EP0975808A2 - Topologically fixed matrix bonded nucleic acid molecule - Google Patents

Topologically fixed matrix bonded nucleic acid molecule

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Publication number
EP0975808A2
EP0975808A2 EP98963380A EP98963380A EP0975808A2 EP 0975808 A2 EP0975808 A2 EP 0975808A2 EP 98963380 A EP98963380 A EP 98963380A EP 98963380 A EP98963380 A EP 98963380A EP 0975808 A2 EP0975808 A2 EP 0975808A2
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acid molecule
deoxyribonucleic acid
dna
binding
molecule
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Claas Junghans
Burghardt Wittig
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA

Definitions

  • the invention relates to a device consisting of one or a plurality of linear double-stranded nucleic acid molecules which are attached at both ends to a matrix, e.g. a solid surface, are attached and fixed in such a way that the ends of the double strands cannot rotate relative to one another in the arrangement, that is to say the double strands are topologically fixed.
  • the invention further relates to a method with which the activity of enzymes or proteins can be determined, which can change the topology of nucleic acids, and also the effectiveness of substances which change the enzyme activities mentioned can be determined.
  • Double-stranded DNA consists of a right-handed helix. In its relaxed, low-energy conformation, the DNA helix has a pitch of 10.4 base pairs per Heiix pass. A deviation from this pitch, i.e. overcoming or underwinding equates to an increase in the amount of conformational energy in DNA.
  • DNA is usually subdued in cells.
  • the degree of this underwinding is strictly regulated and serves, among other things, as an energy store for strand-separating processes.
  • the topology of the processed strand i.e. the winding density of the helix in space, changed significantly.
  • enzymes so-called topoisomerases, must act on the processed DNA and normalize its topology.
  • the cell can only transcribe or replicate to a limited extent or not at all; in the latter case, a eukaryotic cell initiates programmed cell death.
  • Substances that inhibit enzymes that regulate the topological state of DNA in certain cells or organisms have found widespread use as cytostatics and antibiotics. A simple, automatable process for finding inhibitors of such enzyme activities would be very advantageous for finding new antibiotics or cytostatics.
  • the topological state of a DNA molecule is described by the sum of the number of turns of the DNA double helix around itself and the number of twists of the helical axis in space (Crick, Proc.Natl.Acad.Sci.USA 73, 2639-2640 ( 1976)).
  • a molecule can assume a very large number of such topological states, all of which have a different conformational energy. If the molecule is fixed at both ends, it can still assume many different states, which, however, are characterized in that the sum of the turns of the helix around itself plus the turns of the helix axis in space is always the same.
  • Circular DNA molecules that have two covalently closed phosphate-sugar chains are topologically fixed. This type of molecule can be used to study the effect of topology-changing enzymes and their inhibitors.
  • DNA rings of different topological states are generated by the action of enzymes and characterized by their migration behavior by an agarose or polyacrylamide gel in the electric field.
  • a disadvantage of this, however, is that the set-up of such an experiment, although in principle simple, comprises a series of handouts which cannot be easily automated.
  • topology-dependent phenomena In addition to the migration of circular topoisomers in the electric field, there are a number of other approaches to make topology-dependent phenomena visible.
  • dyes such as ethidium bromide in the helix, which is topology-dependent, and the different sensitivity of the DNA to DNA-modifying or degrading enzymes or reagents, the binding of antibodies to topology-dependent DNA forms such as Z-DNA is particularly important here to be mentioned (Lafer et al., Proc.Natl.Acad.Sci.USA 78, 3546-3550, 1981).
  • a number of proteins are postulated to bind to the DNA differently depending on the topological state of the DNA.
  • One method of topologically fixing DNA molecules that are not based on circular molecules would be to bind the DNA to a matrix with both ends in such a way that the strands of the phosphate backbone are no longer freely rotatable around one another at the ends. This would be achieved by fixing each strand of the double strand at both ends, or else closing the strand at the ends via a short loop of single-stranded oligonucleotides, and connecting this loop to the matrix via at least two anchor points.
  • the topology of the molecule is not defined, the DNA is not bound to a solid surface and the fixation, because such fixation can be demonstrated, is caused by the size of the molecule and the binding to DNA-binding proteins.
  • Strick et al. (Science 271, 1835 (1996)) describe how they attach a DNA molecule to a slide at one end and pull it at the other end to determine the elasticity of the molecule.
  • the molecules are linear, not covalently closed, and the type of attachment and the number of anchor points are undefined.
  • Quake et al. Describe a similar experimental setup. (Nature 388, 151 (1997)), but here there is neither covalently closed DNA, nor is the molecule topologically fixed. Although both approaches are brilliant in their experimental setup they do not enable the quick and uncomplicated investigation of topological phenomena.
  • DNA molecules in tissue are known from our own application (DE 196 48 625).
  • Binding modes but especially the condition that both ends of the molecule must be bound with a defined winding state, are not disclosed there. In the sense of the technical solution disclosed there, it is the desorption of the bound
  • the object outlined in the introduction is achieved in such a way that double-stranded DNA molecules are bound to a matrix with both ends in such a way that the strands of the phosphate backbone at the ends are no longer freely rotatable about one another by either
  • subdued matrix-bound DNA is produced by winding the DNA molecules before binding to the matrix, for example by intercalation of a suitable ligand such as ethidium bromide or chloroquine. After binding and washing out the ligand, the DNA is submerged. Conversely, it is possible to produce overcome DNA by binding other ligands, such as netropsin.
  • the DNA is bound to the matrix in that chemically modified DNA building blocks which are present at the ends of the DNA molecules are bound to the surface of the matrix via covalent or quasi-covalent interactions.
