EP1226272A2 - Double-strand nucleic acid probes and the use thereof - Google Patents

Abstract

The invention relates to electronically-isolable double-strand nucleic acid probes and use thereof for rapid and easy detection of interactions between double-stranded nucleic acids and factors which interact with them either by mediated or direct means. The invention further relates to the production of said double-strand nucleic acids.

Description

Double-stranded nucleic acid probes and their use

description

The present invention relates to electronically readable double-stranded nucleic acid derivatives and their use for the quick and easy detection of interactions between double-stranded nucleic acids and factors which interact directly or indirectly with them, in particular with proteins, peptides, single-stranded or double-stranded nucleic acids or substances which damage nucleic acids.

Double-stranded nucleic acids play a crucial role in the living cell and also in many viruses, especially as carriers of the hereditary system. They are subject to natural systems such as B. a cell of various internal and external influences. Interactions between proteins and double-stranded DNA are of particular interest, since such interactions have a decisive influence on the transcription or repression of individual genes and thus on the phenotype of the corresponding organisms. But also the replication of the hereditary system in mitosis or meiosis, the restriction z. B. viral nucleic acids or the packing and unpacking of eukaryotic nucleic acids in the chromosomes are other important processes in living cells that are controlled by the complex interplay of proteins and nucleic acids. In addition, other chemical substances can also interact with double-stranded nucleic acids, only the class of carcinogenic intercalating or nucleic acid-damaging substances should be mentioned here. Nucleic acids themselves, whether single-stranded or double-stranded, can with their double-stranded relatives, e.g. B. in the recombination, insertion or transposition, interact.

Knowledge of molecule-molecule interactions plays a central role in the investigation of such processes, regardless of whether it is about the identification of the individual biologically active building blocks or the elucidation of the mechanisms, i.e. the interaction of these building blocks. Developing new more efficiently S

Detection methods for such interactions are therefore the focus of interest.

In Napier, M.E. et al. "Probing Biomolecule Recognition with Electron Transfer: Electrochemical Sensors for DNA Hybridization" Bioconjugate Chem. (1997), 8 (6), 906-913 have already described measurement arrangements that allow hybridization events to be measured directly on single-stranded nucleic acids. This detection method is based on this that guanosine-bound DNA is oxidized via ruthenium complexes and that this process can be demonstrated by means of cyclic voltammetry.

In the publications WO 95/15971, WO 96/40712 and DE 19901761 A1, methods are described which utilize the electrical conductivity of double-stranded nucleic acid hybrids compared to single-stranded nucleic acids. Both use their method to detect only single-stranded nucleic acid sequences. For this purpose, complementary single-stranded nucleic acid probes which contain at least one electron-donor unit and at least one electron-acceptor unit, preferably designed as an electrode, are used to detect the presence of single-stranded nucleic acid target sequences. In the electrically conductive probe-target sequence hybrid is thermally or via a current-inducing signal, such as. B. light, generates a detectable electron flow.

The methods described are limited to the detection of hybridized single-stranded nucleic acids. The detection is limited to the mere presence of a target sequence in the medium to be examined and, moreover, does not allow any statements about the biological activity of the interacting nucleic acids.

If interactions of double-stranded nucleic acid sequences are to be investigated, other methods, e.g. B. fall back on gel electrophoretic methods. J

In the gel shift method (Fried, M. & Crothers, DM "Equilibrium and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis" (1981) Nucleic Acid Res. 9, 6505-6525; Garner, MM & Revzin, A. "A gel elecrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system" (1981) Nucleic Acid Res. 9, 3047-3060) becomes a radioactive DNA fragment incubated a protein extract to be examined. If the protein extract contains a DNA fragment binding protein, two bands can be detected by gel electrophoresis, one represents the free DNA fragment, the second, shifted band contains the DNA fragment-protein complex.

Another in vitro method for the detection of DNA-protein bonds is the footprint technique (Watson, J.D. et al, Recombinant DNA, 2nd edition 1993, pp. 143-146, Spektrum Akademischer Verlag). A radioactively labeled DNA sequence is an incomplete restriction degradation, eg. B. subjected to DNAse I. The sequence is protected from degradation at the corresponding site by a bound protein; the corresponding bands are missing in the electrophoretic fragment detection compared to the free sequence.

These methods include the preparation of the DNA fragments and the protein extracts, the labeling of the DNA fragments, the formation of a reaction mixture and the gel electrophoretic separation. The implementation of these indispensable verification steps is very labor-intensive and time-consuming. In addition, the detection cannot be carried out consistently under native conditions and the use of reaction mixture components separated by gel electrophoresis for further investigations is difficult.

Proceeding from this, the object of the present invention is to provide novel probes for the detection and analysis of interactions of double-stranded nucleic acids as well as methods for the rapid and simple detection of such interactions. The task is accomplished with the help of new, electronically readable double-strand

Solved nucleic acid probes.

The probes contain at least partially double-stranded nucleic acids or single-stranded nucleic acids with self-recognizing domains which are attached to a conductive surface, preferably to an electrode, e.g. B. are bound to field effect transistors. The nucleic acid section present as a duplex is linked to at least one electron donor unit or alternatively to at least one electron acceptor unit, with at least a portion of the nucleic acid region to be investigated between the electrode and the electron acceptor unit or electron donor unit lies. This sub-area forms the detection point. The binding of the individual probe subunits can take place covalently or via stable supramolecular interactions, such as van der Waals interactions, dipole interactions, especially hydrogen bonds, or ionic interactions.

All single-stranded or double-stranded nucleic acids of any sequence can be used to construct the probe. It is irrelevant whether natural, synthetic or modified nucleic acid sequences are used. When binding single-stranded nucleic acid, the corresponding double strand can be formed simply by hybridization with a complementary single-stranded nucleic acid (e.g. McCarthy, BJ et al., "Specificity of Moiecular Hybridization Reaction" Annu. Rev. Biochem. (1970), 39 , 131-150), the formation of a double strand in the case of self-complementary single-stranded nucleic acids being caused by intramolecular refolding. In the synthesis of such single-stranded nucleic acids, two domains with more complementary sequences are advantageously covalently linked via a bridge comprising at least one nucleotide, preferably one from 4 to A bridge consisting of 6 nucleotides, in particular of thymine nucleotides, or linked via an artificial, at least one atomic bridge, such as disulfide bridges (Hetian Gao et al., “Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges ", Nucleic Acid Research, 1995, Vol. 23, No. 2, "85-292), stilbene dicarboxyamide bridges (Lewis, FD et al.," Distance-depending electron transfer in DNA Hairpins "Book of abstract 215 ^th ACS National Meeting, Dallas, Vol. 29, S. Physik 255, 1988), Ru complex bridges (Lewis, FD et al.,

"Synthesis and spectroscopy of Ru (II) -bridged DNA hairpins", Chem. Commun .. Vol.

4, p. 327, 1999), hexaethylene glycol bridges (Durand, M. et al., "Circular dichroism

Studies of an Oligonucleiotide containing Hairpin Loop made of a Hexaethylene Glycol Chain: Conformation and Stability. ", (1990) Nucl. Acids Res. 18, 6353-6359), aromatic terephthalimide bridges (Salunkhe, MS et al., (1992) , Control of

Folding and Binding of Oligonucleotides by Use of a Non-Nucleotide Linker ", J. Am.

Chem. Soc. 114, 8768-8772), unbranched or branched diol bridges, 3 ^' -amino-

Modifier-CPG (GlenResearch), which is preferably built on a thiol support, or branched phosphoramidite bridges. A great advantage of such nucleic acids containing self-complementary sequence segments lies in the simple, fully synthetic accessibility of the single-stranded nucleic acid and the simple manufacture of the double-stranded probe from a single molecular nucleic acid subunit. In addition, it has been shown that in the manufacture of the double-stranded nucleic acid probes, the intramolecular

Hybridization of the single-stranded self-complementary sequence segments containing nucleic acids is independent of the nucleic acid concentration. Another advantage of this process is that it can be produced enzyme-free. Another advantage is therefore that the double-stranded nucleic acid probes can also be prepared under denaturing conditions. A hairpin-forming single-stranded sequence with a free 3 ^' OH end can also be obtained using a DNA or RNA polymerase, e.g. B. a Klenow or Taq polymerase to the corresponding nucleic acid duplex are completed.

