WO2009037511A1

WO2009037511A1 - Assay for dna supercoiling/relaxation

Info

Publication number: WO2009037511A1
Application number: PCT/GB2008/050845
Authority: WO
Inventors: Haris Jahic; Adam Shapiro
Original assignee: Astrazeneca Ab; Astrazeneca Uk Limited
Priority date: 2007-09-21
Filing date: 2008-09-19
Publication date: 2009-03-26

Abstract

The present invention provides methods for assaying the activity of topology-changing molecules and methods for screening for compounds that modulate the activity of these molecules.

Description

ASSAY FOR DNA SUPERCOILING/RELAXATION

FIELD OF THE INVENTION

The present invention relates to methods for assaying the topological state of a double- stranded DNA molecule and methods for identifying compounds that modulate the activity of substances/molecules, e.g., enzymes, that affect the topological state of the DNA molecule.

BACKGROUND

Double-stranded DNA that does not have at least one free end, as in circular plasmids for example, can have a range of supercoiling topologies, from positively supercoiled through relaxed to negatively supercoiled. The linking number is the sum of the degree of twisting of the DNA double helix and the degree of supercoiling. A change in the number of supercoils in a circular DNA molecule requires that the backbone of one or both of the DNA strands be broken. This may occur by physical or chemical damage, or by the action of enzymes. In living cells, the supercoiling topology of DNA is controlled by enzymes called DNA topoisomerases.

DNA topoisomerases all share the property of catalyzing interconversion between different topological forms of double-stranded DNA. DNA topoisomerases have been isolated from plasmid, viral, prokaryotic, and eukaryotic sources. There are different classes of topoisomerase enzymes (termed type I and type II) that are distinguished by an operational difference; the type I enzymes catalyze DNA interconversion during which the linking number changes in steps of one, while the type II enzymes perform reactions during which the linking number changes in steps of two. Negatively supercoiled DNA is more easily unwound, allowing RNA polymerase to bind more readily to the DNA, hence promoting the transcription of certain genes (Reece & Maxwell, 1991, Crit. Rev. Biochem. MoI. Biol, 26:335-375). Assays of topoisomerase activity are important for the identification of compounds that modulate topoisomerase activity. Topoisomerases in bacteria are targets for antibacterial agents. For example, fluoroquinolones such as ciprofloxacin target the essential bacterial enzyme DNA gyrase. The DNA gyrase introduces negative supercoils into closed circular DNA molecules, including the bacterial chromosome. Topoisomerases in eukaryotes are targets for anti-cancer drugs. In the search for compounds that act on topoisomerases, assays are needed that measure the topological state (supercoiled v. relaxed) of the DNA substrate. Typically, low-throughput gel electrophoresis separation of DNA topoisomers has been employed. This technique is unsuitable for screening large compound libraries and is slow and awkward for routine testing of new compounds produced by topoisomerase-targeted antibacterial drug discovery programs.

Maxwell et al. recently reported a potentially high-throughput, microplate-based assay for DNA supercoiling/relaxation activity by DNA {Nucleic Acids Research 34, elO4, 2006). This assay is based on the principle that supercoiled and relaxed DNA plasmids have different propensities, under certain conditions, to form triplexes by Hoogsteen base pairing with particular pyrimidine- rich single-stranded DNA oligonucleotides if the same sequences are also present in the plasmid.