  • the ends of the DNA molecules can be linked with short single-stranded loops produced by automated chemical synthesis, with individual bases being biotin-modified. These molecules bind to a streptavidin-coated matrix with a strength close to that of covalent bonds.
  • amino linker-modified DNA molecules can be bound to a matrix which contains activated carboxylic acid residues or epoxy-activated residues. The binding of thiol-modified DNA to gold surfaces is also conceivable.
  • the length of the matrix-bound DNA molecules according to the invention can be varied and, depending on the task of the enzyme activity to be investigated, can be between twenty base pairs and many kilobases.
  • the topology-dependent binding of the anti-Z-DNA antibody Z22 (Stollar et al .: J.Biol.Chem 257, 12801-12085 (1982); ibid: 261, 468-476 (1986); ibid.:265, 18608- 18614 (1990)) was shown for DNA constructs up to ⁇ 200 bp, smaller constructs were not examined. However, it is conceivable, e.g. B. to investigate topology-dependent sequence recognition phenomena also bind much shorter constructs.
  • topology-specific DNA structures or the topological state can be done in several ways. Above all, there are methods that can be easily detected in automated systems, especially enzymatic ones
  • Secondary antibody-coupled color reactions can be made visible, or the fluorimetrically detectable intercalation of ethidium bromide, propidium iodide or other dyes.
  • the intercalation of these dyes winds up the DNA helix and thus increases the conformational energy contained in a topologically fixed DNA molecule, since the
  • topologically fixed molecules bind measurably less dye than relaxed molecules; this difference can be measured directly.
  • topology-dependent parameter changes in the investigation with surface plasmon resonance or the detection of bonds or enzyme activities by this method on matrix-bound topologically fixed DNA may also be mentioned.
  • enzyme activities which can change the topological state of DNA are investigated in such a way that matrix-bound topologically fixed DNA of a defined topological state is incubated with the relevant enzyme activity and the change in the topological state of the DNA either after a controlled termination of the experiment or in a real-time process is followed by one of the methods mentioned.
  • the choice of the appropriate measurement method and parameters will depend heavily on the mechanism of the enzyme activity to be investigated.
  • plasmid bluescript SK (Stratagene) was cut completely with EcoRI and then with a 200-fold molar excess of 5 ' -phosphorylated oligodeoxynucleotide AATTGGCCGGCCGGCXTTXGCCGGCCGGCC (X corresponds to biotin-modified uracil), TIB ) ligated with T4 DNA ligase (MBI, Heidelberg). After the excess oligodeoxynucleotides have been separated off, the covalently closed, approximately 3 kb long DNA molecule is obtained, each with two biotin modifications at both ends.
  • Covalently closed DNA from step 1 was taken up in Tris buffer (pH 8.0) in a concentration of 2 ⁇ g / ml; 2 ng each of the construct was incubated in 75 ⁇ l phosphate-buffered physiological saline (PBS) in the presence of 20 ⁇ M ethidium bromide on streptavidin-coated microtiter plates (Boehringer Mannheim). It was then washed three times with PBS.
  • PBS phosphate-buffered physiological saline
  • microtiter plate wells coated with submerged DNA are incubated with 4 u topoisomerase I (Life) for between 0 and 30 min.
  • topoisomerase I Life
  • the topology-dependent change in conformation under the influence of topoisomerase is verified by incubation with anti-Z-DNA antibody Z22 (A.Rich, MIT, Cambridge, MA, USA) and subsequent incubation with secondary peroxidase-conjugated anti-mouse IgG (Boehringer Mannheim) Color reaction detected with tetramethylbenzidine.
  • FIG. 1 schematically shows a covalently closed, topologically fixed DNA molecule which is bound to a solid surface.
  • topology-induced Z-DNA can be detected by antibody binding (left side); Topoisomerase exposure (top right) relaxes the DNA, causing the Z-DNA structure to disappear; this prevents the antibody signal (bottom right)

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Abstract

The invention relates to nucleic acid molecules which are bonded on a surface in such a way that coiling energy or superhelicity cannot escape from said molecules. The invention also relates to a method with which such enzyme actions can be examined, said enzyme actions changing the topological state of DNA. Such nucleic acid molecules and methods can be utilized in pharmaceutical research in the automated search for cytostatics or antibiotics.

Description

Topologisch fixiertes, matrixgebundenes NukleinsäuremolekülTopologically fixed, matrix-bound nucleic acid molecule
Beschreibungdescription
Die Erfindung betrifft eine Vorrichtung, bestehend aus einem oder einer Vielzahl von linearen doppelsträngigen Nukleinsäuremolekülen, welche mit beiden Enden an einer Matrix, z.B. einer festen Oberfläche, befestigt und so fixiert sind, daß die Enden der Doppelstränge nicht in der Anordnung relativ zueinander rotieren können, das heißt die Doppelstränge topologisch fixiert sind. Femer betrifft die Erfindung ein Verfahren, mit welchem die Aktivität von Enzymen oder Proteinen bestimmt werden kann, welche die Topologie von Nukleinsäuren verändern können, sowie auch die Wirksamkeit von Stoffen ermittelt werden kann, die die genannten Enzymaktivitäten verändern.The invention relates to a device consisting of one or a plurality of linear double-stranded nucleic acid molecules which are attached at both ends to a matrix, e.g. a solid surface, are attached and fixed in such a way that the ends of the double strands cannot rotate relative to one another in the arrangement, that is to say the double strands are topologically fixed. The invention further relates to a method with which the activity of enzymes or proteins can be determined, which can change the topology of nucleic acids, and also the effectiveness of substances which change the enzyme activities mentioned can be determined.