To produce the probes according to the invention, e.g. B. natural or synthetic DNAs, cDNAs or RNAs can be used. Their hybrids and their modified derivatives can also be used. The modified derivatives include, in particular, nucleic acids which contain nucleotides modified on the sugar, such as 2 ^, O-methyl nucleotides or 2 ^' O-allyl nucleotides. A modification of the phosphate group, such as. B. to phosphoramide, phosphorothioate or

Methylphosphonate is also possible. It is also possible to use nucleic acids which contain no sugar or a sugar which does not occur in natural nucleic acids, such as. B. the peptidyl nucleic acids or the pyranosyl nucleic acids. In addition to those modified on the sugar-phosphate backbone έ> Nucleic acids can also be used as probe components that contain non-naturally occurring nucleotides such as xanthine, hypoxanthine or inosine.

Double-stranded DNA or RNA nucleic acid sequences are preferably incorporated into the probes. Of particular interest are naturally occurring proteins or peptides binding DNA sequences, in particular DNA sequences involved in gene regulation, such as. B. operator or promoter sequences. Depending on the application problem, the use of consensus sequences or allele-specific or organism-specific sequences can be expedient.

The length of the nucleic acid sequence between the electrode and the electron donor units or electron acceptor units is preferably between 2 and 100 base pairs, particularly preferably between 5 and 50 base pairs. The double-stranded area must extend to the nucleic acid electrode linker in order to ensure an electron flow. Single-stranded areas can extend beyond the double-stranded detection region lying between the electrode and the electron donor units or electron acceptor units. The double-stranded detection region can contain single-strand breaks in the sugar-phosphate backbone.

The formation of a double strand can by chemical modifications that lead to inter- or intramolecular bridge bonds, such as. B. disulfide bridges, or other non-natural strand subunits can be chemically stabilized. Another possibility of stabilizing the nucleic acid duplex sections is the targeted introduction of thymine nucleotides at two opposite positions in the duplex and subsequent radiation-induced thymidine bridge formation. Analogously, azido nucleotides or psoralen derivatives can also be used for photoinduced bridging (Fabrega, C. et al., “Studies on the Synthesis of Oligonudeotides Containing Photoreactive Nucleosides: 2-Azido-2 ^' -Deoxyinosine and 8-Azido-2 ^' - Deoxyadenosine ", Biol. Chem., Vol. 379, 527-433, 1998; Pieles, U. et al., Nucleic Acid Research 1989, 17, 285). For example, the degradation of the nucleic acid duplex by exonucleases can be prevented. The sensitivity of the double-stranded nucleic acids to undesired, ? Process-related influences such as the temperature of the medium to be examined are also reduced.

All molecules, parts of molecules or surfaces which are able to donate electrons from their ground state or from an excited state or to take up electrons in the ground state or in an activated state can act as electron donor units or electron acceptor units. The excitation of an electron donor unit can e.g. B. done by exposure to light or ionization, the activation of an electron acceptor z. B. by ionization also by means of light or by chemical ionization. Proven electron donors and acceptors are e.g. B. metal complexes or organic redox-active compounds, as described in patent application WO 96/40712 or naturally occurring or modified photo-inducible redox-active reaction centers such as z. B. can be used in the patent application DE 19901761 A1. However, electrically conductive surfaces, in particular electrodes, can also be used as donor or acceptor units. In particular, the use of ferrocene or PQQ as electron donor units has proven itself experimentally.

The covalently bound electron donor units or electron acceptor units can be integrated in multimolecular electron transfer systems. On the one hand, the electron donor unit (electron acceptor units) acting in the double-stranded nucleic acid probe can be dissolved electron donors such as e.g. Anions (cations) can be charged in a regenerable manner, with a closed circuit being built up using the double-stranded nucleic acid probes as a resistor. On the other hand, analogous to the naturally occurring reaction centers, such as. B. the chlorophyll, other redox-active electron transfer units may be associated with the covalently bound electron donor or acceptor unit.

Inducible and / or regenerative donor or acceptor units are preferred. Regenerative systems enable a continuous flow of electrons through the duplex nucleic acid, while in inducible systems the flow of electrons through the inductor can be controlled over time. 3

Are z. B. chlorophyll, bacteriochlorophyll or its modified derivatives used as electron donor systems, the electron flow can be induced analogously to the natural photosynthesis processes by exposure to light. About suitable electron donating dissolved substances, such as. B. Fe (CN) ^{3 "} or Fe (CN) β ^4" , the reaction centers can be regenerated again. Induction can also take place directly via chemical substances which transfer electrons to the electron donor units or withdraw electrons from the electron acceptor units.

The connection of the nucleic acids to the electrode can e.g. via a thiol group (Chidsey, C.E.D., Sience, V. 251, p. 919, (1991)) or via phosphorothioates or phosphorodithioates on a gold surface. However, a connection via highly conductive molecule linkers (e.g. described by Kayyem et al. In WO 98/20162), which preferably contain conjugated double bond systems and have a higher conductivity than the bound nucleic acid, is also possible. The use of linkers with conjugated double bond systems allows electron conduction along the linker structure to the electrode surface. This elegantly enables passivation of the free electrode surface. The double-stranded nucleic acid probes can be connected to an unstructured, coherent electrode surface or to a structured, e.g. B. matrix-shaped electrode array surface. The structuring takes place in such a way that the individual array positions of the electrode can be read independently of one another. The individual array positions can be assigned nucleic acid probes with specific sequences, or the reaction space can be compartmentalized. A high degree of parallelization can thus be achieved in the electronic reading of the electrode.

It has surprisingly been found that the double-stranded nucleic acid probes according to the invention offer the possibility of detecting factors interacting with nucleic acid double-strands by changing the conductivity. Interacting factors are chemical substances or radiation that interact with a nucleic acid duplex and trigger a change in the primary, secondary, tertiary or quaternary structure of the duplex. These structural changes lead to a significant change in the electrical resistance of the duplex structure and thus to a change in the electron flow along the double-stranded nucleic acid. Presumably, in addition to the structural changes in the double-stranded nucleic acids due to interacting factors, other effects such as B. the isolating effect of proteins or peptides bound to nucleic acids or the direct participation of intercalating substances, such as. B aromatic molecules, on the electron line through the nucleic acid double strand also has an influence on the resulting electron flow. As a rule, aggregates form between the probes and the interacting substances, the aggregates being stabilized via intermolecular forces or even via covalent bonds.

Changes in the electrical conductivity of double-stranded nucleic acids are e.g. B. by the binding of proteins or peptides such as antibodies, polymerases, transcription factors, enhancers or repressors, the activity of which in turn by indirectly acting substances such as. B. can be modulated via inductors or modifying enzymes. Organic molecules such as some hormones can also be used directly or in conjunction with proteins or peptides such as e.g. B. hormone receptors, interact with double-stranded nucleic acids. Single-stranded nucleic acid can also, for. B. interact with triplex formation with double-stranded nucleic acids. But also saline solutions, mimetics or substances intercalating into the nucleic acid duplex, including cytostatics like the anthracyclines, cause measurable changes in the structure of the double strand.

The effects described so far leave the covalent bonds in the double strand intact. Other proteins or substances damaging nucleic acids directly attack the primary structure of the nucleic acids. Endonucleases cleave nucleic acids, exonucleases break down nucleic acids from a free strand end, ligases form covalent bonds between terminal nucleotides during

Topoisomerases cause single strand breaks and linkages. But also mimetics, nucleic acid-damaging substances and cytostatics that z. B. alkylating on nucleic acids or cross-linking, such as platinum complexes, for. B. cisplatin, methanesulfonates, e.g. B. busulfan, or n-nitroso compounds, for example Carmustine can attack their covalent bonds and / or new covalent ones

Make ties. Radioactive and electromagnetic radiation, such as. B. α-, ß-, γ- or UV radiation can also lead to such structural changes and thus to a measurable change in the electron flow within the double-stranded nucleic acid.