SUMMARY OF THE INVENTION

The present invention provides, in part, a method for assaying the extent of supercoiling of a double-stranded DNA molecule and methods for identifying compounds that modulate the activity of enzymes that affect the extent of supercoiling of DNA. The method is a solution- based method. The method includes providing (i) a DNA substrate for a topology-changing enzyme, wherein the DNA substrate has a particular topological state and comprises a triplex- forming sequence, (ii) a topology-changing enzyme; and (iii) a test agent; contacting (i) the DNA substrate, (ii) the topology-changing enzyme; and (iii) the test agent to form a mixture and incubating the mixture for a sufficient amount of time to allow the topology-changing enzyme to change the topology of the substrate; providing (iv) a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a triplex-forming sequence that complements the triplex-forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains it's fluorescence at pH less than 6.0 and (v) a triplex-forming buffer; contacting the mixture with (iv) the single-stranded oligonucleotide and (v) the triplex-forming buffer for a sufficient time to allow triplex formation between the substrate and the single stranded oligonucleotide, and detecting a change in flourescence signal compared to a control, wherein none of the components used in the method described above are immobilized on a solid surface. The double-stranded DNA substrate can be in any topological state. In one embodiment, the DNA substrate is supercoiled. In this embodiment, the topology-changing enzyme relaxes the DNA substrate. In another embodiment the DNA substrate has a relaxed topology and the topology-changing enzyme (such as DNA gyrase) causes the DNA substrate to supercoil. Any topology-changing molecule such as an enzyme can be used in the invention. Examples of enzymes include type II topoisomerase (DNA gyrase or topoisomerase IV (ParCE)), a type I typoisomerase, an endonuclease, a restriction enzyme or any process that causes single- stranded nicks or double stranded breaks in DNA. The single-stranded oligonucleotide is typically labeled. The oligonucleotide can be labeled at the 3' end, the 5' end or internally. In one embodiment, the fluorescent dye is at the 5' end of the single stranded oligonucleotide. The oligonucleotide is typically labeled with a fluorescent dye such as one of the Bodipy-Fl series, e.g., Bodipy-FL or one of the Alexa series, e.g., AlexaFluor 488. The single-stranded oligonucleotide also typically contains a triplex- forming sequence.

For example, the triplex forming sequence can be (TTC)_n where n > 1. In one embodiment, the triplex-forming sequence is TTCTTCTTCTTCTTCTTCTTCTTCTTC (SEQ ID NO:1) and the single-stranded oligonucleotide comprises the sequence of TTCTTCTTC or TTCTTCTTCT. In one embodiment of the invention the trip lex- forming buffer has low magnesium (II) concentration, e.g., of less than 1OmM. In this embodiment, a lower fluorescence signal is detected if the DNA is supercoiled. In another embodiment, the triplex-forming buffer has high magnesium (II) concentration, e.g., 50 mM. In this embodiment, a higher fluorescence signal is detected if the DNA substrate is supercoiled.

The test agent can be any molecule such as a compound or a polypeptide (e.g., an antibody), or it can be a form of treatment such as radiation. In one embodiment the test agent is an inhibitor of an enzyme that affects DNA topology.

In another aspect, the invention includes a method for determining the effect of a topology-changing molecule on the topological state of a double-stranded DNA molecule. The method includes providing a double-stranded DNA molecule that serves as a substrate for a topology-changing molecule such as an enzyme, wherein the DNA molecule has a particular topological state and comprises a triplex- forming sequence; providing a topology-changing molecule; contacting the DNA molecule and the topology-changing molecule to form a mixture and incubating the mixture for a sufficient amount of time to allow the topology-changing molecule to change the topology of the substrate; providing a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a trip lex- forming sequence that complements the triplex- forming sequence present on the double-stranded DNA substrate and further comprises a fluorescent dye that retains its fluorescence at pH less than or equal to 6.0; contacting the mixture with the single-stranded oligonucleotide for a sufficient time to allow triplex formation between the substrate and the single stranded oligonucleotide, and detecting a change in fluorescence signal compared to a control.

In yet another aspect, the invention includes a kit. In one embodiment, the kit can include: a double-stranded DNA molecule that serves as a substrate for a topology-changing enzyme, wherein the DNA molecule comprises a triplex-forming sequence; a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a triplex- forming sequence that complements the triplex-forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains it's fluorescence at pH less than < 6; and printed instructions on how to perform the solution-based method.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts a line graph showing a change in anisotropy of BODIPY-FL-labeled

TTCTTCTTC triplex-forming single-stranded DNA due to binding at pH 3.5 to either relaxed or supercoiled plasmid containing the triplex- forming sequence TTCTTCTTCTTCTTCTTCTTCTTCTTC. (R - SC; relaxed minus supercoiled plasmid anisotropics.)

