DE19950969A1 - Double-stranded nucleic acid probes and their use - 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

The present invention relates to electronically readable double-strand Nucleic acid derivatives and their use for quick and easy Detection of interactions between double-stranded nucleic acids and factors interacting directly or indirectly with them, in particular with Proteins, peptides, single-stranded or double-stranded nucleic acids or nucleic acid damaging substances.

Double-stranded nucleic acids play in the living cell and also in many Viruses, especially as carriers of the hereditary system, play a crucial role. there they are subject to natural systems such as B. a cell more diverse inner and external influences. Interactions between proteins and double-stranded DNA are of great interest because of such interactions decisive influence on the transcription or repression of individual genes and thus have on the phenotype of the corresponding organisms. But also the Replication of the genes in mitosis or meiosis, the restriction z. B. more viral Nucleic acids or the packing and unpacking of eukaryotic nucleic acids in the chromosomes are other important processes in living cells that controlled by the complex interplay of proteins and nucleic acids become. In addition, other chemical substances can also be used double-stranded nucleic acids interact, here is representative only the class of carcinogenic intercalating or substances harmful to nucleic acid. Even nucleic acids themselves, whether single-stranded or double-stranded, can with their double-stranded Relatives, e.g. B. in the recombination, insertion or transposition, in Interaction.  

When examining such processes, whether it is the identification of the individual biologically active building blocks or to clarify the mechanisms, the interaction of these building blocks, the knowledge of molecular Molecule interactions play a central role. Developing new more efficiently The focus is on detection methods for such interactions 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 been described measuring arrangements that allow it Hybridization events directly on single strand of nucleic acids to capture. This detection method is based on the fact that guanosine-bound DNA is oxidized via ruthenium complexes and this process is carried out using Cyclovoltametry can be demonstrated.

In the publications WO 95/15971, WO 96/40712 and DE 199 90 701 A1 Described procedures which double the electrical conductivity Use nucleic acid hybrids against single-stranded nucleic acids. Both use their process to only use single-strand Detect nucleic acid sequences. For this purpose, to prove the Existence of single-stranded nucleic acid target sequences complementary Single-stranded nucleic acid probes used that are covalently bound at least one electron donor unit and at least one electron Acceptor unit, preferably configured as an electrode. I'm electric conductive probe-target sequence hybrid is thermal or over a current-inducing signal, such as. B. light, a detectable electron flow generated.

The methods described are based on the detection of hybridized single-stranded nucleic acids limited. The proof is limited to that mere presence of a target sequence in the medium to be examined and leaves  moreover, no statements about the biological activity of the interacting Nucleic acids too.

Interactions of double-stranded nucleic acid sequences are to be investigated must be based on other methods, e.g. B. on gel electrophoretic methods be resorted to.

In the gel shift method (Fried, M. & Crothers, D. M. "Equilibrium and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis "(1981) Nucleic Acid Res. 9, 6505-6525; Garner, M.M. & 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) is a radioactive DNA fragment with a incubated protein extract to be examined. Does the protein extract contain a DNA Fragment-binding protein can be detected by two bands 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 forms the footprint technique (Watson, J.D. et al. Recombined DNA, 2nd edition 1993, Pp. 143-146, Spektrum Akademischer Verlag). In doing so, a radioactively marked DNA sequence an incomplete restriction degradation, e.g. B. with DNAse I subject. The sequence on the appropriate place protected from degradation, against the free sequence The corresponding bands are missing in the electrophoretic fragment detection.

These methods include the preparation of the DNA fragments and the Protein extracts, the labeling of DNA fragments, the formation of a Reaction approach and the gel electrophoretic separation. The implementation These indispensable verification steps are very labor-intensive to use and time consuming. In addition, the proof can not be consistently under native conditions are performed and use  gel-electrophoretically separated reaction mixture components for further Investigations are difficult.

Proceeding from this, the present invention is based on the object Detection of interactions of double-stranded nucleic acids of novel probes for To provide and procedures for quick and easy to perform Provide evidence 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 ones Nucleic acids or single-stranded nucleic acids with self-recognizing Domains attached to a conductive surface, preferably to an electrode are bound. The nucleic acid section which is present as a duplex is included at least one electron donor unit or alternatively with at least one Electron acceptor unit linked, with at least a portion of the investigating nucleic acid region between the electrode and the electron Acceptor unit or electron donor unit. This section forms the Detection point. The binding of the individual probe subunits can be covalent or via stable supramolecular interactions, such as from the Waals Interactions, dipole interactions, in particular Hydrogen bonds or ionic interactions take place.

