CA2374438A1 - Detection system for studying molecular interactions and its preparationand use - Google Patents

Abstract

The invention relates to a detection system comprising: a) a support (components (a) and (b)), at least one detection unit (component (b)), preferably a nucleic acid, bonded to the support, wherein said detection unit (component (b)) includes a region (A) having a constant structure and a region (B) adjacent to region (A) having a variable structure. The invention also relates to a method for separating individual components in a sample, wherein at least one detection unit (component (b)) that is bonded to the support is bonded to at least one component of the sample under appropriate conditions.
The invention further relates to the utilization of the inventive detection system and method for locating and/or identifying or characterizing nucleic acids and/or proteins in a sample or for locating and/or identifying cellular or artificial binding partners.

Description

WO 00!71749 PCT/EP00/04791 Detection system for studying molecular interactions and its preparation and use Description The present invention relates to a detection system (figure 1 ) comprising a support (component (a)) and a detection unit component (b) which is bound to the support and which comprises a constant region (A) and an adjacent variable region (B), and a component (c) which includes a region complementary to region (A) and (B) and an effector unit bound thereto, where appropriate via a suitable linker; the invention relates furthermore to a method for preparing such detection systems and to methods for studying molecular interactions by using the detection systems.
In a human cell generally up to approx. 30 000 genes which characterize the present state of the cell are active. The state of a cell may represent, for example, increased cell division in the case of a cancer cell or generally modified metabolic activities in the diseased state. The activity of a gene may be determined via its mRNA as transcriptional product or via the corresponding protein as translational product. The mRNA or protein profile of a cell thus reflects its present state.
Very recently, the characterization of a healthy or diseased cell on the basis of its protein profile has become very important for investigating diseases and finding pharmacologically active compounds. Various methods which make it possible to characterize the protein profile of a cell have already been developed.
In order to directly characterize the protein profile in a cell, the cellular proteins have to be fractionated. An efficient method is, for example, a two-dimensional polyacrylamide gel electrophoresis (2DE). In this, the proteins are fractionated in the first dimension according to their isoelectric point (1P) and in the second dimension according to their molecular weight.
The proteins are then visualized in the 2D gel by staining, approx.
1 000-2 000 proteins being stained as spots in typical 2D gels. The result is a protein pattern or profile which is significant for each cell and which reflects the particular state of the cell at the protein level. Each protein of a CONFIRMATION COPY

cell has on the 2D gel a position specific for said protein, so that it is possible to produce a "proteome map" for the cell and, consequently, for the whole organism. In order to determine the molecular causes of cellular modifications, for example during disease processes, the protein profiles of healthy and diseased cells are compared in order to detect possible differences. This comparison may be carried out, for example, in an auto-mated way by means of suitable computers (see, for example, Wilkins, M.R. et al. (eds.) Proteome Research: New Frontiers in Functional Genomics. Springer Verlag, Heidelberg (1997) or Humphrey-Smith, I. et al.
(1997) Electrophoresis 18, 1216-1242). This method, however, has the substantial disadvantage that for further analysis of the detected proteins it is necessary to sequence said proteins at the protein level, and this procedure is at the moment still time-consuming and expensive.
Alternative methods for determining possible differences in the protein profile of a cell use "arrays", i.e. matrix systems. Arrays are arrangements of immobilized detection species which play an important part in the simul-taneous determination of analytes in analytics and diagnostics. Examples are nucleic acid arrays (see, for example, Southern et al. Genomics (1992) 13, 1008; U.S. Patent No. 5,632,957, W097/27317 or EP-A1-0 543 550) or peptide arrays (Fodor et al., Nature 1993, 364, 555). W096/01836, for example, describes an array of DNA molecules of different sequence, which served to detect gene sections and thus, for example, led to the diagnosis of pathogenic bacteria. The patent publications U.S. 5,605,662, W096101836, U.S.5,632,957 and W097/12030 describe semiconductor chips and methods which can be used to carry out specific binding reactions of biological compounds such as nucleic acids or proteins to specific addressable sites in the form of an array in an electronically controllable way. For example, nucleic acids in a sample are hybridized to a nucleic acid array on a semiconductor chip with the aid of an electric field and then nucleic acids which are not bound or bound unspecifically are removed by simply reversing the polarity of the electric field. It is possible here to detect a mismatch of a single base pair through precise adjustment of the electrical field strength.
It was an object of the present invention to provide a detection system which is assembled from a population of pairing system-effector fusion molecules and which can assist in identifying and characterizing said population. Another object of the present invention is a method for studying effector-binding partner interactions.
Surprisingly, it has now been found that an appropriate collection of detection units comprising in each case a region (A) having a constant sequence and a region (B) adjacent to region (A) and having a variable sequence is suitable, by means of specific hybridization to pairing system-effector fusion molecules, for characterizing the effector profile and then identifying effector-binding partner interactions.
The present invention relates to a detection system (figure 1 ) comprising i) a support (component (a)) and ii) at least one detection unit (component (b)), preferably a pairing system, bound to the support, wherein said detection unit comprises a region (A) having a constant sequence and a region (B) adjacent to region (A) and having a variable sequence, and iii) a pairing system-effector fusion molecule (component (c)) linked thereto and comprising a sequence complementary to the detection unit (component (b)).
Preferably, a plurality of detection units (components (b)) form on the support (component (a)) an array in which each array position can be assigned to a defined variable region B of component (b). Starting from the different detection units on the array surface, the particular position-specific detection systems (components (a)-(c)) can be assembled.
The term detection unit (component (b)) means according to the present invention nucleic acids or analogs thereof which in particular contain pentoses, preferably a pentopyranose or pentofuranose. Generally, the pentose is selected from a ribose, arabinose, lyxose or xylose. Examples of suitable nucleic acids or analogs thereof are DNA, RNA, in particular mRNA or p-RNA (pyranosyl RNA, see for example W099/15539), aminocyclohexyl nucleic acids (CNA, see for example W099/15509), peptidic nucleic acids (PNA, see, for example, W092/20702) or Science (254), 1999, 1497-1500) or non-helical supramolecular nanosystems, as described, for example, in W098I25943.
In this connection, the detection unit (component (b)) hybridizes specifically with the regions of component (c) which are complementary to its region A
and B. Examples of component (c) are in particular nucleic acid-protein acceptor derivatives, preferably nucleic acid-puromycin derivatives, or fusagenes such as nucleic acid-protein fusion molecules, in particular nucleic acid-puromycin-protein fusion molecules. Particular preference is given to fusion molecules made of the nucleic acids RNA and DNA, preferably fused to puromycin and a protein. The term protein in accordance with this invention includes proteins and protein structures which are synthesized from posttranslationally or chemically modified amino acids such as, for example, glycosylated, phosphorylated, halogenated, lipid-esterified amino acids, etc., and also shorter peptidic amino acid sequences.
The term "constant sequence" (region A) means according to the present invention a sequence, preferably a nucleic acid sequence on RNA, DNA or cDNA basis, or else sequences of nucleic acid analogs such as, for example, a p-RNA, CNA or PNA sequence, which is identical in all detection units (components (b)).
The term "variable sequence" (region B) means according to the present invention a sequence, preferably a nucleic acid sequence, whose sequence in the particular regions B is different but known. The variable sequence of a nucleic acid, for example, is its nucleic acid sequence formed by random events or permutation of the individual nucleotides.
The length of region (A) andlor region (B) is, preferably independently of one another, approx. 5 to approx. 80 nucleotides, preferably approx. 5 to approx. 30 nucleotides, and in particular approx. 10 to approx. 30 nucleotides for region (A) and in particular approx. 7-8 nucleotides for region (B), the nucleotides in a particularly preferred embodiment being deoxyribonucleotides (c~, ribonucleotides (r) or 2-hydroxymethylribo-nucleotides (hmr). In the embodiments below, the nucleic acid sequences are stated without their specific backbones. The nucleic acid sequences stated therefore comprise iri any case the embodiments (c~, (rj and (hmrj.
In addition, RNAs according to the present invention may be synthesized not only from ribonucleotides but also from 2-hydroxymethylribo-nucleotides.
A protein profile of a protein population, for example a protein population of a cell, is successfully characterized, for example, by selecting proteins by means of suitable nucleic acid-protein fusions. W098I31700, for example, describes a system in which a protein acceptor, for example a puramycin, is bound to the nucleic acid, preferably mRNA, via a suitable linker. This makes it possible, shortly before mRNA translation into the corresponding protein has finished, to bind the synthesized protein covalently to its encoding mRNA and thus to characterize it in more detail. The linker whose sequence is known is particularly advantageously suited as binding region to region (A) of the nucleic acid of the invention. It is possible, for example, to use a polyT~5 strand as region (A) for binding to a linker of a nucleic acid-protein fusion, which has the sequence A2~CC, for example.
In order to generate the nucleic acid-protein fusion, the linker may contain a protein acceptor, for example a tRNA amino acid analog such as puromycin which is particularly suitable. Examples of comparable systems which can be used for the present invention are described in DE19646372C1, W098/16636, W091/05058, U.S.5,843,701, W093/03172 or W094/13623.
In order to be able to cover all the different components of a population of pairing system-effector fusion molecules such as, for example, nucleic acid fusions, in particular nucleic acid-puromycin-protein fusions (fusagenes), the variable sequences of regions (B) of the detection units (component (b)) must contain all possible permutations. The preferably used length of the variable sequence (region B) depends in this case on the complexity of the population, which is, as experience shows, relatively low in a prokaryotic cell and relatively high in a eukaryotic cell. In a human cell, for example, approx. 30 000 genes are active so that in the case of an array containing all pairing system-effector fusion molecules region B
nucleic acid sequences having a length of 7-8 nucleotides in permutated sequence are sufficient in order to cover all active genes of a human cell.
The number of possible permutations for an n-mer oligonucleotide is, as is known, 4n, where n is the number of nucleotides of the oligonucleotide. For identification of all active genes of a human cell, therefore, preference is given to a nucleic acid bound to a support, which has the following formula:

