WO2022200213A1

WO2022200213A1 - Detection of the cleavage and ligation activity of dna topoisomerases

Info

Publication number: WO2022200213A1
Application number: PCT/EP2022/057172
Authority: WO
Inventors: Cinzia TESAURO; Magnus Stougaard; Birgitta R. KNUDSEN
Original assignee: Vpcir.Com Aps
Priority date: 2021-03-22
Filing date: 2022-03-18
Publication date: 2022-09-29

Abstract

The present invention relates to assays for determining the influence of an anti-topoisomerase 1B drug on the cleavage step and/or the ligation step of topoisomerase 1B enzymatic activity. The invention also relates to kits comprising such assays and uses thereof.

Description

DETECTION OF THE CLEAVAGE AND LIGATION ACTIVITY OF DNA TOPOISOMERASES

Technical field of the invention

The present invention relates to assays for determining topoisomerase IB activity. The invention specifically also relates to assays for determining the influence of an anti-topoisomerase IB drug on the cleavage step and/or the ligation step of topoisomerase IB enzymatic activity. The invention also relates to kits comprising such assays and uses of the assays and kits for drug screening.

Background of the invention

Enzymes able to relax DNA through cleavage and ligation reactions (such as Topoisomerases) constitute a class of enzymes that are important in controlling the DNA topology during crucial moments of the life of a cell. Topoisomerases are able to bind, cleave and ligate DNA, changing the DNA topology [1]. Topoisomerases 1 (TOPI) are of particular interest in cancer research. By targeting TOPI, it is possible to selectively kill cancer cells, which are dividing faster than non-cancer cells. TOPI is also of particular interest for research in Molecular and Cellular Biology [2]. In 2008 a new method called Rolling-Circle- Enhanced-Enzyme-Activity-Detection (REEAD), to measure the cleavage-ligation equilibrium activity of human TOPI was developed [3]. With proper and targeted modifications of the DNA-substrates, the method proved to be effective in the analysis of other TOPI enzymes, such as Plasmodium falciparum TOPI, the pathogen causing Malaria [4]. The REEAD technology can be used for measurements in bulk or in small biological samples such as cell lines extract [5], tissue samples [6], blood [7], and saliva [8], or at the single cell level [9].

Rapid, reliable, and high-sensitive assays are essential for the development of novel drug and disease screening systems. DNA topoisomerases are among the DNA binding enzymes that attract researcher interest, being the target of several clinical and experimental small molecule drugs. Topoisomerases are nuclear enzymes that remove supercoil tensions in the DNA and play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells contain two types of Topoisomerases: type I, which make single-stranded breaks in the DNA, and type II which cleave both strands of the double helix and passes a second double helical DNA strand through the gap. The type I topoisomerases are further divided into three mechanistically divided groups: IA, IB, and IC [10].

In eukaryotic cells, TOP1B enzymes relax supercoil tensions in the DNA by a catalytic cycle which comprises: /) TOP1B binding to the backbone of DNA, ii) the cleavage of one strand of the DNA with the formation of a transient TOP1B-DNA intermediate that allows for the rotation of the cleaved strand, ///) the ligation of the cleaved strand to re-establish the duplex DNA iv) the dissociation of TOP1B from the DNA. TOP1B can be inhibited by several small molecule drugs that act through different mechanisms such as prevention of DNA binding, inhibition of DNA cleavage, or inhibition of the ligation, with stabilization of the TOP1B-DNA cleavable complex. For this reason, TOP1B inhibitors are divided into two classes: poisons and catalytic inhibitors. Poisons include clinically used drugs, such as the derivatives of the natural compound camptothecin (CPT) that reversibly bind the covalent TOP1B-DNA cleavable complex, slowing down the ligation of the cleaved DNA strand, thus transforming TOP1B into a poison for the cell and inducing cell death [11]. Two water-soluble CPT derivatives, TPT (topotecan) and Irinotecan, have been approved by the FDA for clinical use in cancer therapy. Catalytic inhibitors act by inhibiting any other step of the TOP1B enzymatic activity.

To measure the activity of TOP1B, a lot of assays have been developed over the years [12]. The TOP1B assays are all based on the ability of TOP1B to bind, cleave, and ligate DNA and on the visualization of these steps of the TOP1B cycle as change in the electrophoretic mobility of DNA in a gel. The most common assay is called a relaxation assay in which a purified TOP1B enzyme is incubated with a supercoiled plasmid DNA. The activity of TOP1B relaxes the supercoiled plasmid stepwise generating relaxed plasmids bands with different mobility in an agarose gel. The relaxation assay visualizes full catalytic cycles of TOP1B and therefore does not allow distinguishing of which steps in the catalytic cycle is affected by a potential TOP1B inhibitor. Other assays have been developed to separate the cleavage and the ligation steps of TOP1B cycle [12]. However, these assays are troublesome, require the use of radiolabelled DNA oligonucleotides, and the preparation and run of time-consuming denaturing gels for the visualization of the cleaved or ligated oligonucleotides. These assays are difficult to master even for a highly skilled researcher, they require a lot of optimizations steps, and they can ^'t be adapted to a high-throughput system. Due to the need for more precise, reliable, easy, and faster methods, in 2008 a novel assay, called Rolling Circle Enhanced Enzyme Activity Detection (REEAD) for the single molecule detection of TOP1B cleavage-ligation equilibrium activity was developed [3]. This assay is based on the use of a DNA oligonucleotide which folds into a dumbbell structure that by the cleavage and ligation activity of TOP1B can be converted into a circularized closed DNA molecule. These circles can be amplified isothermally by Rolling Circle Amplification, mediated by a DNA polymerase such as the Phi29 polymerase. Upon hybridization with fluorescent complementary probes, the TOP1B activity can be quantified upon visualization of the Rolling Circle Products (RCPs) in a fluorescent microscope. This method is highly sensitive, quantitative, and can be multiplexed with activity measurement of other DNA binding enzymes [13]. Moreover, the method proved to be effective in the analysis of different TOP1B enzymes, such as hTOPl, Plasmodium falciparum TOPI (the pathogen causing Malaria) [4,7,8] and Leishmania Donovani TOPI. The REEAD technology can be used for activity measurements both of purified enzymes in drug screening systems [14]and of endogenous enzymes in biological samples such as extract from cell lines [5] , tissue biopsies [6], blood [7], and saliva [8] and it enables measurement at the single- cell level [9]. However, as REEAD quantifies the cleavage-ligation equilibrium activity in a drug screening setting it cannot determine which of these two steps in the catalytic cycle is targeted and therefore neither if the drug is a catalytic inhibitor or a poison.