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 molecular hybridization reaction" Annu. Rev. Biochem. (1970), 39 , 131-150), the formation of a double strand in the case of self-complementary single strand nucleic acids being caused by intramolecular refolding. In the synthesis of such single-stranded nucleic acids, two domains with complementary sequences are advantageously linked covalently via a bridge comprising at least one nucleotide, preferably a bridge consisting of 4 to 6 nucleotides, in particular of thymine nucleotides, or via an artificial, at least one atomic bridge. Such artificial bridges are e.g. B. Disulfide bridges (Hetian Gao et al., "Stabilization of double-stranded oligonucleotides using backbone-linked disulfide bridges", Nucleic Acid Research, 1995, Vol. 23, No. 2, 285-292), stilbene dicarboxyamide bridges ( Lewis, FD et al., "Distance-DEPENDING electron transfer in DNA hairpin" Book of abstract 215 ^th ACS National meeting, Dallas, vol. 29, p Physics 255, 1988), Ru complex bonds (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 (GIe nResearch), which is preferably built on a thiol support, or branched phosphoramidite bridges. A great advantage of such self-complementary sequence sections containing nucleic acids 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 takes place independently 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. Can also be used are their hybrids and their modified derivatives. To the modified Derivatives include in particular nucleic acids that are modified on sugar Contain nucleotides such as 2'O-methyl nucleotides or 2'O-allyl nucleotides. A Modification of the phosphate group, such as. B. phosphoramide, phosphorothioate or methylphosphonate is also possible. It can also contain nucleic acids none or one not used in natural nucleic acids are used occurring sugar, such as. B. the peptidyl nucleic acids Pyranosyl nucleic acids. In addition to those modified on the sugar-phosphate backbone Nucleic acids can also be used as probe components are the non-naturally occurring nucleotides, such as xanthine, hypoxanthine or inosine.

Double-stranded DNA or RNA nucleic acid sequences are preferred in the Probes installed. Naturally occurring are of particular interest Proteins or peptides binding DNA sequences, especially in the Gene regulation involved DNA sequences, such as. B. operator or Promoter sequences. Depending on the application problem, the use of Consensus sequences or allele specific or organism specific Sequences, may be appropriate.

The length of the nucleic acid sequence between the electrode and the electron Donor units or electron acceptor units are preferably between 2 and 100 base pairs, particularly preferably between 5 and 50 base pairs. The double-stranded area must linker to the nucleic acid electrodes are sufficient to ensure an electron flow. Single-stranded areas can be via the double-stranded, between the electrode and the electron Donor units or electron acceptor units lying detection region  reach out. The double-stranded detection region can single-strand breaks in the Sugar-phosphate backbone included.

The formation of a double strand can be achieved through chemical modifications lead to inter- or intramolecular bridge bonds, such as B. Disulfide bridges, or other non-natural strand subunits chemically be stabilized. Another way of stabilizing the Nucleic acid duplex sections offer the targeted introduction of thymine Nucleotides on two opposite positions in the duplex and subsequent radiation-induced thymidine bridge formation. Analog can also azido nucleotides or psoralen derivatives for photoinduced Bridging can be used (Fabrega, C. et al., "Studies on the Synthesis of Oligonucleotides 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). So can for example the degradation of the nucleic acid duplex by exonucleases be prevented. The sensitivity of double-stranded nucleic acids against undesirable, process-related influences such as temperature of the medium to be examined is also lowered.

All can be used as electron donor units or electron acceptor units Molecules or parts of molecules act that are able from their ground state or to release electrons from an excited state or in Ground state or in an activated state to take up electrons. The Excitation of an electron donor unit can e.g. B. by exposure to light or Ionization take place, the activation of an electron acceptor z. B. by Ionization also takes place by means of light or by means of chemical ionization. Proven electron donors and acceptors are e.g. B. metal complexes or organic redox-active compounds as described in the patent application WO 96/40712 are described or naturally occurring or modified photo-inducible redox-active reaction centers such as z. B. in the Patent application DE 199 90 701 A1 can be used.  

The covalently bound electron donor units or electron acceptor Units can be integrated into multimolecular electron transfer systems his. Firstly, the one acting in the double-stranded nucleic acid probe Electron donor unit (electron acceptor units) via dissolved Electron donors such as B. anions (cations) can be charged regeneratively, being a closed circuit with the double-stranded nucleic acid probes is built up as a resistance. On the other hand, analogous to that of course occurring reaction centers, such as. B. chlorophyll, also others redox-active electron transfer units with the covalently bound Electron donor or acceptor unit associated.

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. 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 ions such. B. Fe (CN) ₄ ^3- or Fe (CN) ₆ ^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, for. B. via a Thiolgruppe (Miller, C. et al. J. Phys. Chem. 95: 877 (1991); Chidsey, C.E.D., Sience, V. 251, p. 919, (1991)) or about phosphorothioates or Phosphorodithioates are done on a gold surface. But also 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 one higher conductivity than the attached nucleic acid is possible. The Using linkers with conjugated double bond systems allows one  Electron conduction along the linker structure to the electrode surface. The enables passivation of the free electrode surface. The The double-stranded nucleic acid probes can be connected to an unstructured, coherent electrode surface or on a structured, e.g. B. matrix-shaped electrode array surface. The structuring takes place so that the individual array positions of the electrode are independent of each other can be read out. The individual array positions can be nucleic acid Probes with specific sequences can be assigned or there can be one The reaction chamber is compartmentalized. So at electronic reading of the electrode a high degree of parallelization can be achieved.