3'-(X)~_g-region (A)-5', where X is any nucleotide selected from adenosine, guanosine, cytosine, thymidine or uracil.
The term "support" means in accordance with the present invention material, in particular chip material made of semiconductors, which is present in solid or else gel-like form. Examples of suitable support materials are ceramic, metal, in particular semiconductors, noble metal, glasses, plastics, crystalline materials or thin layers of the support, in particular of said materials, or (bio)molecular filaments such as cellulose and structural proteins. Preference is given to support systems as described in EP-A1-0543550 or W099/15893 and in particular in U.S.5,605,662, W096/01836, U.S.5,632,957 or W097/12030, since these can be used for producing semiconductor chips which can be used in carrying out specific binding reactions of nucleic acids to specific addressable sites in the form of an array in an electronically controllable way. This makes it possible to hybridize in a particularly advantageous way the nucleic acids population of a sample, which is to be fractionated specifically, to a nucleic acid array with the nucleic acids of the invention on the semiconductor chip with the aid of an electric field and then to remove nucleic acids which are not bound or bound unspecifically by simply reversing the polarity of the electric field. A particularly preferred embodiment of the detection system of the invention is therefore an electronic chip.
The support is generally loaded covalently, more or less covalently, supramolecularly or physically as well as magnetically (A.R. Shepard et al.
(1997) Nucleic Acids Res., 25, 3183-3185, No. 15), in an electrical field or via a molecular sieve, preferably according a method as described in U.S.5,605,662, W096/01836, U.S.5,632,957, W097/12030 or W099/15893.
The invention further relates to a method for preparing a pairing system-effector fusion molecule array, for example a fusagene array, comprising the following process steps:

i) preparation of an array by attaching the detection units (components (b)) comprising a region (A) having a constant sequence and a region (B) adjacent to region (A) and having a variable sequence to a support (component (a)), wherein each array position can be assigned to a detection unit having a particular region B, and ii) hybridization of the detection units (components (b)) with pairing system-effector fusion molecules (components (c)) comprising a sequence complementary to the detection unit (component (b)).
The preparation of components (c)) such as, for example, generation of a fusagene library (components (c)) may be carried out following W098/31700 or Roberts and Szostak (Proc. Natl. Acad. Sci. USA, 1997).
In the method of the invention, the array is prepared by binding more than one detection system comprising component (b) and component (c) spatially separated to the support (component (a)), for example in separate cells. In this connection, the detection units (components (b)) may be applied to the support surface directly, for example via adsorption or via spacers known to the skilled worker. Specific preparation methods are described in more detail, for example, in EP-A1-0543550 or W099/15893 and in particular in U.S.5,605,662, W096/01836, U.S.5,632,957 or EP-B 1-0373203, W 097/12030 or W 098/31700.
Preference is given to arrays which include all possible permutations in region B of the detection units (components (b)).
Preferred embodiments of the method of the invention include detection systems which were already described in more detail hereinbefore. In a particularly preferred method of the invention, the detection systems are assembled on an electronic chip, the sample component, for example a nucleic acid fusion, being advantageously bound with the aid of an electrical field to the detection unit (component (b)) bound to a support, for example a nucleic acid region of component (c) with complementary sequence. A detailed description of an electronic chip of this kind and its application are described, fOr example, in EP-A1-0543550 or W099/15893 and in particular in U.S.5,605,662, W096/01836, U.S.5,632,957 or °