Doppelsträngige DNA besteht aus einer rechtsgängigen Helix. In ihrer entspannten, energieärmsten Konformation hat die DNA-Helix eine Ganghöhe von 10,4 Basenpaaren pro Heiixdurchgang. Eine Abweichung von dieser Ganghöhe, d.h. eine Über- oder Unterwindung, kommt einer Energieerhöhung der DNA in ihrem Gehalt an Konformationsenergie gleich.Double-stranded DNA consists of a right-handed helix. In its relaxed, low-energy conformation, the DNA helix has a pitch of 10.4 base pairs per Heiix pass. A deviation from this pitch, i.e. overcoming or underwinding equates to an increase in the amount of conformational energy in DNA.
In Zellen liegt DNA in der Regel unterwunden vor. Der Grad dieser Unterwindung wird streng geregelt und dient unter anderem als Energiespeicher für strangtrennende Prozesse. Bei Ablesevorgängen wie Transkription und Replikation wird die Topologie des prozessierten Stranges, d.h. die Windungsdichte der Helix im Raum, stark verändert. Damit diese lebenswichtigen Prozesse ablaufen können, müssen eine Reihe von Enzymen, sog. Topoisomerasen, auf die prozessierte DNA wirken und ihre Topologie normalisieren.DNA is usually subdued in cells. The degree of this underwinding is strictly regulated and serves, among other things, as an energy store for strand-separating processes. In reading processes such as transcription and replication, the topology of the processed strand, i.e. the winding density of the helix in space, changed significantly. In order for these vital processes to take place, a number of enzymes, so-called topoisomerases, must act on the processed DNA and normalize its topology.
Hemmt man diese Enzyme, so kann die Zelle nur eingeschränkt oder gar nicht mehr transkribieren oder replizieren; im letzteren Falle leitet eine eukaryote Zelle den programmierten Zelltod ein. Stoffe, welche Enzyme hemmen, die den topologischen Zustand von DNA in bestimmten Zellen oder Organismen regulieren, haben weite Verwendung als Cytostatika und Antibiotika gefunden. Ein einfaches, automatisierbares Verfahren, Hemmer solcher Enzymaktivitäten zu finden, wäre sehr vorteilhaft zum Auffinden neuer Antibiotika oder Cytostatika.If these enzymes are inhibited, the cell can only transcribe or replicate to a limited extent or not at all; in the latter case, a eukaryotic cell initiates programmed cell death. Substances that inhibit enzymes that regulate the topological state of DNA in certain cells or organisms have found widespread use as cytostatics and antibiotics. A simple, automatable process for finding inhibitors of such enzyme activities would be very advantageous for finding new antibiotics or cytostatics.
Der topologische Zustand eines DNA-Moleküls wird beschrieben durch die Summe der Zahl der Windungen der DNA-Doppelhelix um sich selbst und der Zahl der Verwindungen der Helixachse im Raum (Crick, Proc.Natl.Acad.Sci.USA 73, 2639-2640 (1976)). Ein Molekül kann abhängig von seiner Länge eine sehr große Zahl solcher topologischen Zustände einnehmen, die alle eine unterschiedliche Konformationsenergie haben. Fixiert man das Molekül an beiden Enden, so kann es immer noch viele verschiedene Zustände einnehmen, die jedoch dadurch charakterisiert sind, daß bei ihnen die Summe aus Windungen der Helix um sich selbst plus der Windungen der Helixachse im Raum immer gleich ist. Vergleichbar ist dies mit dem Bild eines an beiden Enden festgehaltenen Springseils, in dem Schlaufen zwar innerhalb des Seiles verschoben werden können, nicht aber aus dem Seil hinaus. Zirkuläre DNA-Moleküle, welche zwei kovalent geschlossene Phosphat-Zucker-Ketten aufweisen, sind topologisch fixiert. An dieser Art von Molekülen kann man die Wirkung von Topologie-verändernden Enzymen und von deren Hemmstoffen studieren. Prinzipiell werden dabei DNA-Ringe unterschiedlicher topologischer Zustände durch Enzymeinwirkung erzeugt, und durch ihr Wanderungsverhalten durch ein Agarose- oder Polyacrylamidgei im elektrischen Feld charakterisiert. Ein Nachteil dabei ist allerdings, daß der Aufbau eines solchen Experiments, obwohl prinzipiell einfach, eine Reihe von Handreichungen umfaßt, welche nicht ohne weiteres automatisierbar sind.The topological state of a DNA molecule is described by the sum of the number of turns of the DNA double helix around itself and the number of twists of the helical axis in space (Crick, Proc.Natl.Acad.Sci.USA 73, 2639-2640 ( 1976)). Depending on its length, a molecule can assume a very large number of such topological states, all of which have a different conformational energy. If the molecule is fixed at both ends, it can still assume many different states, which, however, are characterized in that the sum of the turns of the helix around itself plus the turns of the helix axis in space is always the same. This is comparable to the picture of a skipping rope held at both ends, in which loops can be moved within the rope, but not out of the rope. Circular DNA molecules that have two covalently closed phosphate-sugar chains are topologically fixed. This type of molecule can be used to study the effect of topology-changing enzymes and their inhibitors. In principle, DNA rings of different topological states are generated by the action of enzymes and characterized by their migration behavior by an agarose or polyacrylamide gel in the electric field. A disadvantage of this, however, is that the set-up of such an experiment, although in principle simple, comprises a series of handouts which cannot be easily automated.