It has surprisingly been found that the double-strand Nucleic acid probes offer the possibility with nucleic acid double strands to detect interacting factors via a change in conductivity. Interacting factors are chemical substances or radiation that are associated with a nucleic acid duplex interact and a change in the primary, secondary, tertiary or quaternary structure of the double strand trigger. These structural changes lead to a significant change the electrical resistance of the duplex structure and thus to a change the electron flow along the double-stranded nucleic acid. Probably have in addition to the structural changes of the double-stranded nucleic acids by interacting factors other effects such. B. the insulating effect of proteins or peptides bound to nucleic acids or the direct Participation of intercalating substances, such as B. aromatic molecules electron conduction through the nucleic acid duplex also has an influence on the resulting electron flow. Usually takes place between the probes and the interacting substances form an aggregate, the aggregates stabilized via intermolecular forces or even via covalent bonds become.  

Changes in the electrical conductivity of double-stranded nucleic acids z. B. by binding proteins or peptides, such as antibodies, Polymerases, transcription factors, enhancers or repressors, their Activity in turn through indirectly acting substances such as. B. via inductors or modifying enzymes that can be modulated. Organic molecules such as some hormones can also be used directly or in Connection with proteins or peptides, such as. B. hormone receptors with double-stranded nucleic acids interact. Single-stranded nucleic acid can also z. B. with triplex formation with double-stranded nucleic acids to interact. But also saline solutions, mimetics or in the nucleic acid Duplex intercalating substances, including cytostatics such as 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 nucleic acids damaging Substances directly attack the primary structure of the nucleic acids.

Endonucleases cleave nucleic acids, exonucleases build nucleic acids from a free strand end, ligases form covalent bonds between terminal nucleotides, while topoisomerases and single strand breaks - create links. But also mimetics, nucleic acid damaging Substances and cytostatics, e.g. B. alkylating on nucleic acids or cross-linking act like platinum complexes, e.g. B. cisplatin, methanesulfonates, e.g. B. Busulfan, or n-nitroso compounds, e.g. B. Carmustine can attack covalent bonds and / or form new covalent bonds.

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

The use of the double-stranded nucleic acid derivatives according to the invention as probes offers many advantages. All interactions with one  Double stranded nucleic acid probe is exposed and which has an effect on that can have electrical conductivity. The measurement can be coulombmetric, impedometric, preferably frequency-dependent resistive or capacitive, voltametric, potentiometric or ampereometric. The disruptive effect of ions directly discharged from the electrode Reaction solution is surprisingly very low. To the The electron flow through the sensor can further increase the sensitivity double-stranded nucleic acid probe can be frequency-modulated. The modulation can over using photo-inducible electron donor units Flashes of light occur. About the number of probes applied or about the Extending the measuring time can also result in very small changes in conductivity can be detected. The number of probes is preferably selected so that the through the interaction between and with the double-stranded nucleic acid resulting current change at least in the nA- Area.

Another advantage of the present probes is their high compatibility with a wide variety of reaction media. In particular, the use of Raw extracts as the reaction medium or the use of reaction media with a salt concentration that corresponds to the natural conditions of interest. Furthermore, the kinetics of double-stranded nucleic acid Interactions examined and equilibrium constants of double-stranded nucleic acids with interacting substances determined become. This offers the possibility of antagonists and allosteric Find interactions with other factors and their activity too determine. It can be used as a multi-step reaction be carried out in which individual factors during the measurement process are pipetted successively to the reaction solution.

Other objects of the invention are methods for the detection of Interactions between and with double-stranded nucleic acids  interacting factors and the use of the invention Double-stranded nucleic acid probes therefor.

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

For this, z. B. known regulatory DNA sequences of a gene in the Probes installed. The probes are applied to an electrode surface, so the sum of the change in electrical conductivity of the double strand DNA probes is measured. In the free, unassociated state, the Reference electron flow through the probes in the presence of a reference solution determined. Alternatively, the reference value can also be electrochemical Standard cell / electrode can be determined. The reference solution contains ideally all components of the reaction solution except those to be tested Factors. By gradually adding the individual substances to be tested the reference electron flow is modulated. The change in electron flow provides information about the interaction between the test substance and the probe sequence, the strength of the change in the electron flow provides information about the strength of the interaction and the equilibrium constant of the Binding reaction. So all direct interactions of the DNA are with the directly interacting substances, such as polymerases, transcription factors or repressors measured. An identification directly with the DNA duplex interacting substances is possible. Especially the quick one  Locating transcription factors with tissue-specific activity is via the Use of probes the known tissue-specific promoter sequences included possible.

The effect of others that influence the activity of these substances indirect factors, can change the equilibrium constants of RNA polymerases, transcription factors or repressors can be determined indirectly. This can be done by either different factors containing reaction solutions are tested or the indirectly acting Factors are gradually introduced into the reaction solution. Thereby can also interactions between individual substances within the regulatory signal chains can be determined.

For example, if an operator-repressor complex becomes a repressor added strong inductor to the reaction solution, goes Equilibrium constant towards zero and the measured electron flow equals the reference electron flow.

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

Another example is the lexA repressor in E. coli, which contains numerous genes regulated that are involved in DNA repair. Through single-stranded DNA bound recA proteins are proteolytically cleaved by lexA and thus by associated operator replaced. The flow of electrons within the double strand As a result, the DNA probe converges to the reference value.  