Improved method for developing and testing drugs targeting DNA relaxing enzymes would be advantageous and in particular, more efficient and/or reliable and/or faster methods would be advantageous.

Summary of the invention

As outlined above, topoisomerase activity has already been used to measure the cleavage-ligation equilibrium without discriminating between these two steps of TOPI catalytic cycle. Anti-TOPIB drugs, with potential anti-cancer effect, should enhance the cleavage or inhibit the ligation step of the TOPI catalytic cycle [11]. This is not addressed by relaxation assays: the current state-of-the-art. For this reason, in here is presented new versions of the REEAD technology, called Cleavage-REEAD and Ligation-REEAD.

These two assays rely on the use of two DNA oligonucleotides, called "first oligonucleotide" (2) (or 'OA" in the examples) and "second oligonucleotide" (6) (or "OB" in the examples) as illustrated in Figure 1 and further explained in Example 1.

Thus, part of the present invention relates to assays for determining the influence of an anti-topoisomerase IB drug on the cleavage step and/or the ligation step of topoisomerase IB enzymatic activity. The invention also relates to kits comprising such assays.

Thus, an object of the present invention relates to method and kits for determining cleavage and ligation efficiency of topoisomerase IB enzymes in the presence or absence of a drug.

In particular, it is an object of the present invention to provide a method allowing for determining the effect of drugs/compounds on the cleavage or ligation activity of DNA relaxing enzymes such as Type IB to po iso me rases that solves the above- mentioned problems of the prior art in relation to separating the effect on cleavage and ligation.

Thus, one aspect of the invention relates to a method for determining the activity of a type IB topoisomerase (1) in a sample, the method comprising: a) providing a first oligonucleotide (2) being a substrate for the type IB topoisomerase (1); said first oligonucleotide (2) comprising a first hairpin structure, comprising: i. a first double stranded stem region (3) comprising a binding and cleavage site for the type IB topoisomerase (i); ii. a first single stranded loop region (4); and iii. a first 5'-single-stranded overhang (5); b) providing a sample comprising the type IB topoisomerase (1); c) incubating the first oligonucleotide (2) with the sample comprising the type IB topoisomerase (1); d) providing a second oligonucleotide (6) comprising a second hairpin structure comprising: i. a second double stranded stem region; ii. a second single stranded loop region (7); and iii. a second 5'-single stranded overhang (8), complementary or (substantially complementary) to the first 5'-single stranded overhang (5) of the first oligonucleotide (2) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1); e) incubating the first oligonucleotide (2) from step c) with the second oligonucleotide (6) from step d); thereby allowing the type IB topoisomerase (1), covalently coupled to the 3'-end of the first oligonucleotide (2), to ligate the 5'-end of the second oligonucleotide substrate (6) to the (new) 3'-end of the first oligonucleotide substrate (2); f) ligating the 3'-end of the second oligonucleotide (6) to the 5'-end of first oligonucleotide substrate (2), preferably using a ligase, thereby forming a circular oligonucleotide of the first oligonucleotide (2) and the second oligonucleotides (6) (except the initial 3'-end of the first oligonucleotide substrate); and g) determining the presence of the circular oligonucleotide, preferably using RCA.

Example 1 provides an overview of an embodiment of the invention.

Example 3 provides data for salt sensitivity of the assay, to validate the assay effectiveness.

Example 4 provides data for specific small molecule drugs, which targets human topoisomerase IB, and shows influence of the compounds on the cleavage and ligation part of topoisomerase IB activity respectively using the method of the invention. Another aspect of the present invention relates to a kit comprising:

- a first container comprising a first oligonucleotide (2) being a substrate for a type IB topoisomerase (1); said first oligonucleotide (2) comprising a first hairpin structure, comprising: i. a first double stranded stem region comprising a binding and cleavage site (3) for the type IB topoisomerase (1); ii. a first single stranded loop region (4); and iii. a first 5'-single-stranded overhang (5);

- a second container comprising a second oligonucleotide (6) comprising a second hairpin structure comprising: i. a second double stranded stem region; ii. a second single stranded loop region (7); and iii. a second 5'-single stranded overhang (8), complementary (or substantially complementary) to the first 5'-single stranded overhang (5) of the first oligonucleotide (2) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1);

- optionally, a third container comprising a type IB topoisomerase (1);

- optionally, a fourth container comprising a ligase;

- optionally, a fifth container comprising a polymerase;

- optionally, a sixth container comprising one or more primers (9), preferably the primer (9) is coupled to a solid support;

- optionally, a seventh container comprising one or more detection oligonucleotides (11); and

- optionally, instruction for using said kit to determine the influence of a drug on the type IB topoisomerase (1), such as the influence of the drug on the DNA cleavage and/or ligation of the type IB topoisomerase (1).