- $ -W 097/12030.
In a preferred embodiment, for example, a suitable nucleic acid linker, for example an A2~CC, is in a first step fused to an mRNA population of a sample preferably chemically or with the aid of a suitable ligase, for example a T4 DNA ligase, the mRNA-linker fusions are bound bound in a second step to nucleic acids of the detection unit (component (b)) having the exemplary formula 3'-(X)~_g-(T~5)-5', preferably with the aid of an electrical field. Where X is any nucleotide selected from adenosine, thymidine, uracil, guanosine or cytosine. In a further step, where appropriate, nucleic acids which are not bound or bound unspecifically are removed, preferably with the aid of an electrical field with reverse polarity and of lower field strength than in the first step.
The invention further relates to a method for fractionating and identifying pairing system-effector fusion molecules, preferably from complex mixtures (Figure 2), comprising the following process steps:
i) preparation of a pairing system-effector fusion molecule library (component (c) library), preferably a fusagene library, ii) hybridization of the pairing system-effector fusion molecules (components (c)) on an array comprising component (a) and components (b) whose region (B) includes all possible permutation, each permutation being specifically assigned to one array position, iii) identification of array positions at which a complex of component (b) and component (c) has formed, iv) characterization of the complexes of component (b) and component (c) identified under iii).
The preparation of components (c)) such as, for example, generation of a fusagene library (components (c)) may be carried out following W098/31700 or Roberts and Szostak (Proc. Natl. Acad. Sci. USA, 1997).
A particular advantage of this method is that the specific construction of the detection units (components (b)) makes it possible to identify also pairing _g_ system-effector fusion molecules, in particular fusagenes, in which the region complementary to component (b) is unknown. This is achieved by on the one hand by the length of the pairing system, which allows for stringent hybridization, and on the other hand by the length of region B by which the hybridization specificity (which is equivalent to fractionation of components (c)) can be adjusted.
The complexes formed during hybridization of the detection units (component (b)) and the particular components (c) can be detected via labeling of components (c). Moreover, when using an electronic chip (component (a)), for example, the complexes can be identified via redox processes in the vicinity of or at the electrode or via physical parameters such as measurements of impedance and direct current or, in the case of a gold chip, for example via surface plasmon resonance measurement.
Generally, the pairing system-effector fusion molecules (components (c)) are eluted sequentially from the individual identified complexes with breaking the hybridization to component (b) by, for example, increasing the temperature, varying the local salt concentration or, preferably, by modulating the electronic binding parameters. These fusagenes are then characterized by molecular biological methods known to the skilled worker, such as RT-PCR, for example, and subsequent DNA sequencing.
The method is preferably used for analyzing fusagene libraries. It is possible, for example, to analyze and/or compare, via the nucleic acid and/or protein profile determined in this way, the state of various cells or tissue samples. In addition, it is possible to detect whether a specific nucleic acid or a specific protein is present in a population. Possible expression states of various cells may also be identified.
The invention further relates to a method for identifying interactions of one or more binding partners (components (d)) which have an affinity for specific effector units of components (c) (Figure 3).
"Affinity" in accordance with the present invention means that a sample component specifically interacts with the effector unit of component (c).
Such interactions may be' in particular specific protein-protein and/or protein-nucleic acid bonds and also the specific binding between a chemical active substance and a protein effector.
The method comprises the following process steps:
i) incubation of a pairing system-effector fusion molecule array with a substance mixture to be analyzed which comprises at least one component (d), ii) identification of array positions at which a complex of component (b), component (c) and component (d) has formed, iii) characterization of the complexes of component (b), component (c) and component (d) identified under ii).
Generally, the complex formed is detected via a labeling [lacuna]
components (d). Moreover, when using an electronic chip (component (a)), for example, the complexes can be identified via redox processes in the vicinity of or at the electrode or via physical parameters such as measurements of impedance and direct current or, in the case of a gold chip, for example via surface plasmon resonance measurement.
Generally, subcomplexes of component (c) and component (d) are eluted sequentially from the individual identified complexes with breaking the hybridization to component (b) by, for example, increasing the temperature, varying the local salt concentration or, preferably, by modulating the electronic binding parameters. The components (d) are then characterized via analytical methods known to the skilled worker.
Examples of methods of labeling nucleic acids, proteins and/or chemical active substances are chemical and/or phyisicochemical, enzyme, protein, radioactive isotope, non-radioactive isotope, toxin, chemiluminescent and/or fluorescent labeling.
Examples of chemical substances known to the skilled worker, which are suitable for chemical labeling according to the invention, are: biotin, fluorescein isothiocyanate (FITC) or streptavidin.
Examples of inventive chemical modifications known to the skilled worker are the transfer of methyl, acetyl, phosphate and/or monosaccharide groups.
Examples of enzymes known to the skilled worker, which are suitable for enzyme labeling according to the invention, for example in the form of an ELISA, are: malic hydrogenase, staphylococcus nuclease, 0-5-steroid isomerase, alcohol dehydrogenase, a-glycerolphosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, ~i-galactosidase, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase, luciferase or acetylcholine esterase.
Examples of proteins or protein fragments known to the skilled worker, which are suitable for a protein labeling of the invention, are an N- or C-terminal (HIS)6, a myc, a FLAG, E tag, Strep tag, a hemagglutinin, glutathione transferase (GST), intein with a chitin, maltose binding protein (MBP) or antibodies or antigen-binding parts of antibodies, for example Fv fragments.
Examples of isotopes known to the skilled worker, which are suitable for a radioactive isotope labeling of the invention, are H, I, I, P, P, 355, 14C, 5lCr, 57To, 58Co, 59Fe, 75Se, 152Eu, 90Y, 67Cu, 217Ci, 211At, 212Pb, 47Sc or 109Pb.
Examples of isotopes known to the skilled worker, which are suitable for a non-radioactive isotope labeling of the invention, are : 2H or 13C.
Examples of toxins known to the skilled worker, which are suitable for a toxin labeling of the invention, are: diphtheria toxin, ricin or cholera toxin.
Examples of chemiluminescent substances known to the skilled worker, which are suitable for a chemiluminescent labeling of the invention, are:
luminol labeling, isoluminol labeling, aromatic acridinium ester labeling, oxalic ester labeling, luciferin labeling, acridinium salt labeling, imidazole labeling or aequorin labeling.
Examples of fluorescent substances known to the skilled worker, which are suitable for a fluorescent labeling of the invention, are: 152Eu, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, Cy3, CyS, green fluorescent protein (GFP) and its variants (YFP, RFP) or fluorescamine.
Bound nucleic acids may also be identified or characterized further via EST
(expressed sequence tags) databases, Northern blot on the detection system of the invention or by sequencing on the detection or after specific release, preferably after prior amplification by means of PCR, RT-PCR or cloning.
The skilled worker is familiar with further labeling or analytical methods not listed here such as, for example, mass spectrometry, NMR spectroscopy, calorimetry or potentiometry, which may also be used for labeling in accordance with this invention.
With the aid of the present invention, it is in a particularly advantageous way possible to identify, characterize and monitor the physiological state of a cell and biological processes of a cell.
The present invention therefore also relates to the use of a detection system of the invention, a method of the invention or an inventive detection unit comprising a region (A) having a constant structure and a region (B) adjacent to region (A) and having a variable structure for finding and/or identifying andlor characterizing at least one sample component (component (d)), in particular nucleic acids and/or proteins of a sample, or for finding and/or identifying cellular or artificial binding partners, preferably proteins, peptides, nucleic acids, chemical active substances, preferably organic compounds, pharmacologically active compounds, crop protection agents, toxins, in particular poisons, carcinogenic and/or teratogenic substances, herbicides, fungicides or pesticides.
In this connection, it is of great interest to study interactions of transcription factors, repressors or enhancers or to study the interactions of enzymes such as, for example, kinases, phosphatases, GTPases, esterases, glycosylases, lipases, oxidases, reductases, hydrolases, isomerases or ligases with their substrates or their regulators such as, for example, inducers, second messengers such as cAMP or cGMP. Further objects studies are receptors and their binding partners such as, for example, hormones and cytokines.
If, for example, the total population of genes expressed in one cell type is represented on the detection system array of the invention by their fusagene (as component (c)), the binding partners of individual proteins (component (d)) can be identified as spots on the array via direct labeling, for example 35S isotope labeling.
Furthermore, it is possible to identify the binding partners of a component (d), preferably a protein, with the aid of a labeled component (d) antibody in the form of a sandwich assay. A particular advantage of this method is the fact it is thereby possible to identify specifically specific interactions of a component (d) from a pool of interacting substances, for example in a cell extract.
Moreover, it is also possible to study the action of cofactors, activators and inhibitors on the interaction of effectors and components (d) by successive additions of the individual factors.
Another important field of application of the detection system arrays is the analysis of enzyme activity patterns. Thus it is possible, for example, to identify with the aid of the array in a simple manner the substrates of a kinase which utilizes AT32P as phosphate donor.
If the array contains, for example, fusagenes with glycogen synthase and/or phosphorylase kinase as effector, it is possible to study in an in vitro assay on the array the action of the effectors as substrate, for example for the protein kinase which creates an important part in glycogen metabolism.
With the aid of such an in vitro assay it is possible to show, for example that the protein kinase is activated with the addition of cAMP, with respect to phosphorylation of the two effectors. Moreover, it is possible to study the removal of the corresponding phosphate groups on the individual effectors, for example by adding protein phosphatase 1.
The invention further relates to conjugates comprising a detection system which includes the components (a) to (c), at least one component (d) and, where appropriate, further substances such as substances interacting with component (d), preferably cbmponent (d) antibodies.