For a given inductor concentration, the degree of approximation of the Electron flow to the reference electron flow is a measure of the strength of the inductive effect. By varying the inductor concentration in contrast, concentration-dependent effects of the inductor on the repressor or a linear dependence of the induction on the inductor concentration, be determined.

If the repressor is inactive in the reaction mixture, the electron flow remains on the Reference value. By adding other substances, their repressions activating effect on the repressor can be tested. Such a Regulatory mechanism lies 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.

The equilibrium constant of binding of RNA polymerases or transcription factors on DNA promoters and the To determine the effect of their cofactors and their modifications. To probes can be used which contain a promoter sequence. It can then a reference value can be determined by adding individual Factors is modulated. A change in the reference value shows one Interaction of the added substance. Then the Identification of the RNA polymerases binding in the promoter area and Transcription factors occur.

If you use e.g. B. the SV 40 promoter for producing the double-stranded Nucleic acid probes can bind specifically to this promoter binding transcription factor Sp1 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, can then activate them or inhibitory influence on the transcription factor-DNA binding measured become.

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 one Tumor suppressing morning. p53 can be added by adding a phosphorylase Presence of a suitable phosphate donor, e.g. B. ATP, phosphorylated and thus be activated. The electron flow changes due to the binding of the phosphorylated p53 to the probe sequence steadily until a concentration dependent saturation value. From the slope of the electron flow-time curve can then, in addition to the pure interaction, information can be obtained via the reaction rate. The greater the slope of the The curve is the higher the reaction speed, in the example the Phosphorylation reaction.

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

A particular advantage of the method described here is the possibility of Investigation of allele-specific interactions between regulatory active substances and the associated DNA regulatory sequences. So can the different activity of DNA sequences and DNA binding proteins  or wild type peptides and mutant DNA sequences, proteins or Peptides in separate batches regardless of phenotypically recognizable Changes are examined. In particular, the study of recessive distinctive features become possible.

In addition, gene product mixtures of different alleles can be one Gens that interact directly or indirectly with double-stranded DNA to be examined. Binds e.g. B. a wild-type transcription factor to one Promoter and its mutant not result in an extract of a transcription factor the largest electron flow change from a homozygous wild-type organism by the double-stranded nucleic acid probe, a transcription factor extract a homozygous mutant organism does not change the conductivity the probe. Heterozygous organisms result in the transcription factor an electron flow through the double-stranded nucleic acid probe between the both extracts from the homozygous organisms, in this case must Extracts with a certain concentration can be used.

The procedure is not based on the identification of substances involved in the Participate in restricted gene regulation, so z. B. also histones, helicases, Bind topoisomerases or ligases to double-stranded DNA. While Helicases and topoisomerases usually block the flow of electrons through the probes can reduce if double-stranded nucleic acid probes are used, the contain one or more bond breaks in the sugar-phosphate backbone that ligated, also lead to an increase in electron flow.

The loss of conductivity is observed in nucleases, the double strand recognize structures, break them down. The flow of electrons through the probes drops steadily and goes to zero if the reaction time is long enough. By adding Such an effect is not observed for substances blocking nucleases.

With the help of bound RNA-DNA hybrid probes, there is the possibility, for. B. To investigate RNAsen that recognizes RNA parts within a double strand  and breaks down. The loss of the RNA building blocks generates a remaining one Single strand, which is now detectable due to the loss of conductivity. To the entire RNA does not have to be broken down, even the loss of one or two Ribonucleotides between the electron donor units or electron Acceptor units and the electrode has a drastic drop in the Conductivity in the double strand results. Analogous can with DNA double strand Probes DNAsen are detected.

The most significant effect on the conductivity of double-stranded nucleic acid Probes have restriction enzymes such as e.g. B. Hind II a smooth Double strand break catalyzes or EcoR I or PST I move the one Double strand break generated, provided a suitable restriction site in the Double strand sequence is present. By separating the two Nucleic acid strands between the electron donor units or electron Acceptor units and the electrode, the electron flow comes through the The double strand completely stops.

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 ⁿ sequences, where n is the number of base pairs, are applied to the electrode in 4 ⁿ separately electronically readable fields. (Electrodes with values of n = 8 can now be manufactured without any problems, but the technical development with regard to the integration density has not yet been completed.) Double-stranded nucleic acid-binding factors modulate the conductivity of the array positions, which 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 which in turn can be used to identify the associated genes using conventional hybridization methods.

This method can also be used for site-specific mutagenesis produced modified nucleic acid sequences or transcription factors be rated. 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 high active transcription factors is possible. Such promoter / transcription factor complexes can be used to construct novel expression vectors be used.

Extracts of regulatory DNA fragments cannot precisely specified sequences of a certain gene into the double-stranded Nucleic acid probes can be installed. With known to these sequences binding factors of certain concentration can then be determined 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 made directly into a double-stranded Nucleic acid probes / probe binding protein can be added. Measured the competitive effect of the nucleic acid fragment extract on the Measuring system.

