo ligand. If the oligo ligand consists of aptamer molecules, it is an oligoaptamer. Methods for creating oligo ligands or oligoaptamers include:
  • oligoligands can be a spacer molecule between the individual mono ligands can be integrated. This can be a linear or branched chain of molecules which carries the corresponding groups at the ends in order to bind modified or unmodified ligands in covalent or non-covalent form. By varying the length or the flexibility of the spacer molecule, an additional variability in the properties of the oligo ligand can be achieved.
  • the properties of the parent libraries and the methods with which they are linked correspond to those of the combinatorics.
  • the use of small building blocks for in vitro selection creates libraries of mono ligands that can be combined with one another in any way to create oligo ligands with new specificities.
  • Affinity ligands can be used in several areas: as specific ligands in affinity chromatography, in binding studies and diagnostic tests or as therapeutic agents. Such ligands show high specificity to their respective target substance or group of
  • Widespread ligands are e.g. Cofactors, substrate analogs or (monoclonal) antibodies. The latter often recognize short sequences of amino acids on the protein surface (epitopes). This enables the use of oligopeptides as immunogens for the production of antibodies against proteins which contain the respective peptide sequence.
  • affinity ligands have only been available or can be produced for a limited number of target molecules, in particular for those target molecules which are either present in purified form or whose structure is at least partially known.
  • a universally applicable process with which Ligands against any macromolecule in principle can also be produced without these prerequisites is not yet known.
  • Oligomers or polymers are known from WO 95/17413. Such "functional elements" are more likely to be obtained by randomly or directionally linking structural domains of natural or functional polymers than by randomly combining all possible monomers
  • the object on which the invention is based is achieved by a process for the production of oligo ligands with binding capacity to macromolecules possibly unknown construction from building blocks, the method being characterized by: a) selection of ligands against a mono- or oligomeric building block, b) selection of ligands against a second mono- or oligomeric building block and optionally against a third to nth mono- or oligomeric building block, c) chemical or enzymatic linking of the selected ligands according to a) and b), in particular with the introduction of spacer molecules, d) screening of the oligoligands obtained for binding to one or more macromolecules (e) to which the oligoligands have binding capacity and e) isolation of the binding oligo ligand according to d) and optionally f) production of the binding oligo ligand according to e) by means of chemical or enzymatic synthesis or by means of cloning,
  • a further embodiment of the invention relates to a process for the preparation of oligo ligands with the desired biological activity, the process being characterized by: a) selection of ligands against a mono- or oligomeric building block, b) selection of ligands against a second mono- or oligomeric building block and optionally against a third to nth mono- or oligomeric building block, c) chemical or enzymatic linking of the selected ligands according to a) and b), in particular with the introduction of spacer molecules, d) screening of the oligoligands obtained for desired biological activity, such as stimulation or inhibition of cell division or inhibition of an enzymatic activity, and e) isolation of the oligoligand (s) with the desired biological activity and optionally f) production of the binding oligoligand (s) according to e) by means of chemical or enzymatic synthesis or by means of cloning.
  • the linked ligands can be RNA oligonucleotides or single-stranded DNA oligonucleotides, and the ligands can be obtained by SELEX (Systematic Evolution of Ligands by Exponential Enrichment). Furthermore, the ligands can be peptides which have been obtained, for example, by a) chemical synthesis and b) expression on the surface of phages (phage display) or on the surface of bacteria.
  • Building blocks can be amino acids, dipeptides, tripeptides, monosaccharides, disaccharides, trisaccharides, mononucleotides, dinucleotides, trinucleotides or fatty acid molecules.
  • a further embodiment of the invention relates to oligo ligands which can be obtained by a process according to the invention.
  • a further embodiment of the invention relates to oligo ligands which are identified, ie screened and isolated, by a method according to the invention and subsequently produced in large amounts, in particular by a) chemical synthesis, b) in vitro transcription by means of purified RNA polymerases, c) in vitro amplification, in particular by means of PCR, LCR and / or 3SR, and / or d) cloning, in particular in E. coli.
  • Oligoligands according to the invention can be characterized in that they are modified or labeled or additionally modified or labeled nucleic acid molecules or peptide molecules which, in a manner known per se for analytical detection methods, involve one or more radioactive groups, colored groups, fluorescent groups, groups for immobilization solid phase, groups for the uptake into cells, groups for an indirect or direct reaction, in particular for an enzymatic reaction, preferably for a reaction with the aid of antibodies, antigens, enzymes and / or substances with affinity for enzymes or enzyme complexes, and / or have otherwise known modifying or modified groups nucleic acid-like structure.