Yet another aspect of the present invention relates to the use of a kit according to the invention for screening of drugs, such as screening the drugs for modulating topoisomerase IB enzyme activity. Brief description of the figures

Figure 1 shows a schematic representation of the reactions occurring during the Cleavage-REEAD and the Ligation-REEAD. Figure 2 shows salt titration curve of the REEAD or Ligation-REEAD assays. hTOPl was incubated with the REEAD assays oligonucleotide or with the Oligonucleotides of the Ligation REEAD assay. The product of the reactions, closed DNA molecule, were amplified by RCA. After hybridization with fluorescent oligonucleotide, the slides were visualized using a fluorescent microscope. The signals were quantified by ImageJ software and plotted in GraphPad prism. The results are plotted as average and standard deviation of four independent experiments.

Figure 3 shows data for Cleavage-REEAD. hTopl was incubated with the OA in presence or absence of CPT or 6d small molecule drug. After the cleavage was performed, the ligation OB was added and the NaCI increased to 500mM. T4 ligation was performed and the product of the reactions, closed DNA molecule, were amplified by RCA. After hybridization with fluorescent oligonucleotide, the slides were visualized using a fluorescent microscope. The signals were quantified by ImageJ software and plotted in GraphPad prism, normalized to the DMSO. The results are plotted as average and standard deviation of four independent experiments. P values were determined using T student test ns: not significant.

Figure 4 shows data for Ligation-REEAD. hTopl was incubated with the OA. After the cleavage was performed, the ligation OB was added in the presence or absence of CPT or 6d small molecule drug and the NaCI increased to 500mM. T4 ligation was performed and the product of the reactions, closed DNA molecule, were amplified by RCA. After hybridization with fluorescent probes, the slides were visualized using a fluorescent microscope. The signals were quantified by ImageJ software and plotted in GraphPad prism, normalized to the DMSO. The results are plotted as average and standard deviation of four independent experiments. P values were determined using T student test ns: not significant.

Figure 5 shows data from a nicking experiment. hTOPl nicking activity in absence (only DMSO) and presence of 50 mM CPT or 50 pM compound 6d. Reactions were mixed with the supercoiled DNA plasmid before adding enzyme at 37 °C for the time indicated. The bands were separated by electrophoresis on a 1% agarose gel with ethidium bromide. C, control (supercoiled DNA only).

Figure 6 shows a schematic representation of an enhanced chemiluminescence (ECL)/colorimetric (TMB) based readout. (I) Enzyme converted DNA circle. (II) The DNA circles, generated by enzymatic reaction, are hybridized to an oligonucleotide primer, which is covalently bound to a solid support (e.g. a microscope slide, a membrane, or a bead). The DNA circles are amplified by Rolling Circle Amplification (RCA) mediated by a phi29 (or other) polymerase with 5 ^'-3 ^'prime polymerization- and strand displacement activity. (Ill) The RCA is performed by incorporation of biotinylated nucleotides. (IV) The rolling circle products (RCPs) are incubated with an anti-biotin antibody conjugated with horse radish peroxidase enzyme (HRP). The antibody binds specifically the biotin molecules incorporated into the RCPs. (V) The signals development is mediated by the HRP enzyme bound to the RCPs. It can be via Enhanced Chemiluminescent (ECL) (a) or via a transformation of a chromogenic substrate into a blue colour (b).

The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Sequence identity

In the present context, the term "identity" is here defined as the sequence identity between oligonucleotides at the nucleotide, base level.

Thus, in the present context "sequence identity" is a measure of identity between nucleic acids at nucleotide level. The nucleic acid sequence identity may be determined by comparing the nucleotide sequence in a given position in each sequence when the sequences are aligned. To determine the percent identity of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) x 100). In one embodiment, the two sequences are the same length. In another embodiment, the two sequences are of different length and gaps are seen as different positions. One may manually align the sequences and count the number of identical amino acids. Alternatively, alignment of two sequences for the determination of percent identity may be accomplished using a mathematical algorithm. Such an algorithm is incorporated into the NBLAST and XBLAST programs of (Altschul et al. 1990). BLAST nucleotide searches may be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to a nucleic acid molecules of the invention. BLAST protein searches may be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecule of the invention.

To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized.

Alternatively, PSI-Blast may be used to perform an iterated search, which detects distant relationships between molecules. When utilising the NBLAST, XBLAST, and Gapped BLAST programs, the default parameters of the respective programs may be used. See http://www.ncbi.nlm.nih.gov. Alternatively, sequence identity may be calculated after the sequences have been aligned e.g. by the BLAST program in the EMBL database (www.ncbi.nlm.gov/cgi-bin/BLAST). Generally, the default settings with respect to e.g. "scoring matrix" and "gap penalty" may be used for alignment. In the context of the present invention, the BLASTN and PSI BLAST default settings may be advantageous.