Preference is given to those conjugates which contain fusagenes as component (c).
The conjugates contained as components (d) are preferably proteins, peptides, in particular transcription factors, receptors, enzymes and/or chemical active substances, preferably organic compounds, pharmacologically active compounds, hormones, crop protection agents, toxins, in particular poisons, carcinogenic and/or teratogenic substances, herbicides, fungicides and/or pesticides.
The following figures are intended to describe the invention in more detail without restricting it.
Description of the figures Figure 1 describes diagrammatically the inventive detection system comprising a support ((1) component (a)) and a detection unit component (b) (2) bound to the support, which can be bound to the support surface via a spacer (3) and which furthermore contains a constant region (A) (4) and an adjacent variable region (B) (5), and a component (c) (6) comprising a region which is complementary to region (A) (4) and (B) (5) and which is formed out of parts of a suitable linker, for example a puromycin (7) containing nucleic acid linker (8), and of parts of the nucleic acid, here RNA (9), bound to the linker. The effector unit (10) is bound to the nucleic acid-puromycin linker (7, 8).
Figure 2 describes diagrammatically the identification of a complex comprising components (a)-(c) with the aid of a label (11 ) contained in component (c).
Figure 3 describes diagrammatically the identification of a complex comprising components (a)-(d) with the aid of a label, for example contained in component (d) (12), shown here by the example of an effector antibody.
The following examples are intended to describe the invention in more detail without restricting it. ' Example 1: Generation of an exemplary FLAG, MYC, STREP fusion glass chip The glass chip surface is silanized by firstly degreasing a standard glass slide in acetone in an ultrasonic bath for 5 minutes (aid: staining troughs for microbiology). After drying in air, the slides are treated with 0.1 M NaOH
solution in an ultrasonic bath for 5 minutes.
This is followed by washing with distilled water in an ultrasonic bath for a further 5 minutes. The last remaining residues of NaOH are removed by repeating the process of washing with distilled water. This is followed by the silanization step. For this purpose, a 2-3% strength (3-glycidyloxy-propyl)trimethoxysilane solution in 95% strength aqueous ethanol (ethanol:water; 95.5 v:v) is prepared. The ethanolic silane solution is adjusted to pH 4.9-5.2 with conc. acetic acid. After addition and hydrolysis for 10 minutes, the glass surfaces are treated with said prepared silanization solution in an ultrasonic bath for 2 minutes.
After washing with 100% ethanol solution in an ultrasonic bath and drying in air, the silanized glass slides are dried at 80°C for 20 minutes. It is then possible to immobilize the amino-modified detection units (components b) on the silanized glass surface.
The detection units (components b) thus immobilized on the chip are synthesized according to standard methods (see below). The RNA coding for the FLAG, MYC and STREP epitope is produced as follows, following Roberts and Szostak (Proc. Natl. Acad. Sci USA, 1997): the DNA template sequence (Seq ID No: 1 ) is amplified with the two primers (Seq ID No:2/3) in a PCR reaction with the aid of Taq polymerase (Promega, Cat. No:
M166F):

5'-GATTACAAGGACGACGACGACAAGGAACAGAAGCTGATCTCCGAAGAGGATC-TGGCAATGTGGAGCCACCCGCAGTTTGAGAAA-3' (Seq ID No: 1 ) 5'-TAATACGACTCACTATAGGGACAATTACTATTTACAATTACAATGGATTACAAG-GAGGACGACGACAAGG-3' (Seq iD No: 2) 5'-AGCGGATGCTTTCTCAAACTGCGGGTGGCTCCAC-3' (Seq ID No: 3) The resulting double-stranded DNA product is transcribed into the corresponding RNA sequence (Seq ID No: 4) by an in vitro transcription (Promega, Cat. No: P 1300):
5'-GGACAAUUACUAUUUACAAUUACAAUGGAUUACAAGGACGACGACGACAAG-GAACAGAAGCUGAUCUCCGAAGAGGAUCUGGCAAUGUGGAGCCACCCGCA-GUUUGAGAAAGCAUCCGCU-3' (Seq iD No: 4).
Furthermore, a linker (Seq ID No: 5) which carries a phosphate group on its 5' terminus and a puromycin residue (Pu) on its 3' terminus is ligated 3'-terminally to the epitope-encoding RNA (Seq ID No: 4): the ligation is carried out using T4 DNA ligase (MBI, Cat. No: EL 0333) and with the aid of two splint molecules (Seq ID No: 617) which are mixed into the reaction in a ratio of 80:20%.
5'- ANFAAAP,AAAACCPu-3' (Sea ID No: 5) 5'-GCGCGC AGCGGATGC-3' (Seq ID No 6) 5'-GCGCGCNTTTTTTTTTAGCGGATGC-3' (Seq ID No: 7) In addition, a fluorescein derivative (NF, Interactiva) is incorporated into the linker by means of standard DNA solid phase synthesis, which derivative makes it possible, in the case of specific hydridization with the complementary components (b), to read out a binding event via the appearing fluorescence.