  • All or part of the oligo ligands according to the invention can consist of PNA.
  • the oligo ligands according to the invention can be protected against nuclease degradation, in particular by incorporating modified nucleotides which are modified in particular by 2'-amino, 2'-fluoro- or 2'-O-methyl groups.
  • Oligoligands according to the invention can consist wholly or partly of L-RNA or L-DNA.
  • one embodiment relates to the use of one or more oligoligands according to the invention for the detection of macromolecules of unknown structure from building blocks, the oligoligands and macromolecules being incubated and oligoligands bound after washing steps being detected.
  • the macromolecules can be extracted in cell extracts or in tissue sections
  • one embodiment relates to the use of oligo ligands according to the invention for therapeutic purposes.
  • a further embodiment relates to the therapeutic use of oligo ligands according to the invention with antibacterial, antiviral or cytostatic activity.
  • one embodiment relates to the therapeutic use of oligo ligands according to the invention by introduction into infected cells, tissues or organisms for combating infections or tumors.
  • Target substance selected selected. After suitable washing steps, bound molecules are eluted and duplicated. This is done by means of PCR (polymerase chain reaction) in the case of DNA ligands or RT-PCR (reverse transcriptase - PCR) in the case of RNA ligands.
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase - PCR
  • flanking regions can contain a promoter to be transformed by in vitro transcription with e.g. bacterial or viral RNA polymerases to enable translation into single-stranded RNA.
  • the amplified double-stranded DNA e.g. can be converted into single-stranded DNA (ssDNA) by asymmetric PCR or by immobilization of a strand and subsequent denaturation. After several rounds of selection and amplification under conditions of increasing stringency, those oligonucleotides dominate in the mixture which have a high affinity for the target substance or a desired activity. Through additional "counter selection" with undesired target molecules e.g.
  • sequences can be removed which have undesirable affinity for these groups / target molecules.
  • Individual aptamers can finally be isolated and characterized by cloning.
  • Oligonucleotide ligands in biological fluids is that in some cases (eg in blood) they are broken down very quickly by nucleases.
  • nucleases for the use of oligonucleotide ligands in liquids containing nucleases, it is therefore advantageous if they are protected against nuclease degradation. This can be achieved by integrating chemically modified nucleotides (e.g. with 2'-amino, 2'-fluoro- or 2'-O-methyl groups) (Jellinek, D; 1995; Eaton, B & Picken, W; 1995 ).
  • Synthetic peptide libraries Oligopeptides with a length of approximately 6-7 amino acids are synthesized by "split synthesis" (Furka, A., Sebestyen, F., Asgedum, M. & Dibo, G; 1988 ). The synthesis is started with, for example, 20 parallel batches, each of these batches starting with a different amino acid. Each of these synthesis approaches is in turn in z. B. 20 synthesis approaches and the synthesis continued with a different one of the 20 amino acids. This is repeated until the desired length (X) is reached.
  • Such a library consists of 20 x peptide sequences.
  • the peptides can either be on a matrix (eg beads) or in a solution
  • Affinity or activity are screened and active peptides are identified by peptide sequencing.
  • SPLC Synthetic Peptide Combinatorial Library
  • SPLC synthetic Peptide Combinatorial Library
  • a library of peptides with a length of approximately 6-7 amino acids is synthesized, the sequence of the first two amino acids being defined.
  • 400 separate peptide mixtures - each with a different dipeptide sequence at the NH 2 terminus - are screened for an affinity or activity.
  • the peptide mixtures which show the highest activities are now varied at the position of the third amino acid, the remaining amino acids again not being determined.
  • the present invention thus relates to combinatorial libraries of oligomeric molecules which have an affinity for one or more target substance (s) or a desired biological, biochemical or chemical activity.
  • monoligands against building block molecules eg monosaccharides, amino acids, nucleotides and short oligomers
  • building block molecules eg monosaccharides, amino acids, nucleotides and short oligomers
  • Two or more of the individual selected mono ligands are subsequently linked together to create new specificity or activity.
  • the oligo ligands are finally tested for the new specificity or activity, for example as follows:
  • building block molecules eg monosaccharides, glycosylated amino acids, nucleotides and short oligomers
  • Suitable spacer molecule are, for example, activated Sepharose beads or the wells of a microtiter plate (MTP).
  • MTP microtiter plate
  • the building blocks are bound by their chemical groups, for example -OH, -NH 2 , -COOH and / or -P0 3 , so that, for example, the side chains of amino acids or the bases of nucleotides are accessible to the potential ligands.
  • Ligands are made from a mixture of degenerate single-stranded oligonucleotides (SELEX) or from a mixture of the peptides expressed on the surface of phages (phage display) or from a mixture of synthetically produced peptides ("Synthetic Peptide Combinatorial Library”) with the immobilized building blocks in vi tro selected.
  • SELEX degenerate single-stranded oligonucleotides
  • phage display a mixture of the peptides expressed on the surface of phages