The percent identity between two sequences may be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. An embodiment of the present invention thus relates to sequences of the present invention that has some degree of sequence variation

Oligonucleotide

In the present context, an oligonucleotide is a sequence of DNA (or RNA) nucleotide residues that form a molecule. Oligonucleotides can bind their complementary sequences to form duplexes (double-stranded fragments) or even fragments of a higher order. Oligonucleotides can be on a linear form, but also exist as circular oligonucleotide molecules, such as single stranded circular DNAs. When referring the length of a sequence, reference may be made to the number of nucleotide units or to the number of bases. Furthermore, the typical DNA or RNA nucleotides may be replaced by nucleotides analogues such as 2'-0-Me-RNA monomers, 2'-0- alkyl-RNA monomers, 2'-amino-DNA monomers, 2'-fluoro-DNA monomers, locked nucleic acid (LNA) monomers, arabino nucleic acid (ANA) monomers, 2'-fluoro-ANA monomers, 1,5-anhydrohexitol nucleic acid (HNA) monomers, peptide nucleic acid (PNA), and morpholinoes. In the present context, the oligonucleotide should enzymatic targets for type IB topoisomerases and enable subsequent amplification by e.g. RCA or PCR. Thus, preferably the oligonucleotide is DNA, but may comprise one or more modified nucleotides.

Stem-loop structure

In the present context the term "Stem-loop structure", refers to intramolecular base pairing that can occur in single-stranded DNA. The structure is also known as a "hairpin" or "hairpin loop". It occurs when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop.

Type IB Topoisomerases

The natural function of type IB topoisomerases is to remove coiling from DNA to maintain DNA integrity during replication. DNA topoisomerases use a tyrosine residue for performing a nucleophilic attach on the DNA backbone thereby forming a temporary covalent link to the scissile DNA. Thus, the type IB topoisomerases form a covalent enzyme-DNA intermediate before self-re-ligating the DNA again, via a 3'-DNA-enzyme covalent intermediate, normally via a tyrosine residue.

Type IB Topoisomerases form a covalent enzyme-DNA intermediate before self- re-ligating the DNA again. Thus, topoisomerase IB activity does not require ligase activity from another enzyme. Type I topoisomerases cleaves only one strand in double stranded DNA,

Examples of specific Type IB topoisomerases are: eukaryotic topoisomerase IB, such as human topoisomerase IB, Plasmodium Topi, such as Plasmodium falciparum TOPI (PfTOPl), vaccinia ToplB, and Leishamia Donovani TOPI (LdTOPl).

Method for determining the activity of a type IB topoisomerase

As outlined above, the present invention discloses a novel method for determining topoisomerase IB activity. Such method is particularly suited for determining which step or steps in the topoisomerase catalytic cycle is influenced by a drug in question. For example, does a drug inhibit the cleavage step and/or the ligation step of the topoisomerase? Thus, an aspect of the invention relates to a method for determining the activity of a type IB topoisomerase (1) in a sample, the method comprising a) providing a first oligonucleotide (2) being a substrate for the type IB topoisomerase (1); said first oligonucleotide (2) comprising a first hairpin structure, comprising i. a first double stranded stem region (3) comprising a binding and cleavage site for the type IB topoisomerase (1); ii. a first single stranded loop region (4); and iii. a first 5'-single-stranded overhang (5); b) providing a sample comprising the type IB topoisomerase (1); c) incubating the first oligonucleotide (2) with the sample comprising the type IB topoisomerase (1); d) providing a second oligonucleotide (6) comprising a second hairpin structure comprising i. a second double stranded stem region; ii. a second single stranded loop region (7); and iii. a second 5'-single stranded overhang (8), complementary (or substantially complementary) to the first 5'-single stranded overhang (5) of the first oligonucleotide (2) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1); e) incubating the first oligonucleotide (2) from step c) with the second oligonucleotide (6) from step d); thereby allowing the type IB topoisomerase (1), covalently coupled to the 3'-end of the first oligonucleotide (2), to ligate the 5'-end of the second oligonucleotide substrate (6) to the (new) 3'-end of the first oligonucleotide substrate

(2); f) ligating the 3'-end of the second oligonucleotide (6) to the 5'-end of first oligonucleotide substrate (2), preferably using a ligase, thereby forming a circular oligonucleotide of the first oligonucleotide (2) and the second oligonucleotides (6) (except the initial 3'-end of the first oligonucleotide substrate); and g) determining the presence of the circular oligonucleotide, preferably using RCA.

Step a)

To enhance subsequent ligation, the 5-end of the first oligonucleotide (2) may be modified. Thus, in an embodiment, first oligonucleotide (2) comprises a 5' phosphorylation. Such modification allows for ligation with e.g. a T4 ligase.

The 3-end of the first oligonucleotide (2) may also be modified to block unspecific ligation and/or prevent degradation. Thus, in another embodiment, the first oligonucleotide (2) comprises a 3' blocking moiety, such as an amino group, a biotin group, a 2-MeORNA group, or a digoxigenin group, preferably an amino group or a 2-MeORNA group. In the example section, a 2-MeORNA group has been used. In the present context "2-MeORNA" may also be named "meA", "2'-0- methyl-RNA" or "2-O-meRNA". Again, such modification blocks for ligation with e.g. T4 DNA ligase if the type IB topoisomerase (1) has not cleaved of the 3'-end (which will then diffuse away). The preferred cleavage site for the topoisomerase may be optimized in the first oligonucleotide (2). Thus, in an embodiment, the cleavage site for the type IB topoisomerase (1) in the first oligonucleotide (2) is positioned between 2 and 6 nucleotides from the 3' end, such as 2-4 nucleotides from the 3'-end such as 3 nucleotides from the 3' end. It is noted that many topoisomerases do not have a specific binding and cleavage sites, but optimized sequences are known.