Neben der Wanderung zirkulärer Topoisomere im elektrischen Feld gibt es eine Reihe anderer Ansätze, topologie-abhängige Phänomene sichtbar zu machen. Neben der Interkalation von Farbstoffen wie Ethidiumbromid in die Helix, welche topologieabhängig ist, sowie der unterschiedlichen Empfindlichkeit der DNA für DNA-modifizierende oder -abbauende Enzyme oder Reagenzien, ist hier vor allem die Bindung von Antikörpern an topologieabhängig auftretende DNA-Formen wie Z-DNA zu nennen (Lafer et al., Proc.Natl.Acad.Sci.USA 78, 3546-3550, 1981). Bei einer Reihe von Proteinen wird postuliert, daß sie in Abhängigkeit des topologischen Zustands der DNA unterschiedlich stark an diese binden.In addition to the migration of circular topoisomers in the electric field, there are a number of other approaches to make topology-dependent phenomena visible. In addition to the intercalation of dyes such as ethidium bromide in the helix, which is topology-dependent, and the different sensitivity of the DNA to DNA-modifying or degrading enzymes or reagents, the binding of antibodies to topology-dependent DNA forms such as Z-DNA is particularly important here to be mentioned (Lafer et al., Proc.Natl.Acad.Sci.USA 78, 3546-3550, 1981). A number of proteins are postulated to bind to the DNA differently depending on the topological state of the DNA.
Eine Methode, DNA-Moleküle topologisch zu fixieren, die nicht auf zirkulären Molekülen beruht, wäre, die DNA mit beiden Enden so an eine Matrix zu binden, daß die Stränge des Phosphatrückgrates an den Enden nicht mehr umeinander frei drehbar sind. Dies würde man dadurch erreichen, daß man jeden Strang des Doppelstranges an beiden Enden fixiert, oder aber den Strang an den Enden über eine kurze Schleife einzelsträngiger Oligonukleotide schließt, und diese Schleife über mindestens zwei Ankerpunkte mit der Matrix verbindet.One method of topologically fixing DNA molecules that are not based on circular molecules would be to bind the DNA to a matrix with both ends in such a way that the strands of the phosphate backbone are no longer freely rotatable around one another at the ends. This would be achieved by fixing each strand of the double strand at both ends, or else closing the strand at the ends via a short loop of single-stranded oligonucleotides, and connecting this loop to the matrix via at least two anchor points.
Matrix- oder Oberflächen-gebundene DNA ist aus der Literatur bekannt. Es gibt allerdings nur wenig Hinweise darauf, daß DNA zur Untersuchung ihres topologischen Verhaltens fixiert worden wäre. Lee und Morgan (Can. J. Biochem 56 (1978), 585-591) beschreiben die Vernetzung unterschiedlicher zirkulärer DNA-Moleküle in polymerisierender Agarose; dabei handelt es sich allerdings nur um eine Methode zur selektiven Rückhaltung zirkulärer Moleküle. Lineare Moleküle werden nicht der Untersuchung zugänglich gemacht. Aus Nicoud et al. (EMBO J. 3 (1984), 721-731) ist die Untersuchung des topologischen Zustands von Teilbereichen sehr großer Chromosomenmoleküle bekannt, die zwischen Glasplatten eingebettet sind. In diesem System ist die Topologie des Moleküls allerdings nicht definiert, die DNA ist nicht an eine feste Oberfläche gebunden und die Fixierung, so denn eine solche Fixierung nachgewiesen werden kann, wird durch die Größe des Moleküls sowie die Bindung an DNA-bindende Proteine hervorgerufen. Strick et al. (Science 271 , 1835 (1996)) beschreiben, wie sie ein DNA-Molekül an einem Ende auf einem Objektträger festmachen und am anderen Ende ziehen, um die Elastizität des Moleküls zu ermitteln. Die Moleküle sind allerdings linear, nicht kovalent geschlossen und die Art der Befestigung sowie die Anzahl der Ankerpunkte Undefiniert. Einen ähnlichen Versuchsaufbau beschreiben Quake et al. (Nature 388, 151 (1997)), aber hier liegt weder kovalent geschlossene DNA vor, noch ist das Molekül topologisch fixiert. Obwohl beide Ansätze in ihrem experimentellen Aufbau brillant sind, ermöglichen sie nicht das schnelle und unkomplizierte Untersuchen topologischer Phänomene.Matrix- or surface-bound DNA is known from the literature. However, there is little evidence that DNA was fixed to investigate its topological behavior. Lee and Morgan (Can. J. Biochem 56 (1978), 585-591) describe the crosslinking of different circular DNA molecules in polymerizing agarose; however, this is only a method for the selective retention of circular molecules. Linear molecules are not made available for investigation. From Nicoud et al. (EMBO J. 3 (1984), 721-731) the investigation of the topological state of partial areas of very large chromosome molecules which are embedded between glass plates is known. In this system, however, the topology of the molecule is not defined, the DNA is not bound to a solid surface and the fixation, because such fixation can be demonstrated, is caused by the size of the molecule and the binding to DNA-binding proteins. Strick et al. (Science 271, 1835 (1996)) describe how they attach a DNA molecule to a slide at one end and pull it at the other end to determine the elasticity of the molecule. However, the molecules are linear, not covalently closed, and the type of attachment and the number of anchor points are undefined. Quake et al. Describe a similar experimental setup. (Nature 388, 151 (1997)), but here there is neither covalently closed DNA, nor is the molecule topologically fixed. Although both approaches are brilliant in their experimental setup they do not enable the quick and uncomplicated investigation of topological phenomena.
