WO1999036430A1

WO1999036430A1 - Oligoligands with a binding capacity

Info

Publication number: WO1999036430A1
Application number: PCT/EP1999/000120
Authority: WO
Inventors: Alexander Gasch; Douglas Friday; Kornelia Berghof; Lutz MÜLLER-KUHRT
Original assignee: BioteCon Gesellschaft für Biotechnologische Entwicklung und Consulting mbH
Priority date: 1998-01-13
Filing date: 1999-01-12
Publication date: 1999-07-22
Also published as: DE19800899A1; DE19980033D2; AU2616399A

Abstract

The invention relates to a method for producing oligoligands which can bind to macromolecules with an optionally unknown composition of building blocks. The inventive method is characterised by the following: a) the selection of ligands against a mono- or oligomeric building block; b) the selection of ligands against a second mono- or oligomeric building block and optionally against a third to nth mono- or oligomeric building block; c) the chemical or enzymatic linking of the ligands selected according to a) and b), especially with the insertion of spacer molecules; d) the screening of the oligoligands obtained for binding to one or several macromolecules to which the oligoligands should be able to bind; and e) the isolation of the binding oligoligand(s) according to d) and optionally, f) the production of the binding oligoligand(s) according to e) by chemical or enzymatic synthesis or by cloning. The macromolecule(s) are not present in steps a) to c).

Description

Oligoligands with binding capacity etc.

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.

Definitions In the present patent application and in particular for the invention described below, the following terms are used throughout as described here:

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.

Furthermore, an oligonucleotide according to the invention can be characterized in that it is single-stranded or has a complementary strand.

Furthermore, 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.

Furthermore, 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. This activity can be, for example, the affinity for a target molecule, the inhibition of an enzyme or else enzymatic 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:

1) the incorporation of complementary bases at the 5 'end of one aptamer and at the 3' end of the other aptamer, which allow hybridization of the two aptamers,

2) the incorporation of interfaces for restriction enzymes which produce complementary ends, which enables the ligation of the double-stranded aptamers after restriction cleavage,

3) the incorporation of chemical groups at the ends of the mono ligands, which allow a direct or indirect connection of the mono ligands, and

4) a direct fusion during the synthesis of the mono ligands. At a certain distance between the domains of one

To achieve 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.

background

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

Target substances. 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.

So far, however, 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.

From WO 95/34575 AI a process for the production of oligomeric substances with high affinity for macromolecules is known. By gradually optimizing each position of the oligomer, the sequence is successively identified from sets of oligomer libraries. To use this method, however, the macromolecule is required to be present. Another method of making functional

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

Building blocks. However, this method does not open a universal route for the production of ligands for any macromolecule.

From US-A-5 637 459 and WO-A-96/04403 it is known to select ligands against a first and a second target molecule or ligands against different epitopes of the same target molecule in the presence of the target molecules and then to link them. It is also known from Science, 278 (1997) 497-499 to select ligands with affinity for epitopes of the same target molecule and to link them to one another. These processes also require the presence of the target molecule (s) to produce affinity ligands.

Description of the invention

According to one embodiment, 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,

the macromolecule (s) not being present in stages a) to c).

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.

Another embodiment of the invention relates to the use of one or more of the invention

Oligo ligands in affinity chromatography with the

Steps: a) immobilization of the oligo ligands on a gel matrix, b) contact of a mixture of substances with the immobilized oligo ligands, c) removal of non-specifically bound substances and d) elution of the bound substances.

Furthermore, 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

(in vitro) or on / in living cells, tissues or organisms

Detect (in vivo).

Furthermore, 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.

Finally, 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.

The invention described here is therefore based on the principle that ligands against various building blocks are first selected in vitro by, for example, one of the following methods and then combined with one another:

1. "Systematic Evolution of Ligands by Exponential Evolution" (SELEX) (Tuerk, C. & Gold, L .; 1990): Ligands for almost everyone Possible target substances (e.g. proteins, amino acids, peptides, nucleotides, carbohydrates) (L. Gold; 1995) are created from a starting mixture of 10 ¹³ - 10 ¹⁵ highly degenerate single-stranded nucleic acids or nucleic acid analogues (approx. 20 - 100 degenerate bases) by binding to one

Target substance 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. The PCR is controlled by constant

Primer binding sites that flank the randomized bases enables. One of these 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. Alternatively, the amplified double-stranded DNA (dsDNA) 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. From the matrix on which the target molecule is immobilized, with closely related target molecules or with the "wrong" chemical groups (such as α-amino group of an amino acid), sequences can be removed which have undesirable affinity for these groups / target molecules. Individual aptamers can finally be isolated and characterized by cloning.