The ligation product comprising RNA (Seq ID No: 4) and linker (Seq ID No: 5) is then purified of the nonligated RNA via a denaturing 6%
strength TBE urea gel.
The fusagene comprising RNA (Seq ID No: 4), epitope-encoding peptide (Seq ID No: 4) and linker (Seq ID No: 5) is synthesized following Roberts and Szostak (Proc. Natl. Acad. Sci USA, 1997) in an in vitro translation (Promega, Cat. No: L 4960) and subsequent incubation of the translation mixture with MgCl2 (150 mM) and KCI (530 mM).
MYC
MDYKDODDKEQKL1SEEDLAMWSHPQFEKASA (Seq !D No:4) IFLaG STREP
The thus synthesized fusagene is then purified to homogeneity via oligo-d(T) cellulose (Amersham Pharmacia Biotech. Cat. No: 27-5543-02) and subsequently by means of Strep-Tactin Sepharose (IBA.
Cat. No: 2-1202-005).
The detection units (components (b)) used comprise in each case a constant region having the sequence 5'-Tt~-3' (region A) and a variable region (region B) composed of eight nucleotides. For components (b) the following sequences were selected (Seq ID No: 8/9):
Komponente (b)-1: 5'- TTTAGCGGATG-3' (Seq ID No: 8) Komponente (b)-2: 5'-TTTTTlTTTTTTTTTGTAGGCGA-3' (Seq 1D No: 9) Here, component (b)-1 has within the variable region B the nucleotide sequence complementary to the molecule comprising RNA (Seq ID No: 4) and linker (Seq ID No: 5), whereas component (b)-2 has no specificity for the target sequence of said molecule, with respect to the variable region B.
The components (b) were prepared by standard solid phase DNA
synthesis. For immobilization via the 3' terminus, a 3'-amino modified C3 CPG support (Glen Research, Cat. No: 20-2950-10) was used and for a 5'-terminal attachment to the glass surface a 5'-amino modifier C6 phosphoramidite (Glen Research, Cat. No: 10-1906-90) was used. For immobilization, as 50 ~M (in 0.1 M NaOH) solution of components (b)-1/2 is applied to the silanized glass surface at positions 1 and 2, respectively.
After incubation for at least 2 hours, the glass surface is washed with warm water for approx. 5 minutes. This is followed by washing the chip with 5 x SSC buffer for 10 minutes.
The fusagene which has been fluorescently labeled with fluorescein in the polyA region is hybridized with the complementary target sequence of component (b)-1 (at position 1 ) by taking up the fusagene in 5 x SCC
buffer and transferring it to the chip, covering it with a cover slip and incubating it at 4°C for 5 minutes. The chip is then washed 3 x with 5 x SSC buffer at room temperature and the fluorescein fluorescence is read out. Finally, washing with 0.5 x SSC buffer is repeated and the fluorescence intensity is read out again. It was shown that only in the case of perfect hybridization with component (b)-1 it was possible to detect a fluorescence signal and that no interactions whatsoever of the fusagene with the unspecific sequence of component (b)-2 took place.
Example 2: Generation of an exemplary FLAG, MYC, STREP fusion glass chip for detecting protein-protein interactions (detection of the FLAG
epitope using a fluorescently labeled specific anti-FLAG antibody) The glass chip surface is silanized by firstly degreasing a standard glass slide in acetone in an ultrasonic bath for 5 minutes (aid: staining troughs for microbiology). After drying in air, the slides are treated with 0.1 M NaOH
solution in an ultrasonic bath for 5 minutes.
This is followed by washing with distilled water in an ultrasonic bath for a further 5 minutes. The last remaining residues of NaOH are removed by repeating the process of washing with distilled water. This is followed by the silanization step. For this purpose, a 2-3% strength (glycidyloxy-propyltrimethoxysilane solution in 95% strength aqueous ethanol (ethanol:water; 95.5 v:v) is prepared. The ethanolic silane solution is adjusted to pH 4.9-5.2 with conc. acetic acid. After addition and hydrolysis for 10 minutes, the glass surfaces are treated with said prepared silanization solution in an ultrasonic bath for 2 minutes.
After washing with 100% ethanol solution in an ultrasonic bath and drying in air, the silanized glass slides are dried at 80°C for 20 minutes. It is then possible to immobilize the amino-modified detection units (component b) on the silanized glass surface.
The components (b) immobilized on the chip were synthesized according to standard methods (see below). The RNA coding for the FLAG, MYC and STREP epitope is produced as follows, following Roberts and Szostak (Proc. Natl. Acad. Sci USA, 1997): the DNA template sequence (Seq ID No: 1 ) is amplified with the two primers (Seq ID No:2/3) in a PCR
reaction with the aid of Taq polymerase (Promega, Cat. No: M166F).
The resulting double-stranded DNA product is transcribed into the corresponding RNA sequence (Seq ID No: 4) by an in vitro transcription (Promega, Cat. No: P 1300).
Furthermore, a linker (Seq ID No: 10) which carries a phosphate group on its 5' terminus and a puromycin residue (Pu) on its 3' terminus is ligated 3'-terminally to the epitope-encoding RNA (Seq ID No: 4): the ligation is preferably carried out using T4 DNA ligase (MBI, Cat. No: EL 0333) and with the aid of two splint molecules (Seq ID No: 6/7) which are mixed into the reaction in a ratio of 80:20%.
5 - CCPu-3' (Seq ID No' 10) The ligation product comprising RNA (Seq ID No: 4) and linker (Seq iD No: 5j is then purified of the nontigated RNA via a denaturing 6%
strength TBE urea gel.
The fusagene comprising RNA (Seq ID No: 4), epitope-encoding peptide (Seq ID No: 4) and linker (Seq ID No: 10) is synthesized following Roberts and Szostak (Proc. Natl. Acad. Sci USA, 1997) in an in vitro translation (Promega, Cat. No: L 4960) and subsequent incubation of the translation mixture with MgCl2 (150 mM) and KCI (530 mM).
The thus synthesized fusagene is then purified to homogeneity via oligo-d(T) cellulose (Amersham Pharmacia Biotech. Cat. No: 27-5543-02) and subsequently by means of Strep-Tactin Sepharose (IBA.
Cat. No: 2-1202-005).