Figure 2 depicts a line graph showing a change in anisotropy of BODIPY-FL-labeled

TTCTTCTTC triplex-forming single-stranded DNA due to binding at pH 3.5 to relaxed plasmid containing the triplex- forming sequence TTCTTCTTCTTCTTCTTCTTCTTCTTC, after various lengths of time of supercoiling of the plasmid by 4 nM Escherichia coli DNA gyrase in the presence of ATP.

Figure 3 depicts a line graph showing measurement of inhibition of E, coli DNA gyrase by ciprofloxacin, using the fluorescence anisotropy assay. DETAILED DESCRIPTION

The supercoiling/relaxation assay of the invention can be used, in part, for screening compound collections for inhibitors of a variety of molecules, such as enzymes, that affect DNA topology, for measurement of compound IC50s (the compound concentration at which the activity of the target enzyme is inhibited by 50%), and for investigation of modes of inhibition of the enzyme targets by compounds. The assay may also be used to investigate or quantify the function of these enzymes, or optimize the conditions for their reactions.

In one example, the method includes contacting a double-stranded DNA molecule that serves as a substrate for a topology-changing enzyme (the DNA molecule has a particular topological state and comprises a triplex-forming sequence); a topology- changing enzyme and a test compound for a sufficient amount of time to allow the topology changing enzyme to change the topology of the substrate. The mixture is then contacted with a single-stranded oligonucleotide. The single stranded oligonucleotide has a triplex-forming sequence that complements the triplex- forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains it's fluorescence at pH less than or equal to 6.0. The single-stranded oligonucleotide is contacted with the DNA substrate/enzyme/compound mix for a sufficient time to allow triplex formation between the substrate and the single stranded oligonucleotide. To determine if the compound affects the enzyme's ability to change the topology of the substrate the flourescence signal is determined and compared to a control. In one embodiment, the flourescence signal being determined is fluorescence polarization or fluorescence anisotropy. Fluorescence polarization or fluorescence anisotropy measurement is based on the differential affinity of supercoiled and relaxed double-stranded DNA substrates containing triplex- forming sequences for a single-stranded oligonucleotide containing the same triplet-rich triplex forming sequence under particular buffer conditions. The fluorescence polarization or anisotropy is higher when the single-stranded oligonucleotide is bound in a triplex with the DNA substrate than when it is free.

The present method has many advantages including: (1) it is completely homogeneous, eliminating the need for a separation step, i.e., washing unbound DNA from the plate; and (2) it does not require immobilization of the oligonucleotide and can therefore use inexpensive, standard, multiwell assay plates rather than expensive streptavi din-coated plates. Where fluorescence polarization (or fluorescence anisotropy) is being detected, this replaces a single fluorescence intensity measurement, which greatly increases measurement precision.

The Topology-Changing Molecule

Any topology-changing molecule such as an enzyme for which a double-stranded DNA substrate can be provided can be used in the present invention. In one example, topoisomerases can be used in the method of the present invention. Topoisomerases have been identified in prokaryotes and eukaryotes. Examples of suitable topoisomerases include type II topoisomerases such as DNA gyrase or topoisomerase IV (ParCE) from bacteria and topoisomerase II from eukaryotes. The topoisomerase from bacteria can be from Haemophilus influenzae, Moraxella catarrhalis, Pseudomonas aeruginosa, Escherichia coli, Chlamydia spp, Legionella spp, Staphylococcus aureus, Staphylococcus saprophyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, Enterococcus faecium, Mycoplasma spp, Bacteroides spp and Clostridium spp. Type I topoisomerases can also be used because they exhibit ATP-independent relaxation of supercoiled DNA.

A topoisomerase enzyme or subunit thereof can be obtained for use in the present invention according to procedures well known to the art. A topoisomerase enzyme or subunit thereof can be obtained by isolation or purification from natural sources or can be expressed using recombinant technology. The enzyme or subunit thereof can be expressed as a single target protein or co-expressed with other proteins. The enzyme or subunit thereof can be expressed with or without peptide tags or fusion proteins. The enzyme or subunit thereof can be isolated as a cell extract, prepared in substantially pure form as a single protein, or prepared as a protein complex. In another example, individual subunits can be reconstituted to form an active multisubunit enzyme. Numerous techniques for obtaining the topoisomerase enzyme proteins, including bacterial topoisomerase enzyme proteins, have been described in the literature. For example, see H. Peng et al. J. Biol. Chem. 268, 24481-24490 (1993); M.H. Barnes et al, Protein Expression and Purification, 29, 259-264 (2003); or X. Pan et al. Antimicrobial Agents and Chemotherapy, 1129-1136 (1999). Catalytically active portions or subunits/fragments of the topoisomerase enzyme can also be used in the assays of the present invention. For example, see S. Bellon et al. Antimicrobial Agents and Chemotherapy, 1856-1864 (2004). In another example, enzymes, such as endonucleases, or processes that cause single- stranded nicks or double-stranded breaks in DNA, thereby causing relaxation of supercoiled DNA, can also be used in the method of the invention.