The length of the loop region and the stem region may also vary. Thus, in an embodiment, the single stranded loop region of the hairpin structure of the first oligonucleotide (2) has a length in the range 4-30 nucleotides such as 10-30 nucleotides, such as 15-25 nucleotides or such as 15-20 nucleotides.

In another embodiment, the stem region of the hairpin structure of the first oligonucleotide (2) has a doubled stranded region of a length in the range 10-30 nucleotides such as 15-30 nucleotides, or such as 20-30 nucleotides.

In a further embodiment the first 5'-single stranded overhang (5) of said first oligonucleotide (2) has a length in the range 5-20 nucleotides such as 10-20, or such as 10-15 nucleotides.

The overall length of the first oligonucleotide (2) may also vary. Thus, in yet an embodiment, said first oligonucleotide (2) has a length in the range 50-70 nucleotides, such as 60-70 nucleotides.

The first oligonucleotide (2) can also be given with specific sequences. Thus, in an embodiment, said first oligonucleotide (2) is selected from the group consisting of: a) a polynucleotide selected from the group consisting of SEQ ID NO: 2, 3, 10, 14, 17, 18, and 19 or; b) a polynucleotide having at least 80% sequence identity with the polynucleotides of a), with the proviso that the binding site for the type IB topoisomerase (1) and the stem-loop structure is maintained.

In the example section, SEQ ID NO 14, has been tested. In yet another embodiment, the first oligonucleotide (2) is hybridized to a third oligonucleotide (9) in the loop region. Such coupling may provide coupling to a solid support and/or the oligonucleotide (9) may function as a primer later in the method. Thus, in an embodiment, the third oligonucleotide (9) is coupled to a solid support, preferably at the 5'-end.

In yet another embodiment, the third oligonucleotide (9) may function as a primer for rolling circle amplification (RCA) or PCR in step g).

Step b)

The sample comprising the type IB topoisomerase (1) may be of different origin or type. Thus, in an embodiment, the sample in step b) comprises purified and/or isolated type IB topoisomerase (1).

In an embodiment, the sample is a biological sample such as selected from the group consisting of tissue samples, saliva, blood, food samples, surface swipes, such as from catheters, environmental sample, such as liquid, water, soil, air, and plant samples, such as seeds. In another embodiment, the biological sample is from an eukaryotic cell, such as human, parasite etc.

Type IB topoisomerases has a distinct mechanism of function. Thus in an embodiment, the type IB topoisomerase (1) forms a covalent enzyme-DNA intermediate before self-re-ligating the DNA again, via a 3'-DNA-enzyme covalent intermediate such as via a tyrosine residue.

The topoisomerase IB may have different origins. Thus, in an embodiment, the type IB topoisomerase (1) is selected from the group consisting of eukaryotic topoisomerase IB, such as human topoisomerase IB, Plasmodium Topi, such as Plasmodium falciparum TOPI (PfTOPl), vaccinia virus ToplB (vTopl), Leishmania such as Leishmania Donovani TOPI (LdTOPl), and other eukaryotic parasites such as Trypanosoma brucei TOPI.

It is noted that the first and second oligonucleotides can be the same for several eukaryotic TOP1B. Vaccinia Topoisomerase IB has a specific cleavage sequence, so for this one specific sequences can be designed. Step c)

Step c relates to incubating the first oligonucleotide (2) with the sample comprising the type IB topoisomerase (1). In an embodiment, in step c), binding of the type IB topoisomerase (1) to the first oligonucleotide (2) results in covalent coupling of the type IB topoisomerase (1) to the 3' end of the cleavage site and removal of the 3' fragment (10) of the first oligonucleotide (2). Due to the removal of the 3'-end by diffusion, the enzyme is trapped covalently coupled to the first oligonucleotide (2). (see also step II in figure 1 and further explained in example 1).

As outlined above, the method of the invention is particular well-suited for testing drugs targeting topoisomerase IB. Thus, in a preferred embodiment, in step c), a drug to be tested is present. This allows for determination an effect of the drug on the cleavage efficiency of the enzyme in question.

In a related embodiment, the drug is under investigation of affecting the DNA cleavage activity of the type IB topoisomerase (1), such as inhibiting or stimulating the DNA cleavage activity of the type IB topoisomerase (1), preferably for inhibiting the DNA cleavage activity of the type IB topoisomerase di-

Step d)

In an embodiment, the second oligonucleotide (6) does not comprise a 5' phosphorylation.

In an embodiment, the single stranded loop region of the hairpin structure of the second oligonucleotide (6) has a length in the range 10-30 nucleotides such as 15-30, such as 18-25.

In yet an embodiment, the stem region of the hairpin structure of the second oligonucleotide (6) has a doubled stranded region of a length in the range 3-20, such as 3-10 nucleotides or such as 5-10 nucleotides. In a further embodiment, the second 5'-single stranded overhang (8), complementary to the first 5'-single stranded overhang (5) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1) has a length in the range 10-15 nucleotides.

In yet an embodiment, the second 5'-single stranded overhang (8), complementary (or substantially complementary) to the first 5'-single stranded overhang (5) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1) is at least complementary at the first position closest to the 3'-end of the first oligonucleotide (2) after covalent binding of the type IB topoisomerase (1) to the first oligonucleotide (2). As previously explained, covalent binding of the enzyme to the first oligonucleotide results in removal of part of the 3'end, generating a new 3'-end (see also figure 1 step II).

In a further embodiment, said first oligonucleotide (2) and said second oligonucleotide (6) have different or identical loop sequences, preferably different loop sequences. The loop sequences can used e.g. for binding of primer(s) (3) or binding of detection oligonucleotides (11) (see figure 1).