  • Synthetic Peptide Combinatorial Library synthetically produced peptides
  • An optional intermediate step consists in checking which of the ligands against building blocks already have significant affinity for the respective target macromolecule and / or have a desired activity.
  • the test can be carried out, for example, in an MTP or on a membrane: if the macromolecule is present in pure form, it is immobilized, for example, in the wells of an MTP or as a spot on a membrane.
  • Monoligands which are provided with a suitable label such as, for example, radioactive, colored, fluorescent groups or groups which directly or indirectly allow enzymatic detection
  • a suitable label such as, for example, radioactive, colored, fluorescent groups or groups which directly or indirectly allow enzymatic detection
  • the extract can first be separated in an SDS polyacrylamide gel and blotted onto a membrane. Incubation and detection then take place as above. Two or more of the above mono ligands are linked together.
  • the classes of mono ligands that bind the respective macromolecules are used (eg ligands against amino acids for proteins or polypeptides as macromolecules).
  • mono ligands against building blocks of different classes eg oligoligands against, for example, modified proteins can be created (for example against myristoylated, glycosylated or proteins complexed or covalently linked with nucleic acids).
  • the linkage could take place according to one of the following methods: a) Cloning (in the case of aptamers): the DNA product of aptamer 1 contains an interface for restriction enzyme 1 (eg Barn HI) in the region of one of the two flanking primer binding sites and an interface for restriction enzyme 2 (eg Hind III) in the region of the other flanking primer binding sites.
  • Aptamer 2 also contains an interface for restriction enzyme 2 (eg Hind III) in one flanking region and an interface for restriction enzyme 3 (eg Eco RI) in the other flanking region.
  • the aptamers are then fused to one another at the interface for restriction enzyme 2 using methods familiar to the skilled worker (J. Sambrook, EF Fritsch, T. Maniatis, 1989) and ligated into a plasmid vector. Large amounts of the diaptamer can then be obtained by multiplying the plasmid in, for example, Escherichia coli, cleaving with suitable restriction enzymes and
  • This chemical coupling has the advantage that mono ligands can also be linked to one another via non-class-specific bonds, and that at the same time an immense
  • the oligo ligands are then linked to the binding to target substances or to activity e.g. tested in the form of a microtiter plate test.
  • Individual oligo ligands are e.g. each immobilized in a well of a microtiter plate (e.g. using biotin / streptavidin) and incubated with a mixture of macromolecules (e.g. a cell extract).
  • the bound target molecule is detected by a specific activity test.
  • a prerequisite for such an approach is that a suitable test must exist with which the macromolecule sought can be detected.
  • Such a test can be used to identify oligo ligands which are e.g. are suitable as ligands for the affinity chromatography purification of a target macromolecule.
  • the binding or inhibition of at least partially purified target macromolecules can also be investigated directly.
  • a target macromolecule immobilized on a solid phase e.g. a target macromolecule immobilized on a solid phase and those
  • Oligoligands detected (eg via introduced groups, see Section 2), which show the highest affinity for the target macromolecule or exert the greatest inhibitory effect on the target macromolecule.
  • one or more oligo ligands can also be examined for their modulating properties of biological processes by means of an in vivo system.
  • B. A population of different oligo ligands can be systematically examined for suitable cytostatic and antibacterial properties using suitable test systems. Oligoligands with weak activity in such a test system could use the ones described above Strategies are combined until an oligoligand with sufficient activity / specificity is found.
  • monomeric building blocks of macromolecules are used for the in vitro selection of ligands, e.g. Amino acids, monosaccharides, nucleotides or short oligomers (e.g. dimers, trimers etc.), as well as such monomeric building blocks in modified form (e.g. glycosylated, sulfonylated, acetylated amino acids, phosphorylated sugars, methylated nucleotides).
  • ligands e.g. Amino acids, monosaccharides, nucleotides or short oligomers (e.g. dimers, trimers etc.)
  • modified form e.g. glycosylated, sulfonylated, acetylated amino acids, phosphorylated sugars, methylated nucleotides.
  • Ligand libraries are related to the size of the target molecule. For example, there are 20 natural amino acids but already 400 (20 2 ) dipeptides. So, for example, if the goal is an extensive collection of protein binding ligands of different specificity, the preferred one should
  • nuclease-resistant oligonucleotides by incorporating modified nucleotides (for example nucleotides which are chemically modified at the 2 V or 5 position) are described in patent application US08 / 117,991 with the title "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides ".
  • Patent US5637459 and patent application WO96 / 04403, both entitled “Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX”, describe methods in which oligonucleotide molecules from two or more parent libraries are mixed against known target molecules and linked together to form a new one To produce library.
  • the chimeric molecules of these libraries simultaneously bind either two epitopes of one target molecule or two epitopes in different target molecules.