Die Bindung von DNA an Oberflächen per se ist aus einer Fülle von Patentschriften sowie Veröffentlichungen bekannt. Während lange Zeit chemische Methoden wie die Carbodiimid- vermittelte Bindung von Phosphaten an die Hydroxygruppen von Glasoberflächen, oder an die Aminogruppen aminofunktionalisierter Kunststoffoberflächen verwendet wurden, ist heute die Bindung Biotin-modifizierter DNA-Oligomere an Streptavidin-beschichtete Kunststoffplatten oder andere Materialien gebräuchlicher. Zur Bindung von Biomolekülen an Oberflächen zur Erzeugung von Enzymelektroden ist die Bindung Thiol-modifizierter Biomoleküle an Goldoberflächen bekannt (siehe u.a. Willner et al. (Nature 376 (1995):672- 5). Mit der Verbreitung von sog. „Chip-basierten" Techniken zur molekularen Diagnostik und Wirkstoffsuche gewinnen automatisierte Verfahren, z.B. auf photolithographischer Basis, zunehmend an Bedeutung (s. z.B. WO 91/16425).The binding of DNA to surfaces per se is known from an abundance of patents and publications. While chemical methods such as the carbodiimide-mediated binding of phosphates to the hydroxyl groups of glass surfaces or to the amino groups of amino-functionalized plastic surfaces have long been used, the binding of biotin-modified DNA oligomers to streptavidin-coated plastic plates or other materials is more common today. For the binding of biomolecules to surfaces for the production of enzyme electrodes, the binding of thiol-modified biomolecules to gold surfaces is known (see, inter alia, Willner et al. (Nature 376 (1995): 672-5). With the spread of so-called "chip-based" Techniques for molecular diagnostics and drug discovery are gaining in importance in automated processes, for example on a photolithographic basis (see, for example, WO 91/16425).
Die Verwendung doppelsträngiger, durch eine kurze Schlaufe einzelsträngiger DNA Nukleotide endverknüpfter DNA-Moleküle zum Einbringen von gentherapeutisch wirksamenThe use of double-stranded DNA molecules end-linked by a short loop of single-stranded DNA nucleotides for the introduction of gene therapy-effective
DNA-Molekülen in Gewebe ist aus unserer eigenen Anmeldung (DE 196 48 625) bekannt.DNA molecules in tissue are known from our own application (DE 196 48 625).
Dort ist bereits die Bindung solcher Moleküle an kleine Goldpartikel genannt. Die konkretenThe binding of such molecules to small gold particles is already mentioned there. The concrete ones
Bindungsmodi, besonders aber die Bedingung, daß beide Enden des Moleküls mit definiertem Windungszustand gebunden werden müssen, sind dort aber nicht offenbart. Es ist im Sinne der dort offenbarten technischen Lösung, die Desorbtion der gebundenenBinding modes, but especially the condition that both ends of the molecule must be bound with a defined winding state, are not disclosed there. In the sense of the technical solution disclosed there, it is the desorption of the bound
Moleküle im Gewebe durch wenige Bindungsstellen der DNA an der Oberfläche zu erleichtem.To facilitate molecules in the tissue by a few binding sites of the DNA on the surface.
Ausgehend von diesem Stand der Technik ist es Aufgabe der vorliegenden Erfindung, ein System zu entwickeln, welches die Untersuchung topologischer Phänomene auf experimentell einfache Weise möglich macht, vorzugsweise auf eine Art, welche der Automatisierung zum Zwecke der Wirkstoff suche zugänglich ist. Insbesondere ist es die Aufgabe der Erfindung, DNA so an Oberflächen zu binden, daß die Windungsenergie unter- oder überwundener DNA im Molekül erhalten bleibt und experimentell festgestellt werden kann, sowie die Ermittlung von Topoisomeraseaktivitäten, deren Hemmung sowie topologieverändernde andere Enzymaktivitäten und deren Hemmung einem einfachen, automatisierbaren Testsystem zugänglich zu machen.Starting from this prior art, it is an object of the present invention to develop a system which makes it possible to investigate topological phenomena in an experimentally simple manner, preferably in a way which is accessible to automation for the purpose of searching for active substances. In particular, it is the object of the invention to bind DNA to surfaces in such a way that the winding energy of DNA which has been overcome or overcome is retained in the molecule and can be determined experimentally, and the determination of topoisomerase activities, their inhibition and topology-changing other enzyme activities and their inhibition in a simple manner to make automatable test systems accessible.
Erfindungsgemäße technische LösungTechnical solution according to the invention
Erfindungsgemäß wird die in der Einführung geschilderte Aufgabe so gelöst, daß doppelsträngige DNA-Moleküle mit beiden Enden so an eine Matrix gebunden werden, daß die Stränge des Phosphatrückgrates an den Enden nicht mehr umeinander frei drehbar sind, indem man entwederAccording to the invention, the object outlined in the introduction is achieved in such a way that double-stranded DNA molecules are bound to a matrix with both ends in such a way that the strands of the phosphate backbone at the ends are no longer freely rotatable about one another by either
• jeden Strang des DNA-Doppelstranges an beiden Enden fixiert, oder aber• each strand of the DNA double strand is fixed at both ends, or else
• den Strang an beiden Enden über je eine kurze Schleife einzelsträngiger Oligonukleotide schließt und diese Schleife über mindestens zwei Ankerpunkte mit der Matrix verbindet. Erfindungsgemäß wird unterwundene matrixgebundene DNA dadurch hergestellt, daß man die DNA-Moleküle vor der Bindung an die Matrix aufwindet, z.B. durch Interkalation eines geeigneten Liganden wie Ethidiumbromid oder Chloroquin. Nach Bindung und Auswaschen des Liganden liegt die DNA unterwunden vor. Umgekehrt ist es möglich, überwundene DNA durch Bindung anderer Liganden, wie z.B. Netropsin, herzustellen.• closes the strand at both ends via a short loop of single-stranded oligonucleotides and connects this loop to the matrix via at least two anchor points. According to the invention, subdued matrix-bound DNA is produced by winding the DNA molecules before binding to the matrix, for example by intercalation of a suitable ligand such as ethidium bromide or chloroquine. After binding and washing out the ligand, the DNA is submerged. Conversely, it is possible to produce overcome DNA by binding other ligands, such as netropsin.