Substrate for a Topology-Changing Molecule

The method includes the use of any double-stranded substrate of a topology-changing molecule such as an enzyme. The substrate can be provided in a partially or fully relaxed or supercoiled state. Typically the DNA substrate is in the form of a duplex and is in a closed- circular state such as a double stranded plasmid. However, other forms of DNA may also be used such as double stranded DNA tethered at both ends.

The size of the substrate is not critical as long as one skilled in the art can appreciate that a change in topology of the DNA substrate has occurred. Typically the size of the DNA substrate is between 0.01 kb to 50 kb.

The DNA substrate of the invention includes an insert of a triplex-forming sequence. The triplex-forming sequence can be pyrimidine and/or purine rich. Typically the DNA substrate contains multiple repeats of a triplet, for example, at least 3, 6, 9, 12, 18, 24 repeats of a particular triplet. In one example, the triplex- forming sequence can be a TC repeat ((TC)_n) or a CT repeat ((CT)_n). In another example, the triplet-forming sequence is a TTC repeat ((TTC)_n)_, a TCT repeat ((TCT)_n), or a CTT repeat ((CTT)_n). In one embodiment, the DNA substrate includes nine TTC repeats, e.g. TTCTTCTTCTTCTTCTTCTTCTTCTTC (SEQ ID NO: 1).

DNA substrate in a relaxed or supercoiled state for use in the present invention can be bought commercially or can be made by recombinant means. Preferably, a highly purified DNA substrate is used.

In one method of the invention, the method can be used to measure supercoiling of relaxed DNA. In this example, the topology changing enzyme can be DNA gyrase. The substrate is typically a partially or completely relaxed plasmid. The plasmid can be partially or fully relaxed by any means known in the art. For example, the plasmid can be purified from bacterial cells. Plasmid DNA purified from bacterial cells is typically in a supercoiled state. To use this DNA as a substrate for DNA gyrase or Topoisomerase II the plasmid must be as topologically relaxed as possible. This may be accomplished by a variety of methods. For example, the plasmid can be treated with topoisomerase I, followed by repurifϊcation of the DNA. Other methods that can be employed are treatment with DNA gyrase in the absence of ATP or with topoisomerase IV in the presence of ATP followed by repurification, or nicking the DNA with endonuclease followed by religation with DNA ligase and repurification. In every case, it is important to minimize the amount of nicked or linearized plasmid in the relaxed DNA substrate because these cannot be supercoiled.

In another embodiment, the method of the invention can be used to measure the relaxation of supercoiled DNA. In this example, the topology changing enzyme can be a Type II topoisomerase such as DNA gyrase, topoisomerase IV or a Type I topoisomerase. The substrate is typically a partially or completely supercoiled plasmid. The plasmid can be partially or fully supercoiled by any means known in the art. For example, plasmid DNA isolated from bacterial cells is supercoiled. The supercoiled plasmid DNA if not maximally supercoiled can be further supercoiled by contacting the plasmid with, for example, DNA gyrase and ATP, followed by repurification, in order to maximize the signal in the assay.