  • the present invention differs from these as follows:
  • the individual ligands produced by SELEX or by other methods are selected by selection on building blocks of macromolecules. Such ligands bind extremely non-specifically to the respective macromolecules and
  • Binding sites cannot be called "epitopes”.
  • the present invention describes a new technique for identifying high affinity molecules.
  • the greatest advantage is that ligands can be produced for any or almost any macromolecular target substance, even if this target substance has not been characterized so far or is not in pure form.
  • the likelihood of finding ligands with affinity for the respective macromolecule class is significantly increased. This is particularly important when looking for substances with a biological activity in biological tests, since the probability of finding substances with the desired properties is greatly increased.
  • target molecules are: amino acids and modified (e.g. glycosylated, phosphorylated) amino acids (e.g. tyrosine, phosphotyrosine, glucothreonine), sugar (glucose, ribulose phosphate), nucleotides (e.g. thymine, GTP, methyl-GTP) and oligomers such building blocks.
  • the monomeric or oligomeric building blocks are immobilized on a gel matrix (eg Sepharose) and placed in a column.
  • binding buffer for example 10mM Hepes, 150mM NaCl, 5mM KC1, 5mM CaCl 2 ImM MgCl 2 , pH 7.2
  • binding buffer for example 10mM Hepes, 150mM NaCl, 5mM KC1, 5mM CaCl 2 ImM MgCl 2 , pH 7.2
  • the bound oligonucleotides are (for example with free target substance) eluted.
  • the oligonucleotides are RNA molecules, they are converted into cDNA using reverse transcriptase.
  • the eluted ssDNA or cDNA is amplified by PCR and the dsDNA product is converted to either ssDNA or RNA. The process is repeated until an affinity of Kd ⁇ 10 ⁇ 4 M is reached.
  • Example 2 Creation of ligands of higher A inity / specificity by linking two or more mono ligands against monomeric or oligomeric building blocks
  • aptamers against monomeric or oligomeric amino acids are linked to one another by ligation in a plasmid vector.
  • the DNA product of Aptamer 1 contains an interface for restriction enzyme 1 (eg barn HI) in the region of one of the two flanking primer binding sites and an interface for restriction enzyme 2 (eg Hind III) in the region of the other flanking primer binding site.
  • Aptamer 2 also contains an interface for restriction enzyme 2 (eg Hind III) in one flanking region and an interface for restriction enzyme 3 (eg Eco RI) in the other flanking region.
  • the aptamers are then fused to one another at the interface for restriction enzyme 2 using methods familiar to the person skilled in the art and ligated into a plasmid vector. Large amounts of the diaptamer can then be obtained by cleaving the plasmid and denaturing the inserted DNA.
  • the mono- or oligo ligands are then checked for binding to proteins in the form of a microtiter plate test. Individual mono- or oligo ligands are immobilized in a well of a microtiter plate (eg using biotin / streptavidin) and incubated with a mixture of macromolecules (eg a cell extract). The bound target molecule is detected by a specific activity test. In this way, different mono- or oligoaptamers can be identified which have high affinity for the respective target protein.
  • Example 3 Mono- or oligo ligands as ligands for affinity chromatography.
  • Mono- or oligo ligands are immobilized on a matrix and used for affinity chromatography.
  • the macromolecules are purified sequentially.
  • a partially cleaned or unpurified mixture of substances e.g. a cell extract
  • Bound macromolecules eg proteins
  • Example 4 Diagnostic applications: Specific detection from a subset of phosphorylated proteins. Molecules from a parent library of ligands against phosphotyrosine are linked to those from a parent library of ligands against other mono- or oligomeric amino acids. These diligands are e.g. contacted with a cell extract. The presence of the target molecule can be detected by labeling (e.g. direct or indirect labeling with e.g. alkaline phosphatase) the di- or oligo ligand. aD The sample e.g. a cell extract is in an SDS
  • Combinatorial oligo ligands are isolated that have an affinity for a protein that has a role in tumor development.
  • In vitro screening of the combinatorial ligand library isolates molecules that bind to the target protein and possibly also inhibit it. In this way, identified oligo ligands could be used to combat tumors. These can be receptors or tumor antigens (e.g. HER 2), intracellular proteins (e.g. telomerase, protein kinases), secreted proteins (e.g. matrix metalloproteinases, angiogenic factors), which play an essential role in tumor growth or in the development of metastases play. Binding of the di- or oligo ligands inhibits tumor cell proliferation or metastasis.
  • the suitable di- or oligo ligands are obtained by screening combinatorial libraries in in vitro assays, for example in a proliferation assay with tumor cells or in an enzyme assay (protease, telomerase, protein kinase).
  • Example 6 Antibiotic tests on bacterial cells (growth inhibition) or viruses. Di- or oligo ligands are also used to combat bacterial and viral infections. The Di or
  • Oligo ligands bind to surface antigens of bacteria or
  • Viruses and thus block the growth of microorganisms or the binding to host cells. Viruses bind to specific receptors and have suitable proteins for them.
  • Blocking these proteins can prevent the infection.
  • Combinatorial binding assays are used for screening
  • Oligo ligands identified. These molecules can then be used to neutralize the microorganisms. Other di or oligo ligands can be screened by screening libraries in