Die Bindung der DNA an die Matrix erfolgt dadurch, daß chemisch modifizierte DNA- Bausteine, welche an den Enden der DNA-Moleküle vorliegen, über kovalente oder quasi- kovalente Wechselwirkungen an die Oberfläche der Matrix gebunden werden. Zum Beispiel können die Enden der DNA-Moleküle mit kurzen, durch automatisierte chemische Synthese hergestellten einzelsträngigen Schleifen verknüpft werden, wobei einzelne Basen biotinmodifiziert sind. Diese Moleküle binden an eine Streptavidin-beschichtete Matrix mit einer Stärke, die der von kovalenten Bindungen nahekommt. Alternativ können Aminolinker- modifizierte DNA-Moleküle an eine Matrix gebunden werden, die aktivierte Carbonsäurereste oder epoxid-aktivierte Reste enthält. Die Bindung von thiolmodifizierter DNA an Goldoberflächen ist ebenfalls denkbar.The DNA is bound to the matrix in that chemically modified DNA building blocks which are present at the ends of the DNA molecules are bound to the surface of the matrix via covalent or quasi-covalent interactions. For example, the ends of the DNA molecules can be linked with short single-stranded loops produced by automated chemical synthesis, with individual bases being biotin-modified. These molecules bind to a streptavidin-coated matrix with a strength close to that of covalent bonds. Alternatively, amino linker-modified DNA molecules can be bound to a matrix which contains activated carboxylic acid residues or epoxy-activated residues. The binding of thiol-modified DNA to gold surfaces is also conceivable.
Die Länge der erfindungsgemäßen matrixgebundenen DNA-Moleküle ist variierbar und kann je nach Aufgabenstellung der zu untersuchenden Enzymaktivität zwischen zwanzig Basenpaaren und vielen Kilobasen liegen. Die topologieabhängige Bindung des Anti-Z- DNA-Antikörpers Z22 (Stollar et al.: J.Biol.Chem 257, 12801-12085 (1982); ibid: 261 , 468- 476 (1986); ibid.:265, 18608-18614 (1990)) wurde für DNA-Konstrukte bis < 200 bp gezeigt, kleinere Konstrukte wurden nicht untersucht. Es ist allerdings vorstellbar, z. B. zur Untersuchung von topologieabhängigen Sequenzerkennungsphänomenen auch viel kürzere Konstrukte zu binden.The length of the matrix-bound DNA molecules according to the invention can be varied and, depending on the task of the enzyme activity to be investigated, can be between twenty base pairs and many kilobases. The topology-dependent binding of the anti-Z-DNA antibody Z22 (Stollar et al .: J.Biol.Chem 257, 12801-12085 (1982); ibid: 261, 468-476 (1986); ibid.:265, 18608- 18614 (1990)) was shown for DNA constructs up to <200 bp, smaller constructs were not examined. However, it is conceivable, e.g. B. to investigate topology-dependent sequence recognition phenomena also bind much shorter constructs.
Der Nachweis topologiespezifischer DNA-Strukturen oder des topologischen Zustands kann auf mehrerlei Weise erfolgen. Vor allem bieten sich Methoden an, die leicht in automatisierbaren Systemen nachgewiesen werden können, also vor allem enzymatischeThe detection of topology-specific DNA structures or the topological state can be done in several ways. Above all, there are methods that can be easily detected in automated systems, especially enzymatic ones
Nachweismethoden wie die Bindung von Antikörpern oder Proteinen, welche durchDetection methods such as the binding of antibodies or proteins, which by
Sekundär-Antikörper-gekoppelte Farbreaktionen sichtbar gemacht werden können, oder die fluorimetrisch nachweisbare Interkalation von Ethidiumbromid, Propidiumiodid oder anderen Farbstoffen. Die Interkalation dieser Farbstoffe windet die DNA-Helix auf, und erhöht damit die in einem topologisch fixierten DNA-Molekül enthaltene Konformationsenergie, da dieSecondary antibody-coupled color reactions can be made visible, or the fluorimetrically detectable intercalation of ethidium bromide, propidium iodide or other dyes. The intercalation of these dyes winds up the DNA helix and thus increases the conformational energy contained in a topologically fixed DNA molecule, since the
Aufwindung an anderer Stelle im Molekül durch eine gegensinnige Windung kompensiert werden muß, die energetisch ungünstige Konformationen der Phosphatreste und Basen erzwingt. Es binden topologisch fixierte Moleküle ab einer bestimmten Konzentration an Interkalator meßbar weniger Farbstoff als relaxierte Moleküle; dieser Unterschied ist direkt meßbar.Winding elsewhere in the molecule must be compensated for by an opposite turn, which forces energetically unfavorable conformations of the phosphate residues and bases. From a certain concentration of intercalator, topologically fixed molecules bind measurably less dye than relaxed molecules; this difference can be measured directly.
Femer sei der direkte Nachweis von topologieabhängigen Parameterveränderungen bei der Untersuchung mit Oberflächenplasmonresonanz oder der Nachweis von Bindungen oder Enzymaktivitäten durch diese Methode an matrixgebundener topologisch fixierter DNA genannt. Die Untersuchung von Enzymaktivitäten, welche den topologischen Zustand von DNA verändern können, erfolgt erfindungsgemäß so, daß matrixgebundene topologisch fixierte DNA eines definierten topologischen Zustands mit der betreffenden Enzymaktivität inkubiert wird und die Veränderung des topologischen Zustands der DNA entweder nach kontrolliertem Abbruch des Experiments oder im Echtzeitverfahren durch eine der genannten Methoden verfolgt wird. Die Wahl der geeigneten Meßmethode und -parameter wird dabei unter anderem stark vom Mechanismus der zu untersuchenden Enzymaktivität abhängen.The direct detection of topology-dependent parameter changes in the investigation with surface plasmon resonance or the detection of bonds or enzyme activities by this method on matrix-bound topologically fixed DNA may also be mentioned. According to the invention, enzyme activities which can change the topological state of DNA are investigated in such a way that matrix-bound topologically fixed DNA of a defined topological state is incubated with the relevant enzyme activity and the change in the topological state of the DNA either after a controlled termination of the experiment or in a real-time process is followed by one of the methods mentioned. The choice of the appropriate measurement method and parameters will depend heavily on the mechanism of the enzyme activity to be investigated.