A big problem when using Oligonucleotide ligands in biological fluids is that in some cases (eg in blood) they are broken down very quickly by 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 ). An interesting alternative is the selection of aptamers against naturally non-occurring or only occasionally occurring optical isomers of target molecules (eg D-amino acids, L-sugar, L-nucleotides) and the subsequent synthesis of the corresponding L-aptamers. These so-called Spiegelmers bind the natural target substances and are considerably more stable in biological fluids (Nolte, A., Klußmann, S., Bald, R., Erdmann, V. & Fürste, J; 1996).

2. Screening of peptide libraries: a) 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. A An alternative is the "Synthetic Peptide Combinatorial Library" (SPLC) method (Houghten, R., Pinilla, C. et al; 1991). In this case, 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. These, for example, 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 process is repeated until the amino acid sequence of the peptide is defined. b) "Phage Display Libraries": Peptides are expressed on the surface of bacteriophages (Scott, J. & Smith, G .: Science; 1990). A short randomized DNA sequence coding for approx. 6 to 7 amino acids or a longer peptide or protein is inserted at the end of the "minor coat protein" locus (Gen III) of a filamentous phage. The corresponding peptide is expressed as part of the coat protein. Each phage in such a library expresses a defined peptide. The expressed peptides can be screened for affinity or activity and the phages with the desired property can be isolated. To determine the peptide sequence (s), DNA is isolated from the phages and then sequenced. After translating the DNA sequence into amino acid sequence, the free peptide can be synthesized for other uses or it can be used directly on the surface of the phage.

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. To create such a library, monoligands against building block molecules (eg monosaccharides, amino acids, nucleotides and short oligomers) are first selected in vitro. 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:

1. Isolation of affinity ligands for building block molecules First, building block molecules (eg monosaccharides, glycosylated amino acids, nucleotides and short oligomers) are immobilized on a solid phase, optionally using a suitable spacer molecule. Suitable matrices are, for example, activated Sepharose beads or the wells of a microtiter plate (MTP). 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. This increases the chance that the ligands are specific for the respective building block and that they bind to the respective building block even if this is part of an oligomeric or macro-molecule. 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.

2. Preliminary tests 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) are incubated with the immobilized substance. After a washing step, bound ligands are detected via the label. If the target substance is not available in pure form, but only in a mixture, such as a protein extract, 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). However, there is also the possibility of linking mono ligands against building blocks of different classes. In this way, 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

Denaturation of the inserted DNA can be obtained. The creation of oligoaptamers by cloning has the advantage that the sequence of the monoaptamers need not be known, and that spacer sequences of variable length and sequence can easily be inserted during the cloning. b) Cloning (for peptide ligands): If the

If amino acid sequences of two or more mono ligands are known, these can be translated into DNA sequence and fused to one another in any way in an expression cassette. This procedure allows the expression of large amounts of the respective fusion proteins and the insertion of spacer sequences of variable length and sequence. c) Chemical synthesis: Two or more mono ligands, the sequence of which is determined beforehand, can be obtained directly by chemical synthesis, while maintaining the class-specific bonds (eg phosphodiester bond in aptamers), are fused together. This procedure has the advantage that groups can be inserted directly in the synthesis, which are a direct or indirect purification, detection or

Allow coupling (e.g. to cytotoxic agents) of the resulting oligo ligand. As with a) and b), there is also the possibility of introducing spacer molecules of variable size and sequence. Chemical coupling: In the synthesis of the mono ligands, reactive groups are introduced, preferably at the ends of the mono ligands, which enable the covalent linking of two or more mono ligands via an at least bifunctional spacer after the synthesis. Alternatively, two or more mono ligands can be used sequentially directly with an at least bifunctional spacer. Both methods result in the general structure; see. the following scheme.

These syntheses can be carried out in solution or on a solid phase and allow the combinatorial synthesis of defined conjugates in high diversity using standard techniques.