In an embodiment, said second oligonucleotide (2) has a length in the range said second oligonucleotide (2) has a length in the range 40-90 nucleotides, such as 50-80 nucleotides, preferably such as 60-70 nucleotides or 40-60 nucleotides.

In a more specific embodiment, said second oligonucleotide (6) is selected from the group consisting of a) a polynucleotide selected from the group consisting of SEQ ID NO: 4, 5, 15, or b) a polynucleotide having at least 80% sequence identity with the polynucleotides of a), with the proviso that the capability to hybridize to said first oligonucleotide (6) is maintained and the stem-loop structure is maintained.

In yet a further embodiment, said first oligonucleotide (2) and said second oligonucleotide (6) are selected from the following combinations: a) SEQ ID NO: 2 and SEQ ID NO: 4; b) SEQ ID NO: 2 and SEQ ID NO: 5; c) SEQ ID NO: 3 and SEQ ID NO: 4; d) SEQ ID NO: 3 and SEQ ID NO: 5; e) SEQ ID NO: 10 and SEQ ID NO: 4 f) SEQ ID NO: 10 and SEQ ID NO: 5; g) SEQ ID NO: 14 and SEQ ID NO: 15; h) SEQ ID NO: 17 and SEQ ID NO: 4; i) SEQ ID NO: 17 and SEQ ID NO: 5; j) SEQ ID NO: 18 and SEQ ID NO: 4; k) SEQ ID NO: 18 and SEQ ID NO: 5;

L) SEQ ID NO: 19 and SEQ ID NO: 4 m) SEQ ID NO: 19 and SEQ ID NO: 5; or n) oligonucleotides of said combinations of a) to m) having at least 80% sequence identity with SEQ ID NO: 2, 3, 4, 5, 10, 14, 15, 17, 18, 19. Step e)

Step e) relates to incubation of the first oligonucleotide (2) from step c) with the second oligonucleotide (6) from step d).

In a preferred embodiment, in step e), a drug to be tested is present allowing for determination of effect of the drug on the ligation efficiency of the type IB topoisomerase (1) in question.

In another preferred embodiment, the drug is under investigation of affecting the DNA ligation activity of the type IB topoisomerase (1), such as inhibiting or stimulating the DNA ligation activity of the type IB topoisomerase (1), preferably for inhibiting the DNA ligation activity of the type IB topoisomerase (1).

In yet another preferred embodiment, in step c) and/or step e), a drug to be tested is present. Step f)

Step f) relates to ligating the 3'-end of second oligonucleotide (6) to the 5'-end of first oligonucleotide (2). In an embodiment, the ligation step f) is performed using a ligase selected from the group consisting of T4 DNA ligase, Pfu ligase, Taq ligase, T7 ligase, and E. coli ligase, preferably T4 DNA ligase. The skilled person would know that other components may be added to the reaction mixture to improve enzyme activity. Thus, in an embodiment, said ligation step is performed in a reaction environment allowing ligation, such as the presence of ATP and Mg. Step g)

Step g) concerns determining the presence of circularized oligonucleotides formed of the first oligonucleotide (2) (without the removed 3'-end) and the second oligonucleotide. See also figure 1, step IV.

In an embodiment, said determination step g) is performed by a method selected from the group consisting of Rolling Circle Amplification (RCA), PCR, real-time-PCR, Southern blotting, quantitative PCR (qPCR), restriction fragment length dimorphism-PCR (RFLD-PCR), primer extension, DNA array technology, LAMP, and isothermal amplification. In another embodiment, said determination step g) is performed by PCR, where one primer is complementary to at least part of the loop region of the first oligonucleotide and where a second primer is identical to at least part of the loop region of the second oligonucleotide. In a preferred embodiment, said determination step g) is performed by RCA, where a primer (9) is complementary to at least part of the loop region of the first oligonucleotide (2).

In another preferred embodiment, said primer (9) is coupled to solid support. In a related embodiment, said RCA is detected by hybridizing a detection oligonucleotide (11) to the amplification product, wherein said detection oligonucleotide hybridizes to a single stranded region of said RCA product, such as e.g. the detection oligonucleotide (11) being identical to at least part of the loop region of said first oligonucleotide (2) or said second oligonucleotide (6), preferably being identical to at least part of the loop region of the second oligonucleotide (6). As also explained in example 1, various readout formats may be used either by hybridization of labelled oligonucleotides to the RCPs or by incorporation of labelled nucleotides during RCA. The labelling can be fluorescent, allowing direct visualization in a fluorescence microscope or a fluorescent scanner or it can be biotin allowing subsequent visualization using avidine/streptavidin conjugated Horse Radish Peroxidase (HRP) or alkaline phosphatase allowing visualization using a colorimetric readout. The visualization can be performed also upon binding of an HRP-conjugated anti-biotin antibody. In sum, amplification can be detected in different ways. In example 6 and figure 6, quantitatively detection after incorporation of biotin in the RCA product, via Enhanced Chemiluminescent (ECL) or via a transformation of a chromogenic substrate into a blue colour (TMB- colorimetric based readout) is shown.

Thus, in an embodiment, RCA is detection by colorimetric readout, such as via Enhanced Chemiluminescent (ECL) or via a transformation of a chromogenic substrate, such as into a blue colour (TMB-colori metric based readout). In yet an embodiment the readout is quantitatively determined.

Other embodiments

In an embodiment, washing step is performed between one or more of the steps of the method, preferably each step, to remove surplus reactants. This is easily performed, especially of the assay is performed in a solid support format, as illustrated in example 1.