The use of the double-stranded nucleic acid derivatives according to the invention as probes offers many advantages. All interactions to which a double-stranded nucleic acid probe is exposed and which have an effect on the electrical conductivity can be detected. The measurement can be coulombmetric, impedometric, preferably frequency-dependent resistive or capacitive, voltammetric, potentiometric or amperometric. The disruptive effect of ions directly discharged at the electrode from the reaction solution is surprisingly very low. In order to further increase the measurement sensitivity, the electron flow can be frequency-modulated by the double-stranded nucleic acid probe. When using photo-inducible electron donor units, the modulation can take place via flashes of light. Very small changes in conductivity can also be detected via the number of probes applied or by extending the measuring time. The number of probes is preferably selected so that the current change resulting from the interaction between the double-stranded nucleic acid and the factors interacting with it is at least in the nA range. Due to the particularly high sensitivity, cyclovoltammetry, differential pulse voltammetry and square wave voltammetry have proven to be a particularly suitable measurement method.

Another advantage of the present probes is their high compatibility with a wide variety of reaction media. In particular, the use of crude extracts as the reaction medium or the use of reaction media with a salt concentration corresponding to the natural conditions are of interest. Furthermore, the kinetics of double-stranded nucleic acid interactions can be examined and equilibrium constants of double-stranded nucleic acids with interacting substances can be determined. That offers the Possibility to find antagonists and allosteric interactions with other factors and to determine their activity. Investigations can be carried out as a multi-step reaction in which individual factors are pipetted to the reaction solution in succession during the measuring process.

Further objects of the invention are methods for the detection of interactions between double-stranded nucleic acids and factors interacting with them and the use of the double-stranded nucleic acid probes according to the invention therefor.

The probes according to the invention are particularly suitable for the investigation of gene regulation, in particular the transcription or repression of genes. A large number of substances that directly interact with the double-stranded DNA play a role in gene regulation, such as RNA polymerases, transcription factors, repressors or enhancers or which only have an indirect effect, such as, for example, B. inducers, enzymes, such as phosphorylases, second messengers, such as cAMP or cGMP, receptors and their binding partners, such as hormones, play an important role. The signal chains that lead to the transcription or repression of individual genes can be extremely complex. The probes according to the invention can provide important insights in elucidating the interaction of the individual factors.

For this, z. B. known regulatory DNA sequences of a gene incorporated into the probes. The probes are applied to an electrode surface so that the sum of the change in the electrical conductivity of the double-stranded DNA probes is measured. In the free, unassociated state, the reference electron flow through the probes is determined in the presence of a reference solution. Alternatively, the reference value can also be determined using a standard electrochemical cell / electrode. The reference solution ideally contains all components of the reaction solution except the factors to be tested. The reference electron flow is modulated by the gradual addition of the individual substances to be tested. The change in the electron flow then provides information about a successful interaction between the test substance and the probe sequence, the strength of the change in the electron flow provides information the strength of the interaction and the equilibrium constant of the binding reaction. All direct interactions of DNA with directly interacting substances such as polymerases, transcription factors or repressors can be measured. This enables identification of substances that interact directly with the DNA duplex. Consequently, a new possibility for the rapid detection of transcription factors with tissue-specific activity is opened by using probes which contain known tissue-specific promoter sequences.

The effect of other factors influencing the activity of these substances, that is to say acting indirectly, can be determined indirectly by changing the equilibrium constants of RNA polymerases, transcription factors or repressors. For this purpose, either reaction solutions containing different factors can be tested or the indirectly acting factors are successively introduced into the reaction solution. This will also

Interactions between individual substances within the regulatory signal chains can be determined.

If, for example, a strong inductor, which acts on the repressor, is added to the reaction solution in an operator-repressor complex, then this is possible

Equilibrium constant towards zero and the measured electron flow adjusts to the reference electron flow.

The lactose operon from Escherichia coli offers an example of such an induction mechanism. The effect of the lac repressor is canceled by the presence of lactose, the repressor loses its affinity for the operator sequence and the electron flow will adjust to the reference value. If β-galactosidase, a lactose-degrading enzyme, is added to the reaction solution, the lac repressor is reactivated.

Another example is the lexA repressor in E. coli, which regulates numerous genes that are involved in DNA repair. RecA proteins bound by single-strand DNA are proteolytically cleaved and thus from 23 associated operator replaced. The electron flow within the double-stranded DNA probe will consequently adjust to the reference value again.

For a given inductor concentration, the degree of alignment of the electron flow with the reference electron flow is a measure of the strength of the inductive effect. By varying the inductor concentration, on the other hand, concentration-dependent effects of the inductor on the repressor or a linear dependence of the induction on the inductor concentration can be determined.

If the repressor in the reaction mixture is inactive, the electron flow remains at the reference value. By adding other substances, their repressive activating effect on the repressor can be tested. Such a regulatory mechanism is e.g. B. in the tryptophan operon of E. coli. The trp repressor is activated by the presence of tryptophan and the repressor tryptophan complex binds to the associated operator sequence. A deviation of the electron flow within the double-stranded DNA probes from the reference value can be determined.

Analogously, there is the possibility of determining the equilibrium constant of the binding of RNA polymerases or transcription factors to DNA promoters and the effect of their cofactors and their modifications. For this purpose, probes can be used which contain a promoter sequence. A reference value can then be determined, which is modulated by adding individual factors. A change in the reference value indicates an interaction of the added substance. The RNA polymerases and transcription factors that bind in the promoter area can then be identified.

If you use e.g. B. the SV 40 promoter for producing the double-stranded nucleic acid probes, the binding of the transcription factor Sp1 which specifically binds to this promoter can be determined.

By adding further protein or peptide cofactors, second messenger or modifying enzymes, such as. B. phosphorylases, or transcription Influencing molecules, like some antibiotics, their activating or inhibiting influence on the transcription factor-DNA binding is measurable.

In the case of the transcription factor Sp1 z. B. its binding to the SV 40 promoter inhibited by the antibiotic actinomycin D.

Another example is the transcription factor p53, the gene product of a tumor suppressor gene. p53 can by adding a phosphorylase, in the presence of a suitable phosphate donor, such as. B. ATP, phosphorylated and thus activated. The electron flow changes continuously due to the binding of the phosphorylated p53 to the probe sequence up to a concentration-dependent saturation value. In addition to the pure interaction, information about the reaction rate can then be obtained from the slope of the electron flow-time curve. The greater the slope of the curve, the higher the reaction rate, in the example of the phosphorylation reaction.

If the DNA sequence of the probes includes the operator and the promoter, competitive effects between RNA polymerases, promoter-binding transcription factors and repressors can also be measured. Ousted z. B. a repressor an already bound transcription factor, the electron flow changes accordingly. The measured electron flows lie between the values for the reaction solutions containing only one RNA polymerase and / or the transcription factor and only the repressor. So z. B. the direct influence of the lac, trp or lexA repressor on the RNA polymerase binding to the promoter can be investigated.

A particular advantage of the method described here is the possibility of investigating allele-specific interactions between regulatory active substances and the associated DNA regulation sequences. For example, the different activity of DNA sequences and DNA-binding proteins or peptides of the wild type and mutated DNA sequences, proteins or peptides can be carried out in separate batches independently of phenotypically recognizable changes to be examined. In particular, the study of recessive characteristics is possible.

In addition, gene product mixtures of different alleles of a gene that interact directly or indirectly with double-stranded DNA can be investigated. Binds e.g. B. a wild-type transcription factor to a promoter and not to its mutant, an extract of a transcription factor from a homozygous wild-type organism gives the greatest change in electron flow by the double-stranded nucleic acid probe, a transcription factor extract from a homozygous mutant organism does not result in a change in the conductivity of the

Probe. Heterozygous organisms with regard to the transcription factor result in an electron flow through the double-stranded nucleic acid probe which lies between the two extracts from the homozygous organisms, in this case extracts with a certain concentration have to be used.

The method is not limited to the identification of substances that participate in gene regulation. B. also bind histones, helicases, topoisomerases or ligases to double-stranded DNA. While helicases, topoisomerases and chromatin remodulating enzymes (e.g. nucleosomal ATPase ISWI) can generally reduce the flow of electrons through the probes if double-stranded nucleic acid probes are used which contain one or more bond breaks in the sugar-phosphate backbone that are ligated also lead to an electron flux increase.

Loss of conductivity is observed in nucleases that recognize and degrade double-strand structures. The flow of electrons through the probes drops steadily and approaches zero if the reaction time is long enough. Such an effect cannot be observed by adding nuclease-blocking substances.

With the help of bound RNA-DNA hybrid probes, there is the possibility, for. B.