Single-Stranded Oligonucleotide

The single-stranded oligonucleotide used in the method of the invention comprises a triplex- forming sequence that complements the triplex- forming sequence present on the DNA substrate (as described above). For example, the single-stranded oligonucleotide probe sequence contains at least 1, 2, 3, 4, 6, 9 triplet sequences that complement the triplex-forming sequence present on the DNA substrate. In one example, where the DNA substrate contains nine contiguous TTC repeats, the single stranded oligonucleotide can be TTCTTCTTC. In another example, the sequence can be a non- integral number of repeats such as TTCTTCTT. In yet another example, the triplex-forming sequence, e.g., (TTC)n, in the double stranded DNA is base paried by Watson-Crick base pairing with its complementary sequence, for example: 5'-TTCTTC 3'

3'-AAGAAG ...5'

The triplex forming sequence in the single-stranded oligonucleotide is complementary to this double-stranded sequence and forms Hoogsteen base paris in the major groove of the double- stranded sequence. For more on triplex formation see Maxwell et al. (2006) Nucleic Acid Research 34 (15)elO4, which is incorporated herein by reference. The number of triplet repeats needed on the single-stranded oligonucleotide to maximize the signal in the assay can be determined by one skilled in the art. Typically the number of trip lex- forming repeats is not critical as long as the oligonucleotide can bind the DNA substrate in a manner proportional to its supercoiling.

In one embodiment of the invention where the triplex-forming buffer has minimal magnesium (II) (eg., 1OmM), the oligonucleotide will bind a relaxed DNA substrate with greater affinity and specificity than a supercoiled DNA susbtrate. In another embodiment of the invention where the trip lex- forming buffer contains greater than 1OmM magnesium (II) (5OmM), the oligonucleotide will bind a supercoiled DNA substrate with greater affinity and specificity than a relaxed DNA susbtrate. The oligonucleotide is typically labelled. The label can be at the 5' end, 3' end or internally labelled. In one embodiment, the oligonucleotide is covalently modified, e.g. at the 5' end, with a label. Any label can be used such as BODIPY-FL or AlexaFluor 488. Identification of a suitable fluorescent dye is within the capabilities of someone skilled in the art. An important feature of the fluorescent label is that it retains its fluorescence at low pH, e.g., less than 6.0 such as around pH 3.5 because triplex formation occurs at acidic pH. Thus only probes that maintain their fluorescence at low pH are used. Also, to minimize interference with the assay from test compound fluorescence or light scattering by insoluble compounds, it is desirable to use a probe with relatively long-wavelength fluorescence. Excitation at 485 nm has been shown to work successfully, though longer wavelengths should be accessible by using longer wavelength members of the BODIPY or AlexaFluor series, for example.

Detecting Step

The change in topology of the DNA substrate is measured by the method of the present invention by detecting a change in fluorescence signal. In one example, fluorescence polarization or fluorescence anisotropy is used. The fluorescence polarization or anisotropy of the fluorescent label attached to the single-stranded oligonucleotide is greater when the single-stranded oligonucleotide is bound to the double- stranded DNA in a triplex than when it is free. The advantage of using ratiometric anisotropy measurements is that it greatly increases measurement precision. The fluorescence polarization or fluorescence anisotropy measurements make use of the differential affinity of supercoiled and relaxed DNA plasmids containing the triplex- forming sequences for the single-stranded oligonucleotide containing the complement of the trip lex- forming sequence. The fluorescent label is typically attached to the single stranded oligonucleotide. Under the conditions where the triplex- forming buffer has minimal magnesium (II) (less than or equal to 1OmM), the single- stranded oligonucleotide has a higher affinity for relaxed DNA and therefore higher polarization is detected when the DNA substrate is in a relaxed form than in a supercoiled form. Under the conditions where the trip lex- forming buffer has more than 1OmM magnesium (II), the single- stranded oligonucleotide has a higher affinity for supercoiled DNA and therefore lower polarization is detected when the DNA substrate is in a relaxed form than in a supercoiled form. The calculation of fluorescence polarization or anisotropy is known in the art, see Lakowicz et al. Principles of fluorescence spectroscopy, 1983, Plenum Press, New York. Chapter 5 (see especially page 112).