The detection units (components (b)) used comprise in each case a constant region having the sequence 5'-T15-3' (region A) and a variable region (region B) composed of eight nucleotides. For components (b) the following sequences were selected (Seq ID No: 819):
Komponente (b)-1: 5'- AGCGGATG-3' (Seq ID No: 8) Komponente (b)-2: 5'-TZTrTT GTAGGCGA-3' (Seq ID No: 9) Here, component (b)-1 has within the variable region B the nucleotide sequence complementary to the molecule comprising RNA (Seq ID No: 4) and linker (Seq ID No: 10), whereas component (b)-2 has no specificity for the target sequence of said molecule, with respect to the variable region B.
The components (b) were prepared by standard solid phase DNA
synthesis. In the case of immobilization via the 3' terminus, a 3'-amino modified C3 CPG support (Glen Research, Cat. No: 20-2950-10) was used and for a 5'-terminal attachment to the glass surface a 5'-amino modifier C6 phosphoramidite (Glen Research, Cat. No: 10-1906-90) was used. For immobilization, a 50 ~,M (in 0.1 M NaOH) solution of components (b)-1/2 is applied to the silanized glass surface at positions 1 and 2, respectively.
After incubation for at least 2 hours, the glass surface is washed with warm water for approx. 5 minutes. This is followed by washing the chip with 5 x SSC buffer for 10 minutes.
The fusagene is hybridized with the complementary component (b)-1 (position 1 ) by taking up the fusion protein in 5 x SCC buffer and transferring it to the individual chip, covering it using a cover slip and incubating it at 4°C for 5 minutes. The chip is then washed 3 x with 5 x SSC buffer at room temperature and a solution of the anti-FLAG
antibody (Sigma Immunochemicals, Cat. No.: F 3040) which have been fluorescently labeled previously (Cy5 Ab Labelling Kit, Amersham Pharmacia Biotech. Cat. No.: PA 35000) is applied and its fluorescence read out. Finally, washing with 0.5 x SSC buffer is repeated and the fluorescence intensity is read out again. It was shown that only in the case of perfect hybridization with component (b)-1 it was possible to detect a fluorescence signal and that no interactions whatsoever of the fusagene or the fluorescently labeled antibody with the unspecific sequence of component (b)-2 took place.
Example 3: Generation of an exemplary fusagene array comprising two different fusagenes on an electronically addressable chip Two fusagenes (components (c)) which could be unambiguously discriminated from one another and characterized in a hybridization experiment on the addressable electronic chip were synthesized. For this purpose, besides the fusagene (component (c)-1 ) from Example 2, another but different fusagene (component (c)-2) comprising RNA (Seq ID No: 15), epitope-encoding peptide (Seq ID No: 15) and linker (Seq ID No: 10) is synthesized with the aid of a template DNA (Seq ID No: 11), two primers (SEQ ID No: 12/13) and a splint (Seq ID No: 14).
5'-GGTGCGCCGGTGCCGTATCCGGATCCGCTGGAACCGCGTGAACAGAAGCT-GATCTCCGAAGAGGATCTGGCAATGTACAAGGACGACGACGACAAG-3' (Seq ID
No: 11) 5'-TAATACGACTCACTATAGGGACAATTACTA7TTACAATTACAATGGGTGCGC-CGGTGCCGTAT-3' (Seq ID No: 12) 5'-CTTGTCGTCGTCGTCCTTGTACATTGCCAGATCCT-3' (Seq ID No: 13) 5'-T-fTTlT1'TfTCTTGTCGTC-3' (Seq ID No:14) 5'-GGACAAUUACUAUUUACAAUUACAAUGGGUGCGCCGGUGCCGUAUCCG-GAUCCGCUGGAACCGCGUGAACAGAAGCUGAUCUCCGAAGAGGAUCUGG-CAAUGUACAAGGACGACGACGACAAG-3' (Seq ID No 15) MYC
~9GAPVPYPDPLEPREQILLIS~~DL.~.~I~'KDDDD~:~Sa (Seq ID No: 15) E-TAG F LAG
The components (b) immobilized on the electronic chip are biotin-labeled components (b) (Seq ID No: 16-28) which were obtained by standard DNA

synthesis, In this connection, the immobilization may be carried out in each case either 3'-terminally or 5'-terminally. In the example listed, the components (b) are attached to the chip surface by a biotin modification on the 3' terminus. For this, a biotinTEG CPG support (Glen Research, Cat.
No.: 20-2955-10) is used in the chemical DNA synthesis and the following components (b) having the appropriate biotin modification and, in addition, comprising a 15 nucleotide-long thymidine region (region A) and a variable region of 8 nucleotides (region B) are synthesized:
Komponente (b)-3 : 5'-~TTTTTTGGATGCTTBio-3' (Seq ID No: 16) Komponente (b)-4 : 5'-TTTlTT'TTfTTTlTTCTTGTCGTBio-3' (Seq 10 No: 17) 1 p Komponente (b)-5 : 5'- TTfGTAGGCGABio-3' (Seq ID No: '18) The component (b)-3 and component (b)-4 are complementary to the relevant component (c)-1 and, respectively, component (c)-2, with respect to their particular variable region B. The component (b)-5 has no specificity whatsoever to the complementary target sequences of component (c)-1 and, respectively, component (c)-2, with respect to region B.
The components (b)-3-5 are immobilized by preparing a 1 pM solution of the particular components (b) in 50 mM histidine buffer and applying in each case 50 p1 of the appropriate component (b) to a chip made of semi-conducting material, which contains 3 x 3 positions. The immobilization was carried out at room temperature. It was chosen to occupy the positions on the chip (row, column) as follows:
1,1: Component (bj-3; 1,2: component (b)-4; 2,1: component (b)-3; 2,2:
component (b)-4; 3,1: component (b)-5; 3,2: component (b)-5; 1,3:
component (b)-3; 2,3: component (b)-4; 3,3: component (b)-5.
The ligated nucleic acids comprising Seq ID No: 4+10 (component (c)-3) and Seq ID NolS+10 (component (c)-4) or the fusagenes (component (c)-1 and component (c)-2) are hybridized by taking them up in 50 mM histidine buffer and carrying out the particular hybridization experiments on the appropriate chip positions (row, column) at room temperature:
1,1: Component (c)-1; 1,2: component (c)-2; 2.1: component (c)-1; 2,2:
component (c)-2; 3,1: component (c)-1; 3,2: component (c)-2; 1,3:

component (c)-3; 2,3: component (c)-4; 3,3: no component (c).
The hybrization events which took place on the chip are then characterized by applying an anti-FLAG antibody (Sigma Immunochemicals, Cat.
No.: F 3040) which specifically recognizes the FLAG epitope and which has been fluorescently labeled previously (Cy5 Ab Labelling Kit, Amersham Pharmacia Biotech, Cat. No.: PA 35000) to positions 1,1 and 1,2. An E-Tag antibody (Amersham Pharmacia Biotech, Cat. No.: 27 9412-01 ) which recognizes the E-Tag epitope and which has been fluorescently labeled previously (CySAb Labelling Kit, Amersham Pharmacia Biotech.
Cat. No.: PA 35000), is applied to positions 2,1 and 2,2. The fluorescent anti-FLAG antibody or anti-E-tag antibody is applied to chip positions 3,1;
3,2; 1,3; 2,3 and 3,3, in order to detect possible unspecific binding events of the antibody.
In all cases the fluorescence intensity was read out: it was shown with the aid of the fluorescently labeled anti-FLAG antibody as fluorescent probe that, in the case of perfect hybridization of components (b) with the complementary components (c)-1/2 a specifically positive binding event took place on chip positions 1,1 and 1,2. In the case of interactions of the anti-E-tag antibody (on positions 2,1 and 2,2), an increase in fluorescence intensity was detectable only on position 2,2 which carries component (c)-2 which is loaded with the E-tag epitope. These experiments clearly show that initially components (c)-1 and -2 were specifically recognized by the relevant antibodies. A control experiment firstly investigated the possibility, whether the two fuser genes were able to unspecifically interact with a component (b) which in the variable region (region B) has no complementarity whatsoever to the components (c) (position 3,1 and 3,2).
After adding the anti-FLAG antibody, no fluorescence was detectable so that it can be assumed that no unspecific hybridization of components (c) occurred. In addition, another control experiment eliminated the possibility that the anti-FLAG antibody interacts with double-stranded nucleic acid. To this end, the relevant components (c)-3/4 without peptide epitope were applied to positions 1,3 and 2,3, and addition of the anti-FLAG or anti-E-Tag antibodies showed that no fluorescence change occurred.
Finally, it was also possible to show that the fluorescent anti-FLAG or anti-E-Tag antibodies have' no affinity whatsoever to single-stranded components (b) (experiment on position 3,3). As summary, the results are Chip position Component (c)/ Antibody Fluorescence Component (b) signal 1,1 1 /3 FLAG +