To investigate RNAsen, recognize and break down the RNA parts within a double strand. The loss of the RNA building blocks generates a single strand that can now be detected due to the loss of conductivity. Not all of the RNA has to be broken down, even the loss of one or two Ribonucleotides between the electron donor units or electron acceptor units and the electrode result in a drastic decrease in the conductivity in the double strand. Analogously, DNAse can be detected with double-stranded DNA probes.

The most significant effect on the conductivity of double-stranded nucleic acid probes have restriction enzymes, such as. B. Hind IM which catalyzes a smooth double-strand break or EcoR I or PST I which produces an offset double-strand break, provided a suitable restriction site is present in the double-strand sequence. By separating the two

Nucleic acid strands between the electron donor units or electron acceptor units and the electrode completely stop the flow of electrons through the double strand.

The binding sequences of individual nucleic acid-binding substances can also be determined with the aid of the double-stranded nucleic acid probes according to the invention. Electrode arrays are advantageously used for this purpose, in which each field is connected to a group of double-stranded nucleic acids of a precisely defined sequence. In the ideal case, all possible 4 ^π sequences, where n is the number of base pairs, can be read out electronically on the electrode in 4 ⁿ

Fields applied. Double-stranded nucleic acid binding factors modulate the conductivity of the array positions that contain a sequence that is recognized by the factor. The associated recognized sequence is determined via the corresponding array position. With the help of the sequences obtained, probes can be produced, with which in turn over conventional ones

Hybridization methods the associated genes can be identified. With this method, modified nucleic acid sequences or transcription factors produced by site-specific mutagenesis can also be evaluated. A strong nucleic acid transcription factor / RNA polymerase binding is an indication of a highly active transcription-promoting complex. The selection of highly active promoter sequences and the associated highly active transcription factors is thus possible. Such promoter / transcription factor complexes can be used to construct novel expression vectors. Extracts of regulatory fragments of DNA fragments of not exactly specified sequences of a specific gene can also be incorporated into the double-stranded nucleic acid probes. Known factors of certain concentration binding to these sequences can then be used to determine whether homozygous binding sequences, homozygous non-binding sequences or heterozygous sequence mixtures are present in the probes.

Alternatively, a nucleic acid fragment extract can be added directly to a double-stranded nucleic acid probe / probe-binding protein. The competitive effect of the nucleic acid fragment extract on the measuring system is then measured.

In addition to the previously mentioned double-stranded nucleic acid substance classes, single- or double-stranded nucleic acids can also interact with the probe sequences.

The at least partial separation of the nucleic acid double strand of the probe due to the hybridization of the single-stranded nucleic acids competing as binding partners with a complementary probe sequence leads to triplex structures or to a dissolution of the probe duplex by re-hybridization. This will interrupt or change the conductivity of the probe. Hybridization events can thus be detected with this method. If a destabilized double-stranded nucleic acid sequence is used in the probe, hybridization with a single-stranded nucleic acid in solution is facilitated. A destabilization against a DNA double strand can e.g. B. can be achieved through the use of DNA / RNA hybrids. The DNA / RNA hybrid pairing can additionally be weakened by modifying the DNA strand to form the phosphorothioate nucleic acid.

The facilitated hybridization with a single strand in solution can also be achieved by a single strand end overhanging from the double strand probe. The hybridization then takes place with the single strand end and parts of the double strand structure. Is in the recognized double strand structure δ

If a break is introduced into the nucleic acid backbone, the newly formed hybrid can even detach completely from the probe and interrupt the flow of electrons within the probes.

In addition, recombination events can also be detected and factors involved in the recombination, in particular enzymes, can be identified. When a double-stranded nucleic acid is completely recombined with the probe sequence, the electron donor or electron acceptor units can be separated from the electrode. Electrons can no longer flow through the probe. The recombination event does not have to be completed, however, the formation of a triplex structure which affects the double-stranded probe structure is sufficient to measure a reduction in the electrical conductivity. If the reaction solution is set recombination-promoting factors such. B. the recA protein from E. coli, then a faster decrease in electron flow or increased decrease in total electron flow can be measured.

If the probes contain integration points for transposable elements, transposition events can also be determined. Since the strength of the electron flow in the double strand of the probe decreases with increasing distance, that is to say with increasing number of base pairs, between the electron donor unit or electron acceptor unit and the electrode, the electron flow also decreases when a transposon is integrated into the probe sequence , A corresponding increase in the electron flow can be demonstrated when the back reaction proceeds.

The use of the double-stranded nucleic acid probes is also suitable for the detection of nucleic acid-damaging substances or for the detection of the nucleic acid-damaging effect of chemical substances. The damage to a double-stranded nucleic acid can be caused in different ways. Some substances, especially aromatic substances such as ethidium bromide or acridine, can intercalate into the nucleic acid dupiex structure.

Other substances can react with nucleic acids, e.g. B. strong bases that hydrolyze the nucleic acids, benzopyrines that add to the nucleic acids or peroxides that easily form radicals and trigger radical chain reactions within the nucleic acid. The change in the nucleic acid structure these substances cause a change in the conductivity of the double-stranded nucleic acid probes.

Such a test can be carried out in a compartmented reaction vessel in which the bottom surface is formed by the electrode in the form of an array. The probes are applied in a uniform density to the individual array positions. Each reaction compartment can be mixed with a reaction solution containing a different test substance. Reaction solutions can also be prepared which contain the same test substance in different concentrations. In this way, a simple test for identifying DNA-damaging and thus carcinogenic substances can be created.

Double-stranded nucleic acids also interact with substances dissolved in the reaction medium, in particular with destabilizing ionic compounds. Interactions with the solvent itself can also affect the conductivity of the probes. For example, the dehydration of the DNA double helix of the B type changes to the A type. This transition can be measured using the novel probes.

The reaction media or solutions can contain any solvents and additives. Aqueous, ionic buffer solutions which can contain chemical substances which interact with double-stranded nucleic acids are preferably used as reaction solutions. The direct use of raw extracts as a reaction medium is also possible. The detection of the interaction between the double-stranded nucleic acid probe sequences and the substances interacting therewith preferably takes place under physiological conditions in 10 to 200 mM aqueous, saline solutions at pH 7-9. A phosphate buffer is preferably used as the buffer. The measuring temperature is preferably between 4 and 40 ° C. A deviation from these measurement conditions can, however, be appropriate in individual cases. For example, an elevated measuring temperature can be used to find proteins with temperature-stable activity, such as the Taq polymerase used for the PCR. <& 0 this results in a variety of replaceability of the double-stranded nucleic acid

Probes for the analysis of active factors interacting with nucleic acid or active double-stranded nucleic acid sequences.

The double-stranded nucleic acid probes according to the invention can be used in analytical devices with classic separation methods, such as. B. the chromatography can be combined. By separating cell extracts or substance libraries, active components that bind to a double-stranded nucleic acid can be quickly detected. The process can be operated continuously in an "online" measuring process.

In order to clarify the invention, examples of possible production methods of the double-stranded nucleic acid probes according to the invention and individual methods for measuring interactions between double-stranded nucleic acid and factors interacting with it are described below.

Suitable double-stranded nucleic acid probes can be produced using methods known to those skilled in the art. For this purpose, thiol groups are introduced into the nucleotide sequences that are to be incorporated into the probe for connection to a gold electrode and amino groups for connection to the electron donor units.

The oligonucleotides used, if not commercially available, for. B. with a DNA synthesis method, the so-called phosphoramidite method (Beaucage, S. L .; Caruthers, M.H. Deoxynucleoside phosphoramidites. A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett.

(1981), 22 (20), 1859-62).

It is a method with which both unmodified and modified nucleic acids, for example RNA ^' s phosphorothioates and PNAs, can be specifically displayed in high purity and yield and is routinely used today

Commitment comes.

For the synthesis, the desired synthesis building blocks are made on a 1 μmol scale in a predetermined order on a solid CPG carrier material with the aid of a o2l Expedite Synthesizer of the type Expedite 8909 from PerSeptive Biosystems. To do this, the 0.1 M solutions of corresponding

Phosphoramidite building blocks and 0.5 M tetrazole solution used. Due to their sensitivity, the building blocks are dissolved in dry acetonitrile (water content <30 ppm).

The necessary modifications are made with the help of Amino-SH or. Phosphate-

On-phosphoramidite building blocks (Eurogentec, Clontech) and carrier materials ("SH-

CPG "-Chemgenes, Glen Research, 3 ^'phosphates CPG Glen Research) constructed.