In other examples, detection of fluorescence intensity, fluorescence lifetime or resonance energy fluorescence transfer or fluorescence quenching can be used. A change in the quantum yield of a fluorescent dye attached to the single-stranded oligonucleotide may result when the single-stranded oligonucleotide becomes bound in a triplex with the double-stranded DNA substrate. The change in quantum yield can be detected as a change in the fluorescence intensity or fluorescence lifetime of the dye. This effect may be enhanced by the inclusion of a second compound that becomes bound to the double-stranded DNA, such as an intercalator or minor - or major grove-binding compound. The second compound may be a fluorescence quencher, for example, or it may itself be fluorescent. If the fluorescent dye attached to the single-stranded oligonucleotide and the compound bound to the double-stranded DNA can behave as a donor and acceptor pair for fluorescence resonance energy transfer, respectively, then the fluorescence of the dye attached to the single-stranded DNA may be reduced due to its proximity to the compound bound to the double-stranded DNA. If the acceptor itself is fluorescent, an increase in the fluorescence of the acceptor may be measured concomitant with the decrease in the fluorescence of the donor upon binding of the single-stranded oligonucleotide to the double- stranded DNA.

Screening for Compounds that affect DNA Topology

The assay described herein can be used to determine the effect of a compound on a topology-changing molecule such as an enzyme. The presence of magnesium (II) in the triplex forming buffer determines the ability of the single-stranded oligonucleotide to bind to a double- stranded DNA substrate when it is in a particular topological state. For example, when the magnesium (II) is present in the buffer at a low concentration, e.g., less than 10 mM, then the single-stranded oligonucleotide will bind more strongly to a relaxed DNA substrate. Conversely, if the magnesium (II) is present in the buffer at a concentration of greater than 10 mM, e.g., 50 mM then the single-stranded oligonucleotide will bind more strongly to a supercoiled DNA substrate.

In one method of the invention, the method can be used to identify a compound that affects the ability of a topology-changing enzyme such as DNA gyrase to supercoil double- stranded DNA. This method includes providing a double-stranded DNA substrate that is relaxed, a DNA gyrase and a test compound. The mixture is then incubated for a sufficient amount of time to allow the gyrase to supercoil the double-stranded DNA. The mixture is then contacted with a single-stranded oligonucleotide bearing a fluorescent label and a triplex- forming buffer having a concentration of magnesium (II) of less than 10 mM. The mixture is incubated for a sufficient time to allow triplex- forming formation between the substrate and the single stranded oligonucleotide. The ability of the compound to inhibit supercoiling of the DNA substrate can be determined by fluorescence polarization, anisotropy, or other fluorescence-based method. A higher fluorescence polarization (or anisotropy) compared to a control (no test compound) indicates that the compound is an inhibitor of DNA gyrase.

In another method of the invention, the method can be used to identify a compound that affects the ability of a topology-changing enzyme such as topoisomerase I to relax double- stranded DNA. This method includes providing a double-stranded DNA substrate that is supercoiled, a topology relaxing enzyme such as topoisomerase I and a test compound. The mixture is incubated for a sufficient amount of time to allow the topoisomerase to relax the supercoiled DNA. The mixture is contacted with a single-stranded oligonucleotide bearing a fluorescent label and in the presence of a triplex- forming buffer having minimal magnesium (less than 1OmM) incubated for a sufficient time to allow triplex formation between the substrate and the single stranded oligonucleotide. The ability of the compound to inhibit the relaxation of the supercoiled DNA substrate can be determined by fluorescence polarization (or anisotropy) or other fluorescence-based method. A lower fluorescence polarization (or anisotropy) compared to a control (no inhibitor) indicates that the compound is an inhibitor of DNA topoisomerase I. The triplex-forming buffer is typically a buffer having a pH of less than 6.0. An example of triplex- forming conditions is the following: 50 mM sodium acetate and 50 mM sodium chloride, adjusted to pH 3.5 with acetic acid. The triplex-forming conditions serve the additional purpose of quenching the enzyme reaction. At the same time, the triplex- forming oligonucleotide carrying the fluorescent probe is added. After a sufficient time for triplex- formation to reach completion, e.g. one hour, the fluorescence polarization or anisotropy of the fluorescent probe is measured. If the assay is performed in a multiwell microplate, a suitable plate reader, e.g. a Tecan Ultra, may be used to make the measurement. It should be noted that in one example the greatest difference in anisotropy between relaxed and supercoiled DNA, was obtained at pH less than 6, as compared with higher pH, and in the absence of magnesium(II) ion. Under these conditions, the anisotropy was higher with relaxed DNA than with no DNA or supercoiled DNA, indicating that binding of the triplex forming single-stranded DNA was tighter with relaxed DNA than with supercoiled DNA.