Beispiel:Example:
1. Herstellung von linearer kovalent geschlossener DNA: Plasmid Blueskript SK (Stratagene) wurde mit EcoRI vollständig geschnitten und anschließend mit einem 200-fachen molaren Überschuß an 5'-phosphoryliertem Oligodesoxynukleotid AATTGGCCGGCCGGCXTTXGCCGGCCGGCC (X entspricht Biotin-modifiziertem Uracil) (TIB-MolBiol, Berlin) mit T4-DNA-Ligase (MBI, Heidelberg) ligiert. Nach Abtrennen der überschüssigen Oligodesoxynukleotide erhält man das kovalent geschlossene, ca. 3 kb lange DNA-Molekül mit je zwei Biotinmodifikationen an beiden Enden.1. Production of linear covalently closed DNA: plasmid bluescript SK (Stratagene) was cut completely with EcoRI and then with a 200-fold molar excess of 5 ' -phosphorylated oligodeoxynucleotide AATTGGCCGGCCGGCXTTXGCCGGCCGGCC (X corresponds to biotin-modified uracil), TIB ) ligated with T4 DNA ligase (MBI, Heidelberg). After the excess oligodeoxynucleotides have been separated off, the covalently closed, approximately 3 kb long DNA molecule is obtained, each with two biotin modifications at both ends.
2. Kopplung an Streptavidin-beschichtete ELISA-Platten:2. Coupling to streptavidin-coated ELISA plates:
Kovalent geschlossene DNA aus Schritt 1 wurde in Tris-Puffer (pH 8.0) in einer Konzentration von 2 μg/ml aufgenommen; je 2 ng des Konstruktes wurde in 75 μl Phosphatgepufferter physiologischer Kochsalzlösung (PBS) in Anwesenheit von 20 μM Ethidiumbromid 45 min auf Streptavidin-beschichteten Mikrotiterplatten (Boehringer Mannheim) inkubiert. Anschließend wurde dreimal mit PBS gewaschen.Covalently closed DNA from step 1 was taken up in Tris buffer (pH 8.0) in a concentration of 2 μg / ml; 2 ng each of the construct was incubated in 75 μl phosphate-buffered physiological saline (PBS) in the presence of 20 μM ethidium bromide on streptavidin-coated microtiter plates (Boehringer Mannheim). It was then washed three times with PBS.
3. Topoisomerase-Assay3. Topoisomerase assay
Die mit unterwundener DNA beschichteten Mikrotiterplattennäpfe werden mit je 4 u Topoisomerase I (Life) zwischen 0 und 30 min inkubiert. Die topologieabhängige Konformationsänderung unter Einwirkung der Topoisomerase wird durch Inkubation mit anti- Z-DNA-Antikörper Z22 (A.Rich, MIT, Cambridge, MA, USA) und anschließender Inkubation mit sekundärem Peroxidase-konjugiertem Anti-Maus-IgG (Boehringer Mannheim) nach Farbreaktion mit Tetramethylbenzidin nachgewiesen.The microtiter plate wells coated with submerged DNA are incubated with 4 u topoisomerase I (Life) for between 0 and 30 min. The topology-dependent change in conformation under the influence of topoisomerase is verified by incubation with anti-Z-DNA antibody Z22 (A.Rich, MIT, Cambridge, MA, USA) and subsequent incubation with secondary peroxidase-conjugated anti-mouse IgG (Boehringer Mannheim) Color reaction detected with tetramethylbenzidine.
Kurze Beschreibung der Zeichnungen: Fig. 1 zeigt schematisch ein kovalent geschlossenes, topologisch fixiertes DNA-Molekül, weiches an einer festen Oberfläche gebunden ist.BRIEF DESCRIPTION OF THE DRAWINGS: FIG. 1 schematically shows a covalently closed, topologically fixed DNA molecule which is bound to a solid surface.
Fig. 2 zeigt den schematischen Ablauf des Verfahrens: Topologie-induzierte Z-DNA kann durch Antikörperbindung nachgewiesen werden (linke Seite); Topoisomeraseeinwirkung (rechts oben) entspannt die DNA, wodurch die Z-DNA-Struktur verschwindet; das Antikörper-Signal wird dadurch verhindert (rechts unten) 2 shows the schematic sequence of the method: topology-induced Z-DNA can be detected by antibody binding (left side); Topoisomerase exposure (top right) relaxes the DNA, causing the Z-DNA structure to disappear; this prevents the antibody signal (bottom right)

Claims

Ansprüche Expectations
1. Lineares doppelsträngiges Desoxyribonukleinsäuremolekül mit komplementärer Basensequenz, dadurch gekennzeichnet, daß beide Einzelstränge sowohl am 5'- Hydroxylende wie am 3'-Hydroxylende über kovalente oder semikovaiente Bindungen an einer Oberfläche befestigt sind.1. Linear double-stranded deoxyribonucleic acid molecule with a complementary base sequence, characterized in that both single strands are attached to a surface both at the 5 ' hydroxyl end and at the 3 ' hydroxyl end via covalent or semicovaient bonds.