For the introduction of the thiol modification, the desired sequence is built up on the 3 'end of the oligonucleotide on thiol-CPG carrier material (Chemgenes) on a scale of 1 μmol. To avoid the oxidation of the sulfur, a 0.02M iodine solution is used during the DNA synthesis (Kumar, A. et al., Nucleic Acids Research 1991, 19, 4561).

The amino group can be removed using a 0.2 M solution AminoModifier II (Clontech) or 0.1 M solution Amino Modifier C6 dT (Glen Research) in dry acetonitrile (water <30 ppm) at selected positions that are not on the electrode side of the double strand Structure, be introduced in a targeted manner.

The modified oligonucleotides are worked up after completion of the syntheses. For this purpose, the oligomers are split off from the CPG support with aqueous ammonia solution (25-32%) and incubated at 55 ° C. for several hours. In the presence of thiol groups, DTT is added up to a concentration of 50mM. The solution is then concentrated and the

Ammonia freed and coevaporated several times with water. Oligonucleotides that are to contain a modified thymidine building block are coupled longer. After determining the absorption at 260 nm, the raw products are freed from demolition products and split-off protective groups using RP-HPLC (Lichrochart 100 RP-C18). A gradient of acetonitrile in 0.1 triethylammonium acetate buffer of 0-60% in 60 minutes is used as the mobile phase. The main peak is isolated, spectrometrically quantified at 260 nm and lyophilized and dissolved in 500 μl 50mM DTT. The solution is incubated for 30 minutes at 37 ° C. and then desalted using a PD-10 column according to the manufacturer's instructions (Pharmacia) and pipetted onto the gold surface for covalent connection to the electrode. The buffer solutions used are previously saturated with argon.

In an alternative method for modifying the oligonucleotides with a thiol group, the sequence with phosphoamidite nucleotide building blocks is built up in the desired sequence on a 3 ^' phosphate CPG, as described above. An AminoModifier II (Clontech) can be used as an amino-modified component. After the amino-modified 3 ^' -phoshorylated oligonucleotide has been split off, the oligonucleotide is esterified with (HO- (CH ₂ ) ₂ -S) ₂ to give PO- (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -OH to introduce the required thiol function , After esterification of the 3 ^' end of the oligonucleotide, the nucleic acid in a concentration of 1-2X10 ^"4 M in application buffer (10 mM Tris, 1mM EDTA, pH 7.5 with the addition of 0.7 molar TEATFB) with 10 ^ -IO ^{' 1} molar Put 2-hydroxymercaptoethanol on the cleaned electrode gold surface and incubate for 2-24 hours.

To prepare the electrode gold surface z. B. freshly cleaved mica (muscovite platelets) are cleaned in an electrical discharge chamber with an argon ion plasma and then gold (99.99%) is applied by electrical discharge in a layer thickness of approximately 100 nm (Ron, Hannoch; Matlis, Sophie ; Rubinstein, Israel. Self-Assembled Monolayers on Oxidized Metals. 2. Gold Surface Oxidative Pretreatment, Monolayer Properties, and Depression Formation. Langmuir (1998), 14 (5), 1116-1121; Golan, Yuval; Margulis, Lev; Rubinstein , Israel. Vacuum-deposited gold films. I. Factors affecting the film morphology. Surf. Sei. (1992), 264 (3), 312-26.)

The metal surface is then cleaned with a 30% H ₂ O ₂ /70% H ₂ SO solution and then immersed in ethanol for 20 minutes. The surface is covered with bidest. Water rinsed one last time.

The preparation of commercially available precious metal electrodes can alternatively be carried out by polishing the electrode surface and subsequent rinsing. The subsequent electrochemical cleaning of the electrodes can, for. B. cyclic voltammetry in sodium hydroxide solution using a potentiostat, the electrodes several times in the potential range from -2.0 to +1, 2V against an Ag / Ag - Reference electrode (3M NaCI solution). As a counter electrode z. B. serve a platinum rod.

The connection of SH-modified oligonucleotides takes place e.g. B. by incubation of the cleaned Au electrodes with a solution of the corresponding oligonucleotide in phosphate buffer by dripping onto the gold disk and incubation for several hours in a water-saturated atmosphere.

The connection of an electron donor or acceptor unit, such as. B. pyrroloquinolinequinone, methoxatin, PQQ or ferrocene can be carried out on the monolayer-bound or in solution oligonucleotides.

The linkage of the redox units (PQQ, Sigma-Aldrich, Steinheim, D) to the primary amino group of the modified T-building block (aminomodifier dT) in the hairpin on monolayer-bound oligonucleotides is achieved by activating the PQQ carboxylate groups in buffer with a soluble carbodiimide and subsequent amide formation ,

The second method of obtaining oligonucleotide monolayers on gold containing redox units is to modify the oligonucleotides with redox-active electron donor or acceptor units, such as. B. PQQ and ferrocene-acetic acid in solution and subsequent immobilization on the gold surface.

A redox unit is linked by activating the carboxylate functions and then binding them to the primary amino group of the modified T building block (amino modifier dT) in the oligonucleotide.

For this purpose, PQQ is dissolved in water and mixed with N-hydroxysuccinimide and water-soluble carbodiimide. For the modification of the oligonucleotide, the activation solution is mixed with water and triethylammonium hydrogen carbonate solution. After adding the amino-modified oligonucleotide, the

Solution left. The reaction mixture can be worked up by

Size exclusion chromatography is done. The synthesis of the ferrocenacetic acid-modified oligonucleotides is carried out by activating the ferrocenacetic acid (e.g. with o- (benzotriazol-1-yl) -N, N, N \ N ^> - tetramethyluronium tetrafluoroborate (TBTU)) and subsequent coupling with the free one Amino group of the oligonucleotide. The activated ferrocene acetic acid is added to a solution of the modifying oligonucleotide in the presence of a base. After a reaction time of several hours, the oligonucleotide is cleaned up.

The covalent attachment of a Zn bacteriochlorophyll electron donor unit can take place after attachment of the nucleic acid to the electrode gold surface and a further washing step with water. For this purpose, the modified nucleic acid substrate with a solution of 3x10 ^"3 molar quinone 2- (CH ₂ -CH ₂ - CO ₂ H-UQ-50), 10 ^{" 2} molar EDC (3 ^' -dimethylaminopropyl) -N-carbodiimide and 10 ^{" 2} molar N-hydroxysulfsuccinimide in 0.1 M HEPES buffer (2- (4-2-hydroxyethyl) -1-piperazino) -ethanesulfonic acid, pH 7.5) The quinone used is obtained by cleaving a methoxy group by HBr (ether cleavage) as standard The resulting free hydroxyl group is reacted with an equimolar amount of CI-CH ₂ -CH ₂ -CO ₂ H and then purified by chromatography (Moore; HB and Folkers, K, Journal of American Chemical Society, 1966, 88, 5919-23; Daves , G. et al. Journal of American Chemical Society, 1968, 90, 4487-4493).

The modified substrate is washed with bidist. Water, again with an aqueous solution of 3x 10 ^"3 molar Zn bacteriochlorophyll (as free acid), 1, 5x 10 ^{" 1} molar (3-dimethylaminopropyl) carbodiimide, 2, 5x10 ^"3 molar hydrazine monohydrate and 1x10 ^{" 1} molar imidazole incubated for 16 hours at 23 ° C. The Zn bacteriochlorophyll as free acid is produced by incubation with trifluoroacetic acid.

(Hartwich, Gerhard; Fiedor, Leszek; Simonin, Ingrid; Cmiel, Edmund; Schaefer, Wolfgang; Noy, Dror; Scherz, Avigdor; Scheer, Hugo. Metal-Substituted Bacteriochlorophylls. 1. Preparation and Influence of Metal and Coordination on Spectra. J. Am. Chem. Soc. (1998), 120 (15), 3675-3683).

The hybridization or refolding of the nucleic acids to the double-stranded probes can take place before or after application of the modified nucleic acid on the electrode gold surface. The hybridization can e.g. B. according to Methods known to those skilled in the art are carried out in a buffered solution. Hybridized nucleic acid duplexes are preferably used in the manufacture of the probes. The hybridization takes place under controlled conditions with slow cooling in order to avoid mismatches as far as possible. Short-term heating to 95 ° C and slow cooling down to room temperature in a water bath have proven to be effective.