Assays of the present invention are conducted under conditions such that the topology- changing enzyme is catalytically active. These are conditions under which the enzyme is capable of changing the topology of the DNA substrate, in the presence of a nucleoside triphosphate such as ATP if necessary. Such conditions are known to those skilled in the art. Reaction conditions are exemplified below and/or are otherwise known in the art.

Kits

The invention further provides kits for performing the methods of the invention described above.

Typically the kit includes: a double-stranded DNA molecule that serves as a substrate for a topology-changing enzyme, wherein the DNA molecule comprises a triplex-forming sequence; a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a triplex- forming sequence that complements the triplex-forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains it's fluorescence at pH < 6.0; a triplex-forming buffer, and printed instructions on how to perform the method. This kit may also include an enzyme that modifies the supercoiling topology of the DNA substrate and a reaction buffer suitable for use with this enzyme.

The invention is further illustrated by way of the following examples, which are intended to elaborate several embodiments of the invention. These examples are not intended to, nor are they to be construed to, limit the scope of the invention. It will be clear that the invention may be practiced otherwise than as particularly described herein. Numerous modifications and variations of the present invention are possible in view of the teachings herein and, therefore, are within the scope of the invention.

EXAMPLES

Example 1: The ability of the method to distinguish between relaxed and supercoiled plasmids that bear a triplex-forming sequence (Figure 1).

In a black, 384-well polystyrene assay plate, 20 microliters/well of various concentrations of fully relaxed or supercoiled plasmid containing the triplex-forming sequence TTCTTCTTCTTCTTCTTCTTCTTCTTC in assay buffer consisting of 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 4 mM MgCl₂, 2 mM dithiothreitol, 1.8 mM spermidine, 5% (v/v) glycerol, 200 nM bovine serum albumin, and 0.3 mM ATP were mixed with 10 microliters/well of 30 nM oligodeoxynucleotide probe in 3X triplex-forming buffer consisting of 150 mM NaCl, and 150 mM sodium acetate at pH 3.5. The oligodeoxynucleotide probe was 5'-BODIPY-FL-labeled TTCTTCTTC. After 90 min, the fluorescence anisotropy of the BODIPY-FL was measured in a Tecan Ultra plate reader, using 485 nm excitation and 535 nm emission filters equipped with polarizers.

Example 2: The ability to detect DNA gyrase activity (Figure 2). In a black, 384-well polystyrene assay plate, 20 microliters/well of 4 nM Escherichia coli

DNA gyrase and 85 micrograms/ml of topologically relaxed plasmid containing the triplex- forming sequence TTCTTCTTCTTCTTCTTCTTCTTCTTC in assay buffer consisting of 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 4 mM MgCl₂, 2 mM dithiothreitol, 1.8 mM spermidine, 5% (v/v) glycerol, 200 nM bovine serum albumin, 1.25% dimethylsulfoxide, and 0.3 mM ATP was incubated at ambient temperature for various lengths of time. The supercoiling reactions were quenched by the addition of 10 micro liters/well of 30 nM oligodeoxynucleotide probe in 3X trip lex- forming buffer consisting of 150 mM NaCl, and 150 mM sodium acetate at pH 3.5. The oligodeoxynucleotide probe was 5'-BODIPY-FL-labeled TTCTTCTTC. After 60 min, the fluorescence anisotropy of the BODIPY-FL was measured in a Tecan Ultra plate reader, using 485 nm excitation and 535 nm emission filters equipped with polarizers.

Example 3: The ability of the method to measure inhibition of DNA gyrase by a chemical compound (Figure 3). The procedure described in example 2 was carried out with a 55-min reaction time in the presence of various concentrations of ciprofloxacin. The % inhibition of the decrease in fluorescence anisotropy by each ciprofloxacin concentration was calculated by comparison to an uninhibited control having no ciprofloxacin and a fully inhibited control having 10 micromolar ciprofloxacin.