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

The at least partial separation of the nucleic acid double strand of the probe due to the hybridization of those competing as binding partners leads single-stranded nucleic acids with a complementary probe sequence to triplex structures or to a resolution of the probe duplex Umhybridization. The conductivity of the probe is interrupted or changed. Consequently, hybridization events can be detected with this method become.  

A probe destabilizes a double-stranded nucleic acid sequence hybridization with a solution in solution is used single-stranded nucleic acid relieved. A destabilization towards one DNA double strand can e.g. B. achieved by the use of DNA / RNA hybrids become. The DNA / RNA hybrid pairing can also be modified of the DNA strand are weakened to phosphorothioate nucleic acid.

The facilitated hybridization with a single strand in solution can also by an overhang from the double-stranded probe Single strand end can be reached. The hybridization then takes place with the Single strand ends and parts of the double strand structure instead. Is in the recognized Double strand structure can then introduce a break in the nucleic acid backbone the newly formed hybrid even detach itself completely from the probe and the Interrupt electron flow within the probes.

In addition, recombination events can be detected and on the Recombination involved factors, especially enzymes, can be identified. At the complete recombination of a double-stranded nucleic acid with the Probe sequence can the electron donor or electron acceptor Units are separated from the electrode. There can be no more electron flow take place through the probe. However, the recombination event does not have to be completely completed, the formation of a triplex structure, which the Double-stranded probe structure suffices to reduce the to measure electrical conductivity. Put the reaction solution recombination-promoting factors such. B. is the recA protein from E. coli a faster decrease in electron flow or increased total electron flow decrease measurable.

The probes contain integration points for transposable elements transposition events can also be determined. Because the strength of the Electron flow in the double strand of the probe with increasing distance, that is increasing number of base pairs between the electron donor unit or  Electron acceptor unit and the electrode decreases, so does the Electron flow from integrating a transposon in the probe sequence. At the The course of the back reaction is a corresponding increase in the electron flow detectable.

The use of double stranded nucleic acid probes is also for detection of nucleic acid damaging substances or for the detection of Nucleic acid damaging effect of chemical substances suitable. The Damage to a double-stranded nucleic acid can occur in different ways are caused. So some substances, especially aromatic ones Substances like ethidium bromide or acridine in the nucleic acid duplex structure intercalate. Other substances can react with nucleic acids, e.g. B. strong bases that hydrolyze the nucleic acids, benzpyrines that adhere to the Add nucleic acids or peroxides that easily form radicals and radicals Trigger chain reactions within the nucleic acid. The change in Nucleic acid structure caused by these substances changes the Conductivity of the double-stranded nucleic acid probes.

Such a test can be carried out in a compartmented reaction vessel where the bottom surface is formed by the electrode in array form. The probes are of uniform density on the individual array positions upset. Each reaction compartment can have a different one Reaction solution containing test substance are added. It can too Reaction solutions are prepared in the same test substance contain different concentrations. So a simple test for Identification of DNA-damaging and thus carcinogenic substances be created.

Double-stranded nucleic acids also interact with im Reaction medium dissolved substances, especially with destabilizing ionic compounds. But also interactions with the solvent itself can affect the conductivity of the probes. For example, at  Dehydration of the DNA double helix of the B type into the A type. This Transition can be measured using the new types of probes.

The reaction media or solutions can be any solvents and Contain additives. Aqueous, ionic are preferred as reaction solutions Buffer solutions that interact with chemical double-stranded nucleic acids May contain substances used. The direct use of raw extracts as a reaction medium is also possible. Interaction detection between the double-stranded nucleic acid probe sequences and the thus interacting substances takes place preferably under physiological conditions in 10 to 200 mM aqueous, saline solutions at pH 7-9 instead. As A Tris buffer is preferably used for the buffer. The measuring temperature is preferably between 4 and 40 ° C. A deviation from these measurement conditions may be appropriate in individual cases. For example, an increased Measuring temperature for finding proteins with temperature-stable activity, such as the Taq polymerase used for the PCR.

This results in a variety of uses for 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. Via a separation of cell extracts or substance libraries can quickly become active on a double strand Nucleic acid binding components can be detected. The process can be operated continuously in an "online" measuring process.

To clarify the invention, some are illustrative below Given examples.  

example 1 Representation of DNA probes

The following DNA sequences are used, which have a transcription factor have recognizing domain (bold).

DNA domain recognizing Sp1:

DNA domain recognizing Oct2A:

The nucleotide sequences are used for binding to a gold electrode Thiol groups and for connecting the Zn bacteriochlorophyll amino groups built-in.

The oligobucleotides used are with a DNA synthesis method, the 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 (20d), 1859-62). It is a method with which both unmodified and modified nucleic acids, e.g. B. RNAs phosphorothioates and PNAs in high Purity and yield can be displayed specifically and routinely today is used.

For the synthesis, the desired synthesis building blocks in 1 µmol scale a predetermined order on solid CPG carrier material with the help of a Expedite 8909 Synthesizer from PerSeptive Biosystems built up. To do this, the 0.1 M solutions of corresponding Phosphoramidite building blocks and 0.5 M tetrazole solution used. The building blocks are due to their sensitivity in dry acetonitrile (water content < 30 ppm) dissolved.  