Applied single-stranded 5 ^* -phosphorylated (phosphate-on-phosphoramidite building blocks (Hörn, T. and Urdea, M., Tetrahedron Letter, 1986, 27, 4705)) and nucleic acids esterified to the thiol, which only have a short hairpin with free 3 ^' OH end, can be built up enzymatically with the help of a DNA polymerase to a double strand. For this, z. B. incubated a DNA structure with a mixture of the four nucleotide triphosphates with Klenow fragment of DNA I polymerase. The desired nucleic acid duplexes are obtained.

The coating, preferably a monomolecular coating, of the free electrode surface has proven to be advantageous. The surface of a gold electrode can e.g. B. with alkyl disulfides, alkyl thiols or hydroxyalkyl thiols after application of the double-stranded nucleic acid probes.

Individual assays that can be carried out with the aid of the double-stranded nucleic acid probes according to the invention are described below using the figures as examples.

1 describes an assay for the investigation of restriction enzymes or exonucleases.

A double-stranded DNA structure (11) with a restriction site and a light-inducible electron donor unit (12) is covalently bound to a gold electrode (13) as described in the previous examples. The DNA double strand (11) of the probe is in a restriction enzyme z. B. Pst1 (14) containing reaction solution cut into two fragments (17, 18). The activity is measured over time under the influence of light (15) using an evaluation device (16). Conversely, ligation of DNA-Zn bacteriochlorophyll degradation products, e.g. B. from the reaction solution generated above, after the solution has been freed from the restriction enzyme by RP-HPLC, enzymatically linked again. For this purpose, the fragment can be ligated in excess of a ligase in a DNA ligase buffer, the efficiency is detected amperometrically by generating an electron flow through the double-stranded DNA.

2 shows an assay for examining the binding of a transcription factor.

The addition of the transcription factor (21), e.g. B. Oct2A, TATA or Sp1 is carried out in a buffered solution on a template consisting of a gold electrode (22) which has double-stranded nucleic acid probes (23) with the respective binding site specific for the transcription factor. The double-stranded DNA probe sequences (23) are with a covalently bound light-inducible electron donor unit (24), for. B. Zn bacteriochlorophyll provided. After incubation with the respective transcription factor (21), the reading is carried out under light irradiation (25) with an evaluation unit (26). B. DNA fragments (27) not bound to the electrode surface of the respective transcription factor are carried in excess as a competitor.

In a somewhat modified process, e.g. B. by adding the sp1 competitor actinomycin D before and after adding Sp1, the DNA-protein interaction and the change in conductivity can be measured.

3 describes an assay for the detection of a DNA-DNA interaction. "Ä?

A self-complementary DNA (31) with a covalently bound light-inducible electron donor unit (34) is applied to the gold electrode (32) according to the examples listed above. A range of nucleotides is conceptually present at the 3 ^' end as a single strand. By hybridization with a longer oligomer, e.g. B. a 38 oligomer (33), which has a complementarity over a region of the other nucleic acid strand, the single strand structure is specifically filled. For this, z. B. an excess of buffered oligomer (33) is added to the bound probes and the mixture is then incubated. The excess of non-hybridized oligonucleotides is then washed out. After adding a second buffered solution containing complementary oligomeric single-stranded nucleic acids, this can be done in the present case e.g. B. a 38-oligomer (35) complementary to the oligomer (33), the re-hybridization under the influence of light (36) to form a hybrid (37) formed from the oligomers (33) and (35) with the aid of an evaluation unit ( 38) measured.

Fig. 4 describes an assay to investigate the intercalation of chemical substances in double-stranded DNA.

In such an assay, double-stranded nucleic acid probes (41) with a covalently bound light-inducible electron donor unit (46) are applied to a matrix-shaped gold electrode array surface (42) at the individual array positions (47). The bound DNA probes (41) are mixed with differently concentrated solutions of substances A, e.g. B. propidium iodide (43), B, e.g. B. ethidium bromide (44) and C, e.g. B. Syber ^® green (45) incubated. (The second dimension of the array surface is not shown in FIG. 4 for reasons of clarity). After exposure to light (48), the effect of substances A to C (43) - (45) on the double-strand structure is via a

Evaluation unit (49) tracked. The separately readable array positions deliver substance and concentration-dependent signals (50). FIGS. 5 to 13 show the measurement curves determined by means of differential pulse voltammetry in accordance with the following exemplary embodiments.

EXAMPLES

Example 1: Pretreatment of the gold electrodes

The Au electrodes are cleaned by polishing the electrode surface with an 3.3 μm alumina suspension (LECO, St. Joseph, USA) and subsequent rinsing with Millipore water (10 MΩ cm). The subsequent electrochemical cleaning of the electrodes is carried out in 0.2M NaOH using cyclic voltammetry (potentiostat: EG&G PAR 273A, GB), the electrodes first 5 times between potentials of -0.5 and -1.8V (against Ag / AgCI reference electrode ( 3M NaCI) and then 3 times between -0.8 and 1.0 V (feed rate 50 mV sec ^"1 ). A platinum rod serves as the counter electrode.

Example 2: Methods for modifying Au electrodes with redox-labeled oligonucleotides

Double-stranded nucleic acids were used for the modification. The hybridization takes place in the stock solution (1 mmol / l solution of the self-complementary single-stranded nucleic acids in water), which were heated to 95 ° C. in a water bath and slowly cooled to room temperature within 2 to 4 hours.

Method A: Modification of oligonucleotide monolayers with a redox label

Linking 5 ^' -SH oligos to gold Linking the 5 ^' -SH modified oligonucleotides (Interactiva, Ulm, D)

(For sequences, see Table 1), incubate the cleaned Au electrodes with a 100 μM solution of the corresponding oligonucleotide in 1M phosphate buffer (pH 6.6, Calbiochem; add 0.5mM dithiothreitol, (DTT, Sigma-Aldrich, WO 01 (31057 PCT / EPOO / 1020.

Steinheim, D) supports the connection ^"* v9on ferrocene modified oligonucleotides) by spotting 5 ul of this solution onto the gold disc and 4-5 hours of incubation at 4 ^C C in a water-saturated atmosphere. The resulting oligonucleotide monolayers are characterized by blocking the diffusion-controlled Fe (II / III) redox reaction in aqueous K ₃ / K ₄ Fe (CN) ₆ solution (salts from Merck, Darmstadt, D) (20 mM in 20 mM phosphate buffer pH 7) checked by means of cyclic voltammetry (CV).

Filling the monolayer gaps with 1, 6-mercaptohexanol The gaps between the individual oligonucleotide molecules on the monolayer, which are due to steric reasons, are filled by incubating the electrodes in a 1 mM solution of 6-mercaptohexanol (Aldrich, USA) in Millipore. Water (degassed) for 60-90 minutes at room temperature.

Linking a redox label (pyrroloquinoline quinone, methoxatin, PQQ) to the monolayer-bound oligonucleotides

The linkage of the redox label (PQQ, Sigma-Aldrich, Steinheim, D) to the primary amino group of the modified T building block (aminomodifier dT) in the hairpin is activated by activating the PQQ carboxylate groups with a water-soluble

Carbodiimide (EDAC, Sigma-Aldrich) and subsequent amide formation achieved. This reaction takes place in a PQQ solution (approx. 1 mg / ml in 10 mM HEPES buffer (pH 8) with the addition of 50 mM EDAC for 30-60 minutes at room temperature.

Method B: Synthesis of redox-modified oligonucleotides

The second method of obtaining redox-labeled oligonucleotide monolayers on gold is to modify the oligonucleotides with redox-active groups (PQQ and ferrocene-acetic acid) in solution and then immobilize them on the gold surface.

Connection of PQQ: The PQQ redox functions are linked by activation of the carboxylate functions and their subsequent binding to the primary amino group of the modified T building block (amino modifier dT) in the oligonucleotide.