In an embodiment, said method is for in vitro and/or ex vivo use.

In another embodiment, said method is for in vitro use for drug screenings.

In a further embodiment, the first oligonucleotide (2) and/or second oligonucleotide (6) comprises one or more modified nucleotides, such as for minimizing undesired ligation reactions and/or exonuclease activity on the nucleotides. In an embodiment, at least step c) is performed in a NaCI concentration in the range 1-200 mM, such as 50-200 mM, such as 50-150 mM. Preferably, step c) is performed within these ranges.

In another embodiment, at least step e) is performed in a NaCI concentration in the range 100-500 mM, such as 150-500 mM, such as 200-500 mM, such as preferably in the range 300-500 mM or 400-500 mM. Preferably step e) is performed within these ranges. Example 3 shows salt sensitivity of the ligation step (step e)) of the assay of the invention. In sum, the reaction conditions can also be optimized, to improve different steps in the method of the invention. Thus, in an embodiment, the step c is being performed in a salt concentration, preferably NaCI, in the range 1-200 mM, preferably 50-150. In an embodiment at least step e) ToplB ligation, is performed in a NaCI concentration in the range 300-500mM.

Kit of parts

The present invention also relates to a kit of part. Thus, an aspect of the invention relates to a kit comprising:

- optionally, a third container comprising a type IB topoisomerase (1);

- optionally, a fourth container comprising a ligase;

- optionally, a fifth container comprising a polymerase;

In an embodiment, said first oligonucleotide (2) and said primer (9) is present in the same container, such as the primer being coupled to a solid support, preferably the coupling is at the 5' end of said primer (9).

In another embodiment, said kit comprises a ligase, a polymerase, and reagents therefore, e.g. in lyophilized form or in a cryoprotector such as glycerol (which can then be kept at -20°C).

Uses

Yet an aspect of the invention relates to the use of a kit according to the invention for screening of drugs, such as screening the drugs for modulating topoisomerase IB enzyme activity.

In an embodiment, modulation of activity is the cleavage and/or ligation activity of a topoisomerase IB enzyme.

Other aspects of the invention

An aspect of the invention relates to a method of determining the presence of circularized oligonucleotides in a sample using RCA, wherein biotin is incorporated in the RCA and the RCA is subsequently determined by colorimetric readout, such as via Enhanced Chemiluminescent (ECL) or via a transformation of a chromogenic substrate, such as into a blue color (TMB-colorimetric based readout). In yet an embodiment the readout is quantitatively determined.

In yet another embodiment, avidine/streptavidin conjugated Horse Radish Peroxidase (HRP) or alkaline phosphatase is subsequently coupled to the RCA comprising biotin. This allows for visualization using a colorimetric readout.

In example 6 and figure 6 quantitatively detection after incorporation of biotin in the RCA product, via Enhanced Chemiluminescent (ECL) or via a transformation of a chromogenic substrate into a blue color (TMB-colorimetric based readout) is shown.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1 - Concept of the invention

In here is disclosed novel tools to measure TOPI activity which have been called Cleavage-REEAD and Ligation-REEAD. Both these assays rely on the use of two DNA oligonucleotides, called Oligonucleotide A and Oligonucleotide B as illustrated in Figure 1. Thus, example 1 illustrates a specific embodiment of the invention.

By the use of the same assay scheme, it is possible to investigate the effect of a potential novel TOP1B inhibitor on the Cleavage or the Ligation step of the TOP1B catalytic cycle. This novel assay is faster, easier, and has the potential of multiplexing and high throughput.

Briefly with reference to figure 1, where numbers in brackets refer to the numbering in Figure 1. I) The Oligonucleotide A (OA) (2) is hybridized to a primer (9) which is covalently bound to a solid support (e.g. a microscope slide, a membrane, or a bead). OA (2) has a 5 ^'phophorylated end and a 3 ^'modification such as a 2-MeORNA or amine.

II) TOP1B (1) is added together with the OA (2) to the solid support. OA is a so- called "suicide" oligonucleotide, meaning that TOPlB can bind and cleave this DNA molecule but cannot ligate it, since the short 3'-end (10) of OA (2) diffuses away before ligation. Other terms for such substrates could be "entrapment- oligonucleotide", "immobilization- oligonucleotide, or capture oligonucleotide". Thus, the enzyme isn't inactivated just trapped/immobilized. Upon cleavage, TOP1B is covalently bound to OA (hence "suicides" analogy). If a potential TOP1B inhibitor is added to the reaction in this step of the assay, it is possible to investigate the effect of the compound onto the cleavage step of the TOP1B enzyme.

III) After the cleavage reaction, the ligation can occur by adding Oligonucleotide B (OB) (6) and increasing the salt concentration. OB (6) is partially complementary to OA (2) having a 5 ^'single-stranded region (8) complementary to 5 ^'single stranded (5) region of OA (2). Upon cleavage, TOP1B (1) can ligate OA and OB together, leaving a nick in one of the two strands. This ligation step is favoured by the increased salt concentration, which shifts the cleavage-ligation equilibrium towards the ligation. If a potential inhibitor is added in this step of the assay, the effect of the compound can be investigated on the ligation-step only.