The necessary modifications are made with the help of amino, SH or Phosphate-on-phosphoramidite building blocks (Eurogentec, Clontech) and Carrier materials ("SH-CPG" chemicals, Glen Research, 3'-phosphates CPG Glen Research).

Introduction of thiol modification

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

Introduction of amino modification

The amino group is 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 at the Electrode side of the double-strand structure are specifically introduced.

Processing of the modified oligonucleotides

After completing the syntheses, the oligomers become more aqueous Cleave ammonia solution (25-32%) basic from the CPG carrier and over incubated for several hours at 55 ° C. In the presence of thiol groups Add DTT to a concentration of 50 mM. Then the Concentrated solution and freed of ammonia and several times with water coevaporated. Oligonucleotides that are using the modified thymidine building block 1 ml of a methanolic solution (methanol: water, 4: 1) for 17 hours Incubated at room temperature, this removes the oligonucleotide from the CPG carrier cleaved. The supernatant, which contains the oligonucleotide, is mixed with 1.5 ml Neutralized 2 M triethylammonium acetate solution.

After determining the absorption at 260 nm, the raw products are made using RP-HPLC (Lichrochart 100 RP-C18) of demolition products and split off Protection groups exempted. A gradient of acetonitrile in 0.1  Triethylammonium acetate buffer, used from 0-60% in 60 minutes. The main peak is isolated, spectrometrically quantified at 260 nm and lyophilized and in 500 µl 50 mM DTT dissolved. The solution is incubated for 30 minutes at 37 ° C and in Connection using a PD-10 column according to the manufacturer's instructions (Pharmacia) desalted and for covalent connection to the electrode on the gold surface pipetted. The buffer solutions used were previously saturated with argon.

Alternative method for modifying the oligonucleotides with a thiol group

As described above, the sequence with phosphoamidite nucleotide building blocks is built up on a 3'-phosphate CPG in the desired order. An AminoModifier II (Clontech) is used as the 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 1-2 × 10 ^-4 M in application buffer (10 mM Tris, 1 mM EDTA, pH 7.5 with addition of 0.7 molar TEATFB) with 10 ^-4 - Put 10 ^-1 molar 2-hydroxymercaptoethanol on the cleaned electrode gold surface and incubate for 2-24 hours.

Preparation of the gold electrode surface

For this purpose, 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 approx. 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. Sci. (1992), 264 (3), 312-26.) The metal surfaces are cleaned with a 30% H ₂ O ₂ /70% H ₂ SO ₄ solution cleaned and then immersed in ethanol for 20 minutes. The surface is covered with bidest. Water rinsed one last time.

Example 2 Covalent attachment of an electron donor unit

After binding the nucleic acid to the electrode gold surface and a further washing step with water, the modified substrate is washed with a solution of 3 × 10 ^-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 O.1 M HEPES buffer (2- (4-2-hydroxyethyl) -1-piperazino) -ethanesulfonic acid, pH 7.5) wetted. The quinone used is obtained by splitting a methoxy group by HBr (ether cleavage) as standard, the resulting free hydroxyl group is reacted with an equimolar amount of Cl-CH ₂ -CH ₂ -CO ₂ H and then purified by chromatography (Moore; HB and Folkers, K ., Journal of American Chemical Society, 1966, 88, 564-570; 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 3 × 10 ^-3 molar Zn bacteriochlorophyll (as free acid), 1.5 × 10 ^-1 molar (3-dimethylaminopropyl) carbodiimide, 2.5 × 10 ^-3 molar hydrazine monohydrate and 1 × 10 ^-1 molar imidazole for 16 hours at 23 ° C. The representation of Zn bacteriochlorophyll as free acid is made 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).

Example 3 Hybridization to double-stranded nucleic acid

Hybridization can take place before or after application of the modified nucleic acid on the electrode gold surface. In the present example, the double-stranded DNA probe is generated by hybridizing the single strands. For this purpose, the single strands (2 × 10 ^-4 molar solution) in a ratio of 1: 1 for 10 minutes to T <40 ° C. in Tris buffer (tris (hydroxymethyl) aminomethane, 10 mM Tris, 1 mM EDTA, pH 7.5 heated with 0.7 molar addition of tetraethylammonium tetrafluoroborate (TEATFB)) and then cooled to room temperature for a period of at least 2 hours. Correctly hybridized nucleic acids are obtained.

Applied single-stranded 5'-phosphorylated (phosphate-on-phosphoramidite Building blocks (Horn, T. and Urdea, M., Tetrahedron Letter, 1986, 27, 4705)) and how in example 1 to the thiol esterified nucleic acids that only have a short hairpin had a free 3'OH end, using a DNA polymerase built up enzymatically to a double strand. The DNA structure was used for this a mixture of the four nucleotide triphosphates (ea. 20 µM) with 10 U Klenow Incubated fragment of DNA I polymerase for 15 minutes at 25 ° C.