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Abstract

L'invention concerne un procédé pour produire des oligoligands présentant un pouvoir de liaison avec des macromolécules constituées de motifs éventuellement inconnus. Ce procédé est caractérisé par les étapes suivantes: a) sélection de ligands pouvant se lier à un motif monomère ou oligomère; b) sélection de ligands pouvant se lier à un deuxième motif monomère ou oligomère et éventuellement à un troisième ou un énième motif monomère ou oligomère; c) liaison chimique ou enzymatique des ligands sélectionnés selon a) et b), notamment avec introduction de molécules d'espacement; d) criblage des oligoligands obtenus, fondé sur leur pouvoir de liaison avec une ou plusieurs macromolécules; et e) isolation du ou des oligoligands selon d); et éventuellement f) production du ou des oligoligands selon e) au moyen d'une synthèse chimique ou enzymatique ou par clonage. La ou les macromolécules ne sont pas présentes lors des étapes a) à c).
PCT/EP1999/000120 1998-01-13 1999-01-12 Oligoligands a pouvoir de liaison WO1999036430A1 (fr)

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DE19980033T DE19980033D2 (de) 1998-01-13 1999-01-12 Oligoliganden mit Bindungsvermögen etc.
AU26163/99A AU2616399A (en) 1998-01-13 1999-01-12 Oligoligands with a binding capacity

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DE19800899.6 1998-01-13
DE1998100899 DE19800899A1 (de) 1998-01-13 1998-01-13 Oligoliganden mit Bindungsvermögen etc.

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PCT/EP1999/000120 WO1999036430A1 (fr) 1998-01-13 1999-01-12 Oligoligands a pouvoir de liaison

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012089207A3 (fr) * 2010-12-31 2012-10-04 Erdmann Volker A Composition pharmaceutique contenant de l'adn-l

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019813A1 (fr) * 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique
WO1995017413A1 (fr) * 1993-12-21 1995-06-29 Evotec Biosystems Gmbh Procede permettant une conception et une synthese evolutives de polymeres fonctionnels sur la base d'elements et de codes de remodelage
WO1995034575A1 (fr) * 1994-06-15 1995-12-21 S.P.I. Synthetic Peptides Incorporated Banque de peptides combinatoire et procede associe
WO1996004403A1 (fr) * 1994-08-02 1996-02-15 Nexstar Pharmaceuticals, Inc. Molecules chimeriques selectionnees par procede selex (evolution systematique de ligands par enrichissement exponentiel)

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019813A1 (fr) * 1990-06-11 1991-12-26 The University Of Colorado Foundation, Inc. Ligands d'acide nucleique
WO1995017413A1 (fr) * 1993-12-21 1995-06-29 Evotec Biosystems Gmbh Procede permettant une conception et une synthese evolutives de polymeres fonctionnels sur la base d'elements et de codes de remodelage
WO1995034575A1 (fr) * 1994-06-15 1995-12-21 S.P.I. Synthetic Peptides Incorporated Banque de peptides combinatoire et procede associe
WO1996004403A1 (fr) * 1994-08-02 1996-02-15 Nexstar Pharmaceuticals, Inc. Molecules chimeriques selectionnees par procede selex (evolution systematique de ligands par enrichissement exponentiel)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012089207A3 (fr) * 2010-12-31 2012-10-04 Erdmann Volker A Composition pharmaceutique contenant de l'adn-l

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AU2616399A (en) 1999-08-02
DE19800899A1 (de) 1999-07-15
DE19980033D2 (de) 2001-06-21

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