For this purpose, 1.65 mg PQQ (Sigma-Aldrich, Steinheim, D) are dissolved in 25μl Millipore water (0.2M) and with 1.15 mg N-hydroxysuccinimide (NHS, 0.25M, Sigma-Aldrich) and 6 mg of a water soluble carbodiimide, ethyl diaminopropyl carbodiimide

(EDAC, 0.5M). After 30 minutes at room temperature, the resulting

The precipitate was dissolved again by adding 50 μl of DMF. After a further 15 minutes, the solution is diluted with a further 145 μl of DMF. To modify the oligonucleotide, 120 μl of the activation solution are mixed with 3.7 μl of Millipore water and 3.2 μl of triethylammonium hydrogen carbonate solution (TEK buffer, 1 M, Fluka, Buchs, CH). After adding 10 OD of the amino-modified oligonucleotide, the solution is left to stand at room temperature for 24 hours. The reaction mixture is worked up by size exclusion chromatography on a Sephadex G25 M column (Amersham-Pharmacia Biotech AB, Uppsala, Sweden).

The yields of oligonucleotide are usually between 70 and 90%. The oligonucleotides labeled with ferrocene acetic acid are synthesized by activating the ferrocene acetic acid with TBTU (Fluka) and subsequent coupling with the free amino group of the oligonucleotide. For this purpose, 4.9 μg of ferrocene acetic acid (Strem Chemicals, Kehl, D) are dissolved in 100 μl DMF in the first step. 75 μl of this solution are then added to a solution of 6 mg TBTU in 75 μl DMF and activated for 1 hour at room temperature. 10 μl of the oligonucleotide to be modified (as a 1 mM aqueous solution) are mixed with 3.2 μl of TEK buffer. Then 25 μl of the activated are added

Ferrocene acetic acid and 95 ul DMF. After a reaction time of 20 to 24 hours at room temperature, the oligonucleotide is purified as described above.

The electrodes are modified with the redox-labeled oligonucleotides in accordance with the instructions for the unmodified 5 ^' thiol-amino-modified oligonucleotides.

Example 3: Electrochemical experiments The electrochemical characterization of the redox-modified oligonucleotide monolayers takes place by means of cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in 20 mM phosphate buffer pH 7. In the case of PQQ-modified nucleic acid oligomers bound to a gold electrode, a signal of the reversible redox reaction occurs in one Potential of approx. -100 mV (against Ag / AgCI). This shows that the PQQ redox units are covalently bound via the nucleic acid oligomers on the modified electrode. The redox signals for ferrocenacetic acid-modified oligonucleotide monolayers occur at potentials of approx. +200 mV (against Ag / AgCI) and are generally broader and less intense than the corresponding PQQ signals.

Example 4: Experiments with intercalators:

Incubation of PQQ oligo electrodes (manufactured according to Example 2 Method A) in intercalator solutions:

The incubation was carried out for a PQQ-Zi6 oligonucleotide electrode Seq.lD No. 4 (Table 1) in intercalator solution (10 μM Hoechst 33258 (bisbenzimide) (Fluka) in 1M phosphate buffer (pH 6.6) for 20 minutes at room temperature.

Curve (51) in FIG. 5 shows the DPV measurement result of a Zi6-PQQ electrode in 20 mM phosphate buffer at pH6.6, (reductive scan, 10 mV / s, step width 500 ms, pulse width 20 ms, pulse height 25 mV) the incubation with Hoechst 33258 curve (52) shows the DPV measurement result after incubation in 10μM Hoechst 33258 in 1 M phosphate buffer for 20 min at RT.

The electrochemical comparison of the electrode shows a clear decrease in the PQQ oxidation signal to a maximum of 20% (ratio of the peak heights) of the initial value after incubation in the intercalator solution (FIG. 5). The structure of the oligonucleotide is consequently changed by intercalators in such a way that the electron transfer is strongly inhibited. Example 5: Experiments with DNAse I:

Incubation of a PQQ-modified oligonucleotide electrode (prepared according to Example 2, Method A) in a DNAse I solution (2U in 100 μl 1x T7 transcription buffer, as a kit from Promega, Madison, USA) for 15 (FIG. 6 curve (62)) or 30 minutes (FIG. 6 curve (63)) at 37 ° C. gives an almost complete suppression of the PQQ redox signals with subsequent electrochemical characterization with CV and DPV. The comparative measurement shown in Fig. 6 curve (61) was carried out before incubation with DNAse I. The effect clearly shows that the PQQ-modified oligonucleotide strands are cut by the protein, the course of the reaction can be followed, after 30 minutes the redox signal has decreased to 15-20%, after 60 minutes to 5-7% of the reference signal ,

Example 6: Experiments with transcription factors:

Examples 4 and 5 show sequence-unspecific interactions between proteins and DNA. In the present example 6, on the other hand, sequence-specific interactions between redox-labeled DNA oligonucleotides and transcription factors are investigated. The oligonucleotide sequences used are listed in Table 1 together with their specific binding transcription factors (TKF). The SP1 transcription factor (as an extract from E-coli) was kindly provided by Prof. Suske (University of Marburg), the Oct2a-TKF was purchased from Röche as a kit with buffer solutions (DIG Gelshift Kit). HeLa-Scribe Nuclear Extract (Gel Shift Assay Grade, with SP1-TKF as a component), also as a kit (Gel Shift Assay Systems), and TATA binding protein were obtained from Promega. Incubation of a PQQ-Zi-3 oligonucleotide electrode containing an oligomer Seq. ID No. 1 in an Oct2a-TCF solution (with 5 μl Oct2a / 90 μl buffer; composition according to the manufacturer's instructions) did not result in a change in the DPV redox signal after 60 minutes at room temperature (FIG. 7). Curve (71) in FIG. 7 shows the measurement before adding Oct-II, curve (72) shows one Measurement 30 minutes after Oct-Il addition, curve (73) 60 minutes after Oct-Il addition, without significant change in signal level. This means that non-specific interactions can be excluded. Incubation of the same electrode in an SP1-TKF solution (10% Suske-SP1 extract (10μl) in Roche Kit Binding Buffer 90 μl)) resulted in a significant reduction in the redox signal after just 30 minutes of incubation (Fig. 8 curve (81) Oxidation signal before incubation with SP1, curve (82) Oxidation signal after 30 minutes of incubation of the electrode with SP1 solution), which further decreased significantly after a further 90 minutes of incubation (FIG. 8 curve (83)). A quantitative analysis of the peak height and area ratios showed a reduction in the signal to 50% after the first incubation and a further reduction to 30-35% after the second incubation phase. While Oct2a does not change the redox behavior of the SP1-specific oligonucleotide monolayer electrode, the current flow during incubation with the SP1 transcription factor is significantly reduced. Thus the specific interaction between the SP1-TKF and the oligonucleotide strands (Zi-3 Seq. ID No. 1 has an SP1-specific recognition sequence, alternatively Zi-5 Seq. ID No. 3 was used) was detected.

In addition, experiments with ferrocene acetic acid modified DNA probes were carried out. The DNA electrodes were produced according to Example 2 Method B and applied to the gold electrode. The SP1-specific sample (Zi-3 Seq. ID No. 1) was analyzed using HeLa-Scribe Nuclear Extract (5.5 mg / ml Gel Shift Assay Grade, Promega) analogous to the Promega protocol (60 μl water 20 μl 5x gel shift binding buffer , 20 ul nuclear extract) incubated for 90 minutes. As a result, a clear decrease (order of magnitude 45-50%) of the redox signal (Fig. 9 curve (92)) compared to the reference measurement before the incubation (Fig. 9 curve (91)) was detected.

The experiment with TATA binding protein (10 fpu, Promega 10μl)) was carried out analogously. When using double-stranded nucleic acid probes with the sequence Zi-3 (Table 1, Seq. ID No. 1), a slight non-specific decrease in the redox peak (FIG. 10, curve (102)) compared to the reference measurement before the incubation (FIG 10, curve (101)) was determined. When a TATA-specific sequence (Zi-4 Seq. ID No. 2, Table 1) was incubated with the TATA binding protein (10 fpu) for 40 and 70 minutes, there was a significant decrease to 75% and 60% of the 3c, observing the output signal (FIG. 11 curve (112) after 40 minutes of incubation,

Curve (113) after 70 minutes incubation, curve (111) reference measurement before

Incubation with TATA binding protein). The control experiment with nuclear extract and a TATA-specific DNA sample (Zi-4 Seq. ID No. 2 (Table 1)) only led to a change in the peak shape (FIG. 12, curve (121) shows the redox signal before

Incubation, curve (122) shows the redox signal after 30 minutes of incubation with a HeLa cell extract solution) and not to a reduction in the redox signal.