Example 4 Restriction analysis ( Fig. 1)

A double-stranded DNA structure (11), with a Pst 1 restriction site

and a Zn bacteriochlorophyll electron donor unit ( 12 ) is covalently bonded to a gold electrode ( 13 ) as described in the previous examples. The DNA double strand ( 11 ) of the probe is divided into two fragments ( 17 , 18 ) in a 1 × Pst 1 buffer (NEBuffer 3) with 10 U Pst 1 ( 14 ) on 50 µl reaction solution applied at 37 ° C in 90 minutes. cut (New England Biolabs). The activity is measured amperometrically over time under the influence of light ( 15 ) with an evaluation device ( 16 ).

Example 5 Ligation

The DNA-Zn bacteriochlorophyll degradation products of the reaction solution from Example 4 were preparatively freed from the restriction enzyme by RP-HPLC. The isolated fragments have an overhanging end, which has the following structure:

This end of the fragment is complemented with a DNA fragment from Example 4, which is bound to the gold electrode surface and whose end looks as follows:

enzymatically linked. For this purpose, the fragment is (10 × excess) with 10 U T4- Ligase / 50 µl ligated at 37 ° C in 30 minutes in DNA ligase buffer, the efficiency is created by generating an electron flow through the double-stranded DNA detected ampereometrically.

Example 6 Binding of a transcription factor ( Fig. 2)

The transcription factor ( 21 ) Oct2A (25-75 ng / µl, + poly [d (IC)] + poly L-lysine, Roche Molecular Biochemicals) or alternatively the transcription factor Sp1 (50 ng / µl, Promega) was added in 20 or 1 × binding (5 × conc; 100 mM Hepes, pH 7.6, 5 mM EDTA, 50 mM (NH ₄ ) ₂ SO ₄ , 5 mM DTT, Tween®20, 1% (w / v), 150 mM KCl ) - or 9 µl incubation buffer (5 × conc; 20% glycerol 5 mM MgCl ₂ , 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25 mg / ml poly (dI-dC ) .poly (dI-dC) + 1 µg BSA / 10 µl buffer) on a template consisting of a gold electrode ( 22 ) on which the double-stranded DNA probe sequences ( 23 ) with one of a covalently bound Zn bacteriochlorophyll electrons -Donor unit ( 24 ), which contain the respective binding site specific for the transcription factor (see Example 1). After at least 15 minutes of incubation at room temperature with the respective transcription factor ( 21 ), the readout is carried out under the influence of light ( 25 ) with an evaluation unit ( 26 ). In parallel, test approaches are analyzed in which DNA fragments containing unbound binding sites of the respective transcription factor ( 27 ) carried in a 125-fold excess as competitor.

By adding the Sp1 competitor Actinomycin D 0.1-0.5 µM before and after Addition of Sp1 will cause the DNA-protein interaction and that Change in conductivity measured ampereometrically.

Example 7 Detection of a DNA-DNA interaction, hybridization ( FIG. 3)

A self-complementary DNA ( 31 ) with a covalently bound Zn bacteriochlorophyll electron donor unit ( 34 ) is applied to the gold electrode ( 32 ) according to the examples given above. A range of 18 nucleotides (3'-GGG (A) ₁₂ GGG ...) was conceptually at the 3'-end as a single strand. The single-strand structure was deliberately filled by hybridization with a 38-oligomer ( 33 ) which had a complementarity over a range of 18 nucleotides 3'-CCC (T) ₁₂ CCCACACCCAATTCTGAAAATGG-5 '. For this, the 38-oligomer ( 33 ) (2 × 10 ^-3 molar solution) in Tris buffer (tris (hydroxymethyl) aminomethane, 10 mM Tris, 1 mM EDTA, pH 7.5) is added to the bound probes in a 10-fold excess and the mixture is incubated for at least 2 hours at room temperature. The excess of non-hybridized oligonucleotides is then washed out. It becomes a double stranded nucleic acid probe with the sequence

consisting of the self-complementary DNA ( 31 ) and the 38-oligomer ( 33 ) obtained. After adding a complementary second 2 × 10 ^-4 molar solution 38-oligomer single-strand sample ( 35 ) of the sequence 5'-GGG (A) ₁₂ GGGTGTGGGTTAAGACTTTTACC-3 'in a Tris buffer (10 mM Tris, 1 mM EDTA, pH 7.5) the re-hybridization is formed under the influence of light ( 36 ) and the 38-oligomer hybrid ( 37 ) is formed from the oligomers ( 33 ) and ( 35 ) with the aid of an evaluation unit ( 38 ) the changes in the conductivity of the DNA probe are measured ampereometrically.

Example 8 Intercalation of ethidium bromide in DNA ( Fig. 4)

In a further experiment, double-stranded nucleic acid probes ( 41 ) with a covalently bound Zn bacteriochlorophyll electron donor unit ( 46 ) according to Example 1 are placed on a matrix-shaped gold electrode array surface ( 42 ) on the individual array positions ( 47 ) upset. The bound DNA probes ( 41 ) are mixed with different concentrations of propidium iodide solution ( 43 ), ethidium bromide solutions ( 44 ) and Syber®green ( 45 ) (0.5 µg / ml, 1 µg / ml, 10 µg / ml) a Tris buffer (10 mM Tris, 1 mM EDTA, pH 7.5) at room temperature for a period of 2 hours. (The second dimension of the array surface is not shown in FIG. 5 for reasons of clarity). After exposure to light ( 48 ), the effect of propidium iodide ( 43 ), ethidium bromide ( 44 ) and Syber®green ( 45 ) on the double-strand structure is followed by an ampereometry using an evaluation unit ( 49 ). The separately readable array positions deliver substance and concentration-dependent signals ( 50 ).