Claims

We claim:

1. A solution-based method for screening if a test agent affects the ability of a topology- changing molecule such as an enzyme to change the topological state of a double- stranded DNA substrate, the method comprises: providing (i) a double-stranded DNA substrate for a topology-changing enzyme, wherein the DNA substrate has a particular topological state and comprises a triplex- forming sequence, (ii) a topology-changing enzyme; and (iii) a test agent; contacting (i) the DNA substrate, (ii) the topology-changing enzyme; and (iii) the test agent to form a mixture and incubating the mixture for a sufficient amount of time to allow the topology-changing enzyme to change the topology of the substrate; providing (iv) a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a triplex- forming sequence that complements the triplex- forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains it's fluorescence at pH less than 6.0 and (v) a triplex-forming buffer; contacting the mixture with (iv) the single-stranded oligonucleotide and (v) the triplex- forming buffer for a sufficient time to allow triplex formation between the substrate and the single stranded oligonucleotide, and detecting a change in flourescence signal compared to a control, wherein none of the components used in the method are immobilized on a solid surface.

2. The method of claim 1, wherein the DNA substrate is supercoiled.

3. The method of claim 2, wherein the topology-changing enzyme relaxes the DNA substrate.

4. The method of claim 3, wherein the topology-changing enzyme is a type II topoisomerase, a type I typoisomerase, an endonuclease, a restriction enzyme or any process that causes single- stranded nicks or double stranded breaks in DNA.

5. The method of claim 4, wherein the type II topoisomerase is topoisomerase IV (ParCE).

6. The method of claim 1, wherein the DNA substrate has a relaxed topology.

7. The method of claim 6, wherein the topology-changing enzyme causes the DNA substrate to supercoil.

8. The method of claim 6, wherein the topology-changing enzyme is a DNA gyrase.

9. The method of claim 1, wherein the fluorescent dye is at the 5' end of the single stranded oligonucleotide.

10. The method of claim 1 or claim 9, wherein the fluorescent dye is one of the Bodipy series or one of the Alexa series.

11. The method of claim 1 , wherein the triplex-forming sequence present in the plasmid is (TTC)_n where n > 1.

12. The method of claim 10, wherein the triplex-forming sequence is TTCTTCTTCTTCTTCTTCTTCTTCTTC (SEQ ID NO: 1).

13. The method of claim 1, wherein the single-stranded oligonucleotide comprises the sequence of TTCTTCTTC or TTCTTCTTCT.

14. The method of claim 1, wherein the triplex-forming buffer has a magnesium (II) concentration of less than 1OmM.

15. The method of claim 14, wherein a lower fluorescence signal is detected if the DNA is supercoiled.

16. The method of claim 1, wherein the triplex-forming buffer has a magnesium (II) concentration of greater than 1OmM.

17. The method of claim 16, wherein a higher fluorescence signal is detected if the DNA is supercoiled.

18. The method of claim 1, wherein the test agent is an inhibitor of an enzyme that effects DNA topology.

19. A method for determining the affect of a DNA changing substance on the topological state of a DNA molecule, comprising: providing a DNA molecule that serves as a substrate for a topology-changing enzyme, wherein the DNA molecule has a particular topological state and comprises a triplex-forming sequence; providing a topology-changing enzyme; contacting the DNA molecule and the topology changing enzyme to form a mixture and incubating the mixture for a sufficient amount of time to allow the topology changing enzyme to change the topology of the substrate; providing a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a triplex-forming sequence that complements the triplex-forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains its fluorescence at pH less than 6.0; contacting the mixture with the single-stranded oligonucleotide for a sufficient time to allow triplex formation between the substrate and the single stranded oligonucleotide, and detecting a change in fluorescence signal compared to a control.

20. A kit for performing the method of claim 1, wherein the kit comprises a DNA molecule that serves as a substrate for a topology-changing enzyme, wherein the DNA molecule comprises a triplex-forming sequence; a single-stranded oligonucleotide, wherein the single-stranded oligonucleotide comprises a triplex- forming sequence that complements the triplex-forming sequence present on the DNA substrate and further comprises a fluorescent dye that retains it's fluorescence at pH less than 6.0; and printed instructions on how to perform the solution-based method.