In a further experiment, the influence of TATA binding protein (TFIID) on electron transfer in immobilized oligonucleotides with a specific recognition sequence (Zi-4 Seq. ID No. 2) was examined.

For this purpose, a cleaned gold electrode was treated with 10OμM Zi-4-ferrocene acetic acid in 0.9M phosphate buffer (+ 1mM DTT), incubation time: 4 hours at 4 ° C. The monolayer gaps are passivated with mercaptohexanol (1 mM solution in H ₂ O) for 1 hour at RT.

The measurement is carried out in an electrolyte solution (20 mM potassium phosphate buffer pH 7.0) using the DPV (pulse height 25mV, feed rate 10mV / s, pulse duration 20ms, step size 500ms, potential against Ag / AgCI (3M NaCI)).

Incubation of the electrode in pure gel shift binding buffer (Promega) is used to carry out control measurements, the proportion of TFIID protein in the incubation solution is varied (from 1μl TFIID + 90μl binding buffer to 10μl TFIID + 90μl binding buffer) and the corresponding Change in the electrochemical signal determined.)

The influence of the pure binding buffer on the level of the DPV redox peaks is negligible. (Fig. 13, curve (131) shows the ferrocene reduction signal from the reference measurement (after 30 minutes incubation with binding buffer) and curve (132) the redox signal of the electrode after a further 20 minutes incubation with binding buffer), the decrease in the signal levels is very low. After incubation of the electrode in TF II D protein solution (1 μl TFIID + 90 μl binding buffer) for 20 minutes, there is a significant decrease in the signal levels in the DPV (FIG. 13, curve (133)), which indicates specific interactions between the restriction enzyme and the binding site of the oligonucleotide strand is due. An increase in the enzyme concentration results in a greater decrease in the signal with the same incubation time. 13, curve (137) shows the effect of a 20 minute incubation of the electrode in 5 μl TFIID + 90 μl binding buffer on the reduction signal, curve (138) shows the corresponding signal after 20 minutes incubation in 10 μl TFIID + 90 μl binding buffer) ,

In addition to the concentration effect of the transcription factor, it was also possible to measure the time dependence of the connection of the transcription factor TFIID to the double-stranded nucleic acid probes. The reduction signal of a solution of 1 μl TFIID + 90 μl binding buffer continues to decrease after 40 minutes of incubation (Fig. 13, curve (134)). After this, an equilibrium has been established under the given reaction conditions; the signal level does not change further over time (Fig. 13, curve (136) shows the reduction signal of a solution of 1 μl TFIID + 90 μl binding buffer after 70 minutes of incubation, curve (135) after 160 minutes of incubation). FIG. 13, curve (139) shows the increase the binding events from a solution of 10μl TFIID + 90μl Binding Buffer after a total of 40 minutes incubation compared to the 20 minutes incubation of the equally concentrated solution (curve (138)).

O 01/31057

36

Table 1 Sequences of the Oligonucleotides Used (Interactiva, Ulm, D)

In the oligomer sequences in Table 1, X represents a commercially available aminomodifier dT building block (formula I), the nucleotide building block located at the 5 ^' end of the oligomer is modified on the C6 thiol (available from Interactiva).

Formula I.

Claims

O 01 / 31057Patent claims: • 3?

1. Nucleic acid probes containing an at least partially double-stranded nucleic acid sequence bound to a conductive surface to the at least one electron donor unit or at least one electron acceptor

Unity is bound.

2. Nucleic acid probes according to claim 1, characterized in that the attached double-stranded nucleic acid probes are composed of two complementary nucleic acid molecules.

3. Nucleic acid according to claim 1, characterized in that the attached double-stranded nucleic acid probes consist of nucleic acid sequences which contain self-recognizing domains.

4. Nucleic acid probes according to one of claims 1 to 3, characterized in that the double-stranded nucleic acid probes have the same nucleic acid sequence.

5. Nucleic acid probes according to one of claims 1 to 3, characterized in that the double-stranded nucleic acid probes have different nucleic acid sequences.

6. Nucleic acid probes according to one of claims 1 to 5, characterized in that the double-stranded nucleic acid probes are applied to a conductive array surface.

7. Nucleic acid probes according to one of claims 1 to 5, characterized in that the double-stranded nucleic acid probes are applied to an unstructured conductive surface.

8. Nucleic acid probes according to one of the preceding claims, characterized in that the double-stranded nucleic acid probes are stabilized by bridge bonds. 3 <3

9. Nucleic acid probes according to one of the preceding claims, characterized in that the double-stranded nucleic acid probes are stabilized by disulfide bridges.

10. Nucleic acid probes according to one of the preceding claims, characterized in that the double-stranded nucleic acid probes contain double-stranded DNAs, double-stranded RNAs or DNA-RNA hybrids.

11. Nucleic acid probes according to one of the preceding claims, characterized in that regenerable and / or inducible electron donor or electron acceptor units are covalently linked to the nucleic acid.

12. Aggregates containing double-stranded nucleic acid probes according to one of the preceding claims 1 to 11 and substances which are selected from the group of substances which interact directly with double-stranded nucleic acids.

13. Aggregates according to claim 12, characterized in that the directly interacting substance is a protein and / or a peptide and / or a single-stranded and / or a double-stranded nucleic acid and / or a mimetic and / or an intercalating and / or a nucleic acid-damaging substance and / or is a cytostatics.

14. Aggregates according to claim 12 or 13, characterized in that the aggregate contains further indirectly interacting substances.

15. A method for the detection of interactions between double-stranded nucleic acids and interacting substances, characterized in that a) probes according to claims 1 to 11 are exposed to a reaction medium and b) the change in electron flow generated is measured by the probes.

16. The method according to claim 15, characterized in that the reaction medium is a solution which contains at least one interacting substance.

17. The method according to claim 16, characterized in that the reaction medium is a solution containing at least one interacting polymerases, transcription factors, repressors, enzymes and / or their inhibitors and / or activators and / or mimetics and / or intercalating and / or nucleic acid-damaging substances and / or contains cytostatics.

18. The method according to claim 15 to 17, characterized in that the reaction medium contains single and / or double-stranded nucleic acids.

19. The method according to claim 15, characterized in that the

Reaction medium contains DNA-damaging or carcinogenic substances.

20. The method according to any one of claims 15 to 19, characterized in that the reaction medium is an aqueous, saline solution.

21. The method according to any one of claims 15 to 20, characterized in that the reaction solution is buffered.

22. The method according to any one of claims 15 to 21, characterized in that the detection takes place at a temperature between 4 and 40 ° C.

23. Use of the double-stranded nucleic acid probes according to one of claims 1 to 11 for the detection of interactions, of equilibrium constants, of competitive effects or reaction rates, between double-stranded nucleic acids and substances which interact directly and / or indirectly with them. O 01/31057

24. Analytical comprising double-stranded nucleic acid probes according to claims 1 to 11 for the identification of factors which act directly and indirectly on the double-stranded nucleic acid probe.

25. Analytical containing double-stranded nucleic acid probes according to the

Claims 1 to 11 for the identification of nucleic acid sequences which interact with directly or indirectly interacting substances.

26. A method for producing double-stranded nucleic acid detectors comprising: a) the synthesis of a single-stranded nucleic acid, b) the intermolecular hybridization with a nucleic acid containing a complementary domain, c) the covalent attachment of the electron donor units or electron acceptor Units to the nucleic acid and d) the covalent attachment of the nucleic acid to a conductive surface.

27. A method for producing double-stranded nucleic acid detectors comprising: e) the synthesis of a single-stranded nucleic acid containing self-recognizing domains separated by a bridge, f) the intramolecular hybridization of the self-recognizing domains of the single-stranded nucleic acid, g) the covalent attachment of the electron donor Units or electron acceptor units to the nucleic acid and h) the covalent attachment of the nucleic acid to a conductive surface.

28. A method for producing double-stranded nucleic acid detectors according to claim 27, characterized in that the bridge consists of at least one nucleotide.

29. A process for the preparation of double-stranded nucleic acids according to claim 27, characterized in that an artificial at least one-atom bridge is a disulfide, stilbene dicarboxyamide, ruthenium complex, hexaethylene glycol, Terephthalimide, branched or unbranched diol, S ^' -amino modifier-CPG or branched phosphoramidite bridge is introduced.