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

Diversity of different spacer molecules can be introduced. The latter in turn results in different orientation and removal of the linked mono ligands. Together with the flexibility of the spacer molecule introduced and its polyfunctionality, these are important parameters with which the specificity of the resulting oligo ligand is modulated can be.

4. 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.

Alternatively, the binding or inhibition of at least partially purified target macromolecules can also be investigated directly. For this, 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. Alternatively, one or more oligo ligands can also be examined for their modulating properties of biological processes by means of an in vivo system. For example, 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.

In the invention described here, 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). The complexity of the possible

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

Strategy to be the selection of ligands against monomeric amino acids. From the limited number of mono ligands (max. 20), an immense variety of oligo ligands with different binding specificities can be obtained through different covalent linkages or through the introduction of variable spacer molecules. Compared to the alternative strategy - the selection of ligands against eg oligopeptides - the workload is greatly reduced.

Structure of possible conjugates: 5'— ► 5 '

The SELEX method for the production of oligonucleotide ligands with high affinity for target molecules is described in patents WO91 / 19813 and US5270163, both entitled "Nucleic Acid Ligands".

Methods for the production of 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".

^• With the present invention it is possible to produce ligands for almost every macromolecule, even those that are not yet fully characterized or isolated. The mono ligands of the parent libraries are relatively unspecific and the specificity is only achieved through the combinatorics the oligo ligands.

After the individual ligands have been linked, no further selection, e.g. by SELEX or other processes. On the other hand, e.g. the chimeras aptamers in the above Invention as a source of new complexity in further rounds of SELEX.

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. In addition, compared to other combinatorial libraries, 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. Furthermore, the modular

The structure of the oligo ligands described suggests that affinity ligands can also be found against epitopes that are not accessible by conventional methods.

Example 1: Isolation of Apta eren using SELEX

Aptamers are selected against different building blocks: 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. After degenerate, single-stranded oligonucleotides have been applied to the column, they are washed intensively with binding buffer (for example 10mM Hepes, 150mM NaCl, 5mM KC1, 5mM CaCl ₂ ImM MgCl ₂ , pH 7.2), and the bound oligonucleotides are (for example with free target substance) eluted. If 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

If the target macromolecule is a protein, for example, 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) is applied. Bound macromolecules (eg proteins) are eluted after a washing step and examined for their activity and their degree of purity. By using further mono- or oligo ligands, which were identified in Example 2, one or more further purification step (s) of the macromolecule can be carried out if necessary.

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

Polyacrylamide gel separated and then blotted onto a membrane. bD The membrane is incubated with the labeled di or oligo ligand. cD The binding of the di- or oligoligand to proteins on the membrane is shown by a suitable detection reaction (depending on the label introduced, e.g. luminescence).

Example 5: Antitumor applications

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. By

Blocking these proteins can prevent the infection.

Combinatorial binding assays are used for screening

Ligand libraries used and binding di- or

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

Growth assays can be obtained.

Literature Hajduk, J.P., Meadows, R.P. & Fesik, S.W .; 1997 Science 278,

497-499

Tuerk, C. & Gold, L .; 1990: Science 249, 505-510

Gold; 1995: Annu. Rev. Biochem. , 64, 763-797

Jellinek, D; 1995: Biochemistry, 34, 11363-11372 Eaton, B & Picken, W; 1995: Annu. Rev. Biochem., 64, 837-863

Nolte, A., Klussmann, S., Bald, R., Erdmann, V. & Fürste, J.;

1996: Nature Biotechnology, 14, 1112-1115 Houghten, R., Pinilla, C. et al; 1991: Nature, 354, 84-86 Scott, J. & Smith, G; 1990: Science, 249, 386-390, Sambrook, EF Fritsch, T. Maniatis, Molecular Cloning - A laboratory Manual, Cold Spring Harbor Press, ^2nd Ed, 1989 Furka, A., Sebestyen, F., Asgedum, M.. & Dibo, G; 1988: Highlights of Mod.Biochem: Proceedings of the 14 ^h Int. Congress of Biochemistry, 5, 47

Claims

claims

1. A process for the preparation of oligo ligands with

Binding capacity to macromolecules of possibly unknown structure from building blocks, characterized by: a) selection of one or more ligands against a mono- or oligomeric building block, b) selection of one or more further ligands against a second mono- or oligomeric building block and optionally one or more third to nth ligands against a third to nth mono- or oligomeric building block, c) chemical or enzymatic linkage 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 macromolecule (s) to which the oligoligands are said to have binding capacity, and e) isolation of the binding oligoligand according to d) and optionally f) production of the (the ) binding oligo ligands according to e) by means of chemical or enzymatic synthesis or by means of cloning,

the macromolecule (s) not being present in stages a) to c).