Claims (27)

1. Nucleic acid probes containing a conductive surface bound, at least partially double-stranded nucleic acid sequence to the at least one electron donor unit or at least one electron Acceptor unit is bound. 2. Nucleic acid probes according to claim 1, characterized in that the attached double-stranded nucleic acid probes from two complementary Nucleic acid molecules are composed. 3. Nucleic acid according to claims 1, characterized in that the attached double-stranded nucleic acid probes Nucleic acid sequences exist that contain self-recognizing domains. 4. Nucleic acid probes according to one of claims 1 to 3 thereby characterized in that the double-stranded nucleic acid probes are the same Have a nucleic acid sequence. 5. Nucleic acid probes according to one of claims 1 to 3 thereby 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 thereby characterized in that the double-stranded nucleic acid probes on a conductive array surface are applied. 7. Nucleic acid probes according to one of claims 1 to 5 thereby characterized in that the double-stranded nucleic acid probes on a unstructured conductive surface are applied.   8. Nucleic acid probes according to one of the preceding claims characterized in that the double-stranded nucleic acid probes by Bridge bonds, especially disulfide bridges, are stabilized. 9. 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. 10. Nucleic acid probes according to one of the preceding claims characterized in that regenerable and / or inducible, in particular photo-inducible electron donor or electron acceptor units, such as e.g. B. chlorophyll or Zn bacteriochlorophyll on the nucleic acid covalently are connected. 11. Aggregates containing double-stranded nucleic acid probes according to the Claims 1 to 10 and substances selected from the group consisting of double-stranded nucleic acids interacting directly are selected. 12. Units according to claim 11, characterized in that the immediately interacting substance a protein and / or a peptide and / or a single-stranded and / or a double-stranded nucleic acid and / or a Mimetics and / or an intercalating and / or a nucleic acid damaging Substance and / or a cytostatics. 13. Units according to claim 11, characterized in that the unit other indirectly interacting substances, especially cofactors such as Activators or core pressors. 14. A method for the detection of interactions between double-stranded nucleic acids and substances interacting therewith characterized in that

• a) probes according to claims 1 to 10 are exposed to a reaction medium and
• b) the generated electron flow change is measured by the probes.

15. The method according to claim 14, characterized in that the Reaction medium is a solution that is at least one interacting Substances, preferably proteins and / or peptides, in particular Polymerases, transcription factors, repressors, enzymes and / or their Inhibitors and / or activators and / or mimetics and / or intercalating and / or contains nucleic acid-damaging substances and / or cytostatics. 16. The method according to claim 14 or 15, characterized in that the Reaction medium contains single and / or double-stranded nucleic acids. 17. The method according to claim 14, characterized in that the Reaction medium DNA-damaging, especially carcinogenic substances contains. 18. The method according to any one of claims 14 to 17, characterized in that the reaction medium is an aqueous, saline solution, especially one 10 to 100 mM solution. 19. The method according to any one of claims 14 to 18, characterized in that the reaction solution is buffered. 20. The method according to any one of claims 14 to 19, characterized in that the detection takes place at a temperature between 4 and 40 ° C. 21. Use of the double-stranded nucleic acid probes according to one of the Claims 1 to 10 for the detection of interactions, preferably for Determination of connections, equilibrium constants, competitive Effects and reaction speeds, between double-stranded  Nucleic acids and interacting directly and / or indirectly with them Substances. 22. Analytical containing double-stranded nucleic acid probes according to the Claims 1 to 10 for the identification of directly and indirectly on the Double-stranded nucleic acid probe factors. 23. Analytical containing double-stranded nucleic acid probes according to the Claims 1 to 10 for the identification of nucleic acid sequences with interact directly or indirectly interacting substances. 24. A method for making double-stranded nucleic acid detectors comprising:

• a) the synthesis of a single-stranded nucleic acid,
• b) 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.

25. A method for making double-stranded nucleic acid detectors comprising:

• a) the synthesis of a single-stranded nucleic acid containing self-recognizing domains separated by a bridge,
• b) the intramolecular hybridization of the self-recognizing domains of the single-stranded nucleic acid,
• 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.

26. A method for producing double-stranded nucleic acid detectors according to Claim 25 characterized in that the bridge consists of at least one Nucleotide, preferably consists of 2 to 6 nucleotides. 27. A method for producing double-stranded nucleic acids according to claim 25 characterized in that an artificial at least one atom Bridge, preferably a disulfide, stilbenedicarboxyamide, ruthenium complex, Hexaethylene glycol, terephthalimide, branched or unbranched diol, 3'- Amino-Modifier-CPG or branched phosphoramidite bridge is introduced.