2. Desoxyribonukleinsäuremoleküi nach Anspruch 1 , wobei die komplementären Stränge des Moleküls an jedem Ende durch eine kurze Schlaufe einzelsträngiger Desoxyribonukleotide kovalent verbunden sind, und die Verbindung mit einer Oberfläche über die kovalente oder semikovaiente Kopplung von in der kurzen einzelsträngigen Schlaufe enthaltenen chemisch modifizierten Desoxyribonukleotidbausteinen an die Oberfläche herbeigeführt wird.2. The deoxyribonucleic acid molecule according to claim 1, wherein the complementary strands of the molecule are covalently connected at each end by a short loop of single-stranded deoxyribonucleotides, and the connection to a surface via the covalent or semicovaient coupling of chemically modified deoxyribonucleotide components contained in the short single-stranded loop Surface is brought about.
3. Desoxyribonukleinsäuremolekül nach Anspruch 1 oder 2, bei welchem die Bindung des Desoxyribonukleinsäuremoleküls an eine mit Streptavidinmolekülen beschichtete Oberfläche durch an das Desoxyribonukleinsäuremolekül kovalent gekoppelte Biotinreste herbeigeführt wird.3. deoxyribonucleic acid molecule according to claim 1 or 2, wherein the binding of the deoxyribonucleic acid molecule to a surface coated with streptavidin molecules is brought about by biotin residues covalently coupled to the deoxyribonucleic acid molecule.
4. Desoxyribonukleinsäuremolekül nach Anspruch 1 oder 2, bei welchem die Bindung des Desoxyribonukleinsäuremoleküls an eine Goldoberfläche erfolgt und durch an das Desoxyribonukleinsäuremolekül kovalent gekoppelte thiolmodifizierte Basen vermittelt wird. 4. deoxyribonucleic acid molecule according to claim 1 or 2, wherein the binding of the deoxyribonucleic acid molecule takes place on a gold surface and is mediated by covalently coupled to the deoxyribonucleic acid molecule thiol-modified bases.
5. Verfahren zur Untersuchung von den Windungszustand von Desoxyribonukleinsäuren ändernden Enzymaktivitäten, dadurch gekennzeichnet, daß5. A method for examining the winding state of deoxyribonucleic acid-changing enzyme activities, characterized in that
• ein lineares doppelsträngiges Desoxyribonukleinsäuremolekül komplementärer Basensequenz in Lösung gebracht wird, vorzugsweise in Anwesenheit eines in die DNA interkaiierenden oder an die DNA bindenden Stoffes bekannter Konzentration, wobei dieser Stoff den Windungszustand von DNA verändert,A linear double-stranded deoxyribonucleic acid molecule of a complementary base sequence is brought into solution, preferably in the presence of a concentration known to intercalate or bind to the DNA, this substance changing the winding state of DNA,
• das Desoxyribonukleinsäuremolekül aus der den in die DNA interkaiierenden oder an die DNA bindenden Stoff enthaltenden Lösung heraus an einer Oberfläche so gebunden wird, daß beide Einzelstränge sowohl am 5'- Hydroxyiende wie am 3'-Hydroxylende über kovalente oder semikovaiente Bindungen an der Oberfläche befestigt sind, wobei vorzugsweise die Befestigung an die Oberfläche über eine die komplementären Stränge verbindende kurze Schlaufe einzelsträngiger Desoxyribonukleotide, in der chemisch modifizierte Desoxyribonukleotidbausteine enthalten sind, die ihrerseits mit einer Oberfläche kovalent verbunden sind, herbeigeführt wird,• The deoxyribonucleic acid molecule is bound to the surface from the solution intercalating or binding to the DNA in such a way that both single strands are attached to the surface at both the 5'-hydroxyl end and the 3'-hydroxyl end via covalent or semicovaient bonds preferably being attached to the surface via a short loop of single-stranded deoxyribonucleotides connecting the complementary strands, which contains chemically modified deoxyribonucleotide building blocks which in turn are covalently linked to a surface,
• das Desoxyribonukleinsäuremolekül in Anwesenheit der zu untersuchenden Enzymaktivität inkubiert wird und• the deoxyribonucleic acid molecule is incubated in the presence of the enzyme activity to be investigated and
• die Änderung des Windungszustands des Desoxyribonukleinsäuremoleküls durch eine geeignete Meßmethode nachgewiesen wird.• The change in the winding state of the deoxyribonucleic acid molecule is detected by a suitable measurement method.
6. Verfahren nach Anspruch 5, wobei das Desoxyribonukleinsäuremolekül durch an das Molekül kovalent gekoppelte Biotinreste an eine Streptavidin-beschichtete Matrix gebunden ist.6. The method according to claim 5, wherein the deoxyribonucleic acid molecule is bound to a streptavidin-coated matrix by biotin residues covalently coupled to the molecule.
7. Verfahren nach Anspruch 5, wobei das Desoxyribonukleinsäuremolekül über an das Molekül kovalent gekoppelte Thiole oder Disulfide an eine Goldoberfläche gebunden ist.7. The method according to claim 5, wherein the deoxyribonucleic acid molecule is bound to a gold surface via thiols or disulfides covalently coupled to the molecule.
8. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß die Meßmethode der Nachweis der Bindung von Anti-Z-DNA-spezifischen Antikörpern an das Desoxyribonukleinsäuremolekül ist.8. The method according to claim 5, characterized in that the measuring method is the detection of the binding of anti-Z-DNA-specific antibodies to the deoxyribonucleic acid molecule.
9. Verfahren nach Anspruch 5, wobei die Meßmethode der Nachweis der Bindung interkalierender Fluoreszensfarbstoffmoleküle an das Desoxyribonukleinsäuremolekül ist. 9. The method according to claim 5, wherein the measurement method is the detection of the binding of intercalating fluorescent dye molecules to the deoxyribonucleic acid molecule.
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