2. A process for the preparation of oligo ligands with the desired biological activity, characterized by: a) selection of one or more ligands against a mono- or oligomeric building block, b) selection of one or more other ligands against a second mono- or oligomeric building block and optionally one or several third to nth ligands 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.

3. A method according to claim 1 or 2, characterized in that the linked ligands are RNA oligonucleotides.

4. A method according to claim 1 or 2, characterized in that the linked ligands are single-stranded DNA oligonucleotides.

5. A method according to claim 1 or 2, characterized in that the linked ligands are peptides.

6. A method according to any one of the preceding claims, characterized in that the building blocks are amino acids.

7. A method according to any one of claims 1 to 5, characterized in that the building blocks are dipeptides.

8. A method according to any one of claims 1 to 5, characterized in that the building blocks are tripeptides.

9. A method according to any one of claims 1 to 5, characterized characterized in that the building blocks are monosaccharides.

10. A method according to any one of claims 1 to 5, characterized in that the building blocks are disaccharides.

11. A method according to any one of claims 1 to 5, characterized in that the building blocks are trisaccharides.

12. A method according to any one of claims 1 to 5, characterized in that the building blocks are mononucleotides.

13. A method according to any one of claims 1 to 5, characterized in that the building blocks are dinucleotides.

14. A method according to any one of claims 1 to 5, characterized in that the building blocks are trinucleotides.

15. A method according to any one of claims 1 to 5, characterized in that the building blocks are fatty acid molecules.

16. Oligo ligands obtainable by a process according to one of claims 1 to 15.

17. Oligo ligands obtainable by a process according to one of claims 1 to 15 and subsequent production in larger

Amount, in particular by a) chemical synthesis, b) in tro tro transcription by means of purified RNA polymerases, c) in tro tro amplification, in particular by means of PCR, LCR and / or 3SR, and / or d) cloning, in particular in E. coli .

18. Oligo ligands according to claim 16 or 17, characterized 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, contain one or more radioactive groups, colored groups, fluorescent groups, groups for immobilization on a solid phase, groups for the uptake in 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 other modifying agents known per se or modified groups have a nucleic acid-like structure.

19. Oligo ligands according to claim 16 or 17, characterized in that they consist entirely or partially of PNA.

20. Oligo ligands according to claim 16 or 17, characterized in that they are protected against nuclease degradation, in particular by incorporating modified nucleotides which are modified in particular by 2'-amino, 2'-fluoro or 2'-0-methyl groups.

21. Oligo ligands according to claim 16 or 17, characterized in that they consist entirely or partially of L-RNA or L-DNA.

22. Use of one or more oligo ligands according to one of claims 16 to 21 in affinity chromatography with the steps: a) immobilization of the oligo ligands on a gel matrix, b) contact of a substance mixture with the immobilized oligo ligands, c) removing non-specifically bound substances and d) elution of the bound substances.

23. Use of one or more oligoligands according to one of claims 16 to 21 for the detection of macromolecules of unknown structure from building blocks, characterized in that oligoligands and macromolecules are incubated and oligoligands bound after washing steps are detected.

24. Use according to claim 23, characterized in that macromolecules in cell extracts or in tissue sections (in vi tro) or on / in living cells, tissues or organisms (in vivo) are detected.

25. Use of the oligo ligands according to one of claims 16 to 21 for therapeutic purposes.

26. Therapeutic use of oligo ligands according to one of claims 16 to 21 with antibacterial activity.

27. Therapeutic use of oligo ligands according to one of claims 16 to 21 with antiviral activity.

28. Therapeutic use of oligo ligands according to one of claims 16 to 21 with cytostatic activity.

29. Therapeutic use of oligo ligands according to one of claims 16 to 21 by introduction into infected cells, tissues or organisms for combating infections or tumors.