1,2 2/4 FLAG +

2,1 1 /3 E-Tag -2,2 2/4 E-Tag +

3,1 1 /5 FLAG -3,2 2/5 FLAG -1,3 3/3 FLAG -2,3 4/4 E-Tag -3,3 -/5 E-Tag -SEQUENZPROTOKOLL
<110> Aventis Research & Technologies GmbH & Co KG
<120> Erkennungssystem zur Untersuchung von Molekulwechselwirkungen, seine Herstellung and Verwendung <130> 99F030PCT1 <140> PCT/EP00/04791 <141> 2000-05-25 <150> DE 19923966.5 <151> 1999-05-25 <160> 20 <170> PatentIn Ver. 2.1 <210> 1 <211> 83 <212> DNA
<213> Kiinstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:Template <400> 1 gattacaagg acgacgacga caaggaacag aagctgatct ccgaagagat ctggcaatgt 60 ggagccaccc gcagtttgag aaa 83 <210> 2 <211> 70 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kiinstlichen Sequenz:Primere <400> 2 taatacgact cactataggg acaattacta tttacaatta caatggatta caaggacgac 60 gacgacaagg 70 <210> 3 <211> 34 " CA 02374438 2001-11-22 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:Primer <400> 3 agcggatgct ttctcaaact gcgggtggct ccac 34 <210> 4 <211> 120 <212> RNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der k~nstlichen Sequenz:Transkript-Protein <400> 4 ggacaauuac uauuuacaau uacaauggau uacaaggacg acgacgacaa ggaacagaag 60 cugaucuccg aagaggaucu ggcaaugugg agccacccgc aguuugagaa agcauccgcu 120 <210> 5 <211> 29 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:Puromycin-Linker <400> 5 aaaaaaaaaa aaaaaaaana aaaaaaacc 29 <210> 6 <211> 25 <212> DNA
<213> Kunstliche Sequenz <220>
<223% Beschreibung der kunstlichen Sequenz:Splint <400> 6 gcgcgctttt ttttttagcg gatgc . 25 <210>
<2i1> 25 <2i2> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kiinstlichen Sequenz:Splint <400> 7 gcgcgcnttt ttttttagcg gatgc 25 <210> 8 <211> 23 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kUnstlichen Sequenz:Komponente (b)-1 <400> 8 tttttttttt tttttagcgg atg 23 <210> 9 <211> 23 <212> DNA
<213> Kiinstliche Sequenz <220>
<223> Beschreibung der kiinstlichen Sequenz:Komponente (b)-2 <400> 9 tttttttttt tttttgtagg cga 23 <210> 10 <211> 29 <212> DNA
<213> Kiinstliche Sequenz <220>
<223> Beschreibung der kiinstlichen Sequenz:Puromycin-Linker <400> 10 aaaaaaaaaa aaaaaaaaaa aaaaaaacc 29 <210> 11 <211> 96 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:Template <400> 11 ggtgcgccgg tgccgtatcc ggatccgctg gaaccgcgtg aacagaagct gatctccgaa 60 gaggatctgg caatgtacaa ggacgacgac gacaag 96 <210> 12 <211> 63 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:Primer <400> 12 taatacgact cactataggg acaattacta tttacaatta caatgggtgc gccggtgccg 60 tat 63 <210> 13 <211> 35 <212> DNA
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:Primer <400> 13 cttgtcgtcg tcgtccttgt aeattgccag atcct 35 <210> 14 <211> 19 <212> DNA
<213> Kunstliche Sequenz <220>

<_'23> Beschreibung der kiinstlichen Sequenz:Splint <400> 14 tttttttttt cttgtcgtc 19 <210> 15 <211> 123 <212> RNA
<213> Kiinstliche Sequenz <22p>
<223> Beschreibung der kUnstlichen Sequenz:Transkript/Protein <400> 15 ggacaauuac uauuuacaau uacaaugggu gcgccggugc cguauccgga uccgcuggaa 60 ccgcgugaac agaagcugau cuccgaagag gaucuggcaa uguacaagga cgacgacgac 120 aag 123 <210> 16 <211> 23 <212> DNA
<213> Kiinstliche Sequenz <220>
<223> Beschreibung der kiinstlichen Sequenz:Komponente (b)-3 <400> 16 tttttttttt tttttggatg ctt 23 <210> 17 <211> 23 <212> DNA
<213> Kiinstliche Sequenz <220>
<223> Beschreibung der kiinstlichen Sequenz:Komponente (b)-4 ~400> 17 tttttttttt tttttcttgt cgt 23 <210> 18 <211> 23 <212> DNA
<213> Kunstliche Sequenz <220>
<2=3> Beschreibung der kunstlichen Sequenz:Komponente (b)-5 <400> 18 tttttttttt tttttgtagg cga 23 <210> 19 <211> 32 <212> PRT
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:FLAG-MYC-STREP-Epitop <400> 19 Met Asp Tyr Lys Asp Asp Asp Asp Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Ala Met Trp Ser His Pro Gln Phe Glu Lys Ala Ser Ala <210> 20 <211> 36 <212> PRT
<213> Kunstliche Sequenz <220>
<223> Beschreibung der kunstlichen Sequenz:E-TAG-MYC-FLAG-Epitop <400> 20 Met Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Rrg Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Ala Met Tyr Lys Asp Asp Asp Asp 20 ~ 25 30

Claims (35)

Claims

1. A detection system comprising i) a support (component (a)) and ii) at least one detection unit (component (b)) bound to the support, wherein said detection unit inclusively comprises a pairing system with a region (A) having a constant sequence of at least 5 nucleotides and a region (B) adjacent to region (A) and having a variable sequence, and iii) a nucleic acid-protein acceptor fusion molecule or nucleic acid-protein fusion molecule (component (c)) linked thereto and comprising a sequence complementary to the detection unit (component (b)).

2. A detection system as claimed in claim 1, wherein region (A) is 5 to approx. 80 nucleotides or nucleotide analogs in length.

3. A detection system as claimed in claim 1, wherein region (A) is 5 to approx. 30 nucleotides or nucleotide analogs in length.

4. A detection system as claimed in any of claims 1 to 3, wherein region (B) is 5 to 30 nucleotides or nucleotide analogs in length.

5. A detection system as claimed in any of claims 1 to 3, wherein region (B) is 7 or 8 nucleotides or nucleotide analogs in length.

6. A detection system as claimed in claims 1 to 3, wherein the nucleo-tides are deoxyribonucleotides (d), ribonucleotides (r) or 2-hydroxy-methylribonucleotides (hmr).

7. A detection system as claimed in claim 2, wherein the nucleotide analogs are p-RNA, CNA or PNA monomers.

8. A detection system as claimed in any of claims 1 to 6, wherein region (A) comprises a polyT strand.

9. A detection system as claimed in any of claims 1 to 6, wherein, for one, region (A) comprises a T7-15 strand.

10. A detection system as claimed in any of claims 1 to 9, wherein component (c) are selected from nucleic acid-puromycin derivatives and/or nucleic acid-puromycin-protein fusion molecules.

11. A detection system as claimed in any of claims 1 to 10, wherein the support is composed of ceramic, metal, in particular semi-conductors, noble metal, glasses, plastics, crystalline materials or thin layers of the support, in particular of said materials, or (bio)molecular filaments such as cellulose, structural proteins, or of a combination of said materials.

12. A detection system as claimed in any of claims 1 to 11, wherein the detection system is assembled on an electronic chip.

13. A method for preparing a detection system as claimed in any of claims 1 to 12, comprising i) preparation of an array by attaching the detection units (components (b)) comprising a region (A) having a constant sequence of at least 5 nucleotides and a region (B) adjacent to region (A) and having a variable sequence to a support (component (a)), wherein each array position can be assigned to a detection unit having a particular region B, and ii) hybridization of the detection units (components (b)) with nucleic acid-protein acceptor fusion molecules or nucleic acid-protein fusion molecules (components (c)) comprising a sequence complementary to the detection unit (component (b)).

14. Method for fractionating and identifying individual components in a solution comprising pairing system-effector fusion molecules, comprising i) preparation of a pairing system-effector fusion molecule library (component (c) library), ii) hybridization of the pairing system-effector fusion molecule library (components (c) library) on an array comprising component (a) and components (b) whose region (B) includes all possible permutation, it being possible for each individual component (b) whose region (A) comprises at least 5 nucleotides to be specifically assigned to one array position, iii) identification of array positions at which a complex of component (b) and component (c) has formed, iv) characterization of the complexes of component (b) and component (c) identified under iii).

15. A method as claimed in either of claims 13 and 14, wherein components (b) having a region (A) of from 5 to 80 nucleotides or nucleotide analogs in length are used.

16. A method as claimed in any of claims 13 to 15, wherein components (b) having a region (B) of between 5 to 30 in length are used.

17. A method as claimed in any of claims 13 to 15, wherein components (b) having a region (B) of from 7 or 8 nucleotides length are used.

18. A method as claimed in any of claims 10 to 13, wherein a region (A) composed of a polyT strand or of a strand complementary to the ribosomal binding site is used.

19. A method as claimed in any of claims 13 to 18, wherein the support is composed of ceramic, metal, in particular semiconductors, noble metal, glasses, plastics, crystalline materials or thin layers of the support, in particular of said materials, or (bio)molecular filaments such as cellulose, structural proteins, or of a combination of said materials.

20. A method as claimed in any of claims 13 to 19, wherein said method is carried out on an electronic chip.

21. A method as claimed in any of claims 13 to 20, wherein the sample component is bound as component (b) or as component (c) with the aid of an electric field to the detection unit bound to a support.

22. A method as claimed in any of claims 13 to 21, wherein the component (c) used is pairing system-effector fusion molecules composed of a nucleic acid and/or analogs thereof.

23. A method as claimed in any of claims 13 to 22, wherein in a [lacuna]
at least one RNA-protein fusion molecule of a sample [lacuna] to at least one nucleic acid bound to a support and having the formula 3'-(X)7-8-(T7-15)-5', where X is any nucleotide selected from adenosine, thymidine, uracil, guanosine or cytosine, and in a further step nucleic acids which are not bound or bound unspecifically are removed.

24. A method as claimed in any of claims 13 to 23, wherein in a [lacuna]
at least one RNA-protein fusion molecule of a sample is bound to at least one nucleic acid bound to a support and having the formula 3'-(X)7-8-(T7-15)-5', where X is any nucleotide selected from adenosine, thymidine, uracil, guanosine or cytosine, with the aid of an electric field, and in a further step nucleic acids which are not bound or bound unspecifically are removed, with the aid of an electric field with reverse polarity and of a lower field strength than in the first step.

25. A method as claimed in any of claims 13 to 24, wherein in a first step a suitable nucleic acid linker is fused, chemically or with the aid of a suitable ligase, to an RNA population of a sample and then translated, and subsequently formation of the RNA-linker-protein fusion is induced, in a second step the RNA-linker-protein fusion molecules are bound to nucleic acids of the detection system of the invention, and in a further step nucleic acids which have not or not specifically bound are removed.

26. A method as claimed in any of claims 13 to 25, wherein detection unit of the formula 3'(X)7-8-(T27GG)-5', where X is any nucleotide selected from adenosine, thymidine, uracil, guanosine or cytosine, [lacuna].

27. A method for identifying interactions of one or more binding partners (components (d)) which have an affinity for specific effector units of components (c), comprising:
i) incubation of a pairing system-effector fusion molecule array with a substance mixture to be analyzed which comprises at least one component (d), ii) identification of array positions at which a complex of component (b) having a constant region (A) with at least 5 nucleotides and a variable region (b), component (c) and component (d) has formed, iii) characterization of the complexes of components (b), components (c) and component (d) identified under ii).

28. A method as claimed in claim 17 for finding and/or identifying and/or characterizing at least one sample component, in particular nucleic acids and/or proteins of a sample, or for finding and/or identifying cellular or artificial binding partners, preferably proteins, peptides, nucleic acids, chemical active substances, preferably organic compounds, pharmacologically active compounds, crop protection agents, toxins, in particular poisons, carcinogenic and/or teratogenic substances, herbicides, fungicides or pesticides.

29. A method as claimed in either of claims 23 and 28, wherein at least one component (d) is directly labeled.

30. A method as claimed in any of claims 27 to 29, wherein at least one effector-bound component (d) is detected using a labeled, specific binding partner such as, for example, an antibody.

31. A method as claimed in any of claims 27 to 30, wherein the inter-action between effector and component (d) according to step ii) is modified by adding further substances influencing the interaction, such as cofactors, coenzymes, activators or inhibitors, for example.

32. A method as claimed in any of claims 27 to 31, wherein an enzyme activity pattern is determined.

33. A conjugate comprising a detection system (components (a) to (c)) and component (d).

34. A conjugate as claimed in claim 33, wherein component (d) is a fusagene.

35. A conjugate as claimed in either of claims 33 or 34, wherein component (d) is a protein, peptide, in particular a transcription factor, a receptor or an enzyme such as, for example, kinases, phosphatases, GTPases, esterases, glycosylases, lipases, oxidases, reductases, hydrolases, isomerases, isomerases or ligases together with substrates or regulators thereof such as, for example, inductors, second messenger such as cAMP or cGMP, and/or a chemical active substance, preferably an organic compound, a pharmacologically active compound, a hormone, a crop protection agent, a toxin, in particular a poison, a carcinogenic and/or teratogenic substance, a herbicide, fungicide and/or pesticide.