IV) The TOP1B ligated substrate is finally incubated with T4-DNA ligase which will seal the nick between the 3 ^'end of OB and the 5 ^'phosphate end of OA that have been also ligated by TOPI. V) The now fully closed DNA molecule can be amplified by Rolling Circle

Amplification (RCA) mediated by the Phi29 polymerase using the oligonucleotide (9) attached to a solid support as primer. By incorporation of modified nucleotides, the cleavage or the ligation activity can be measured upon visualization of the RCPs. Various readout formats may be used either by hybridization of labelled oligonucleotides (11) to the RCPs or by incorporation of labelled nucleotides during RCA. The labelling can be fluorescent, allowing direct visualization in a fluorescence microscope or a fluorescent scanner or it can be biotin allowing subsequent visualization using avidine/streptavidin conjugated Horse Radish Peroxidase (HRP) or alkaline phosphatase allowing visualization using a colorimetric readout. In sum, amplification can be detected in different ways.

Example 2 - Materials and methods Oligonucleotide sequences for Cleavaae-Liaation REEAD eukaryotic TOPI:

Table 1: Oligonucleotides useful for Fluorescent readout (optimized versions)

Bold: Primer/primer binding sequence. Underlining: loop/ID sequence. [meA]: 2'- O-methyl-RNA (2-O-meRNA).

It is noted that for e.g. SEQ ID NO: 1, the non-bold part is a linker sequence, which in an embodiment could be substituted with another nucleic acid sequence, such (n (...) n) with the same or similar length such as in the range 10-30, or 10- 20, or 14-18 or 16 nucleotides.

Table 2: Oligonucleotides used for Colorimetric readout (optimized versions)

Table 3: REEAD Oligonucleotides (used in examples 3 and 4)

Bold: Primer/primer binding sequence. Underlining: loop/ID sequence.

Table 4: Cieavage-REEAD/Ligation-REEAD Oligonucleotides (used in examples 3 and 4)

Bold: Primer/primer binding sequence. Underlining: loop/ID sequence

Oligonucleotide sequences for Cleavaae-Liaation-REEAD vaccinia TOPI

Table 5: Oligonucleotides useful for Fluorescent readout ( vaccinia TOPI )

Table 6: Oligonucleotides used for Colorimetric readout (vaccinia TOPI)

Bold: Primer/primer binding sequence. Underlining: loop/ID sequence. [meA]: 2'- O-methyl-RNA (2-O-meRNA). Detection of hTOPl bv REEAD

The reactions were carried out onto primer-coupled High Density Glass slides (#DHDl-0023 Surmodics). 25 mm² squared hydrophobic areas were drawn on the glass surface using a mini pap pen (#008877 Thermo Fisher). The 5-amine- antitopo primer (SEQ ID NO: 1) was coupled to the squares of the slide according to the Surmodics manufacturer descriptions. Purified hTOPl [15] was incubated with 0.1 mM REEAD hTOPl substrate (SEQ ID NO: 11) in a standard hTOPl reaction buffer containing 10 mM Tris-HCI, pH 7.5, 5 mM CaC , 5 mM MgCh, and increasing concentration of NaCI (150-300-350-400-500 mM) for 30 min at 37 °C. Circularization reactions were terminated by inactivation for 5 min at 95 °C.

Subsequently, the circularized substrates were hybridized to the primer-coupled slide for 60 min at 37 °C. The slides were washed for 1 min at room temperature in wash buffer 1 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.3% SDS) followed by 1 min at room temperature in wash buffer 2 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.05% Tween-20). Finally, the slides were dehydrated in 99.9% ethanol for 1 min and air-dried.

Rolling circle DNA amplification (RCA) was performed for 60 min at 37 °C in lx Phi29 buffer (50 mM Tris-HCI, 10 mM MgCI₂, 10 mM (NH₄)2S04, 4 mM DTT pH 7.5) supplemented with 0.2 pg/pl BSA, 250 pM dNTP, and 1 unit/pl Phi29 DNA polymerase. The reaction was stopped by washing in wash buffers 1, 2 and 99.9% ethanol and air-dried. The Rolling Circle Products (RCPs) were detected by hybridization to 0.17 pM of REEAD fluorescent probe in a buffer containing 20% formamide, 2xSSC (300mM NaCI, 30 mM Sodium citrate) and 5% glycerol for 30 min at 37 °C. The slides were washed in wash buffers 1 and 2, dehydrated, mounted with with Vectashield (Vector laboratories), and visualized in the Olympus 1X73 fluorescent microscope. 15 pictures for every square of the slide were taken using a 63x objective and the hTOPl activity was quantified counting the fluorescent dots using ImageJ software.

Detection of hTOPl activity bv Cleavaae-REEAD The reactions were carried out onto primer-coupled High Density Glass slides (#DHDl-0023 Surmodics). 25 mm² squared hydrophobic areas were drawn on the glass surface using a mini pap pen (#008877 Thermofischer). The 5-amine-anti- ID33 primer (SEQ ID NO: 13) was coupled to the squares of the slide according to the Surmodics manufacturer descriptions.

Purified hTOPl [15] was incubated with 0.1 mM Cleavage/Ligation-REEAD OA substrate (SEQ ID NO: 14) in a standard hTOPl reaction buffer containing 10 mM Tris-HCI, pH 7.5, 5 mM CaCI₂, 5 mM MgCI₂, lOOmM NaCI for 30 min at 37 °C and in presence of 0.1% DMSO, or 50 pM CPT or 50 pM 6d [16]. The reactions were carried out onto the slide-squares coupled with the 5 ^'-amine anti-ID33 (SEQ ID NO: 13). The slides were washed two times with a buffer containing 10 mM Tris- HCL pH 7.5 and 1 mM EDTA to remove the small molecules drugs.

Subsequently, a buffer containing 1 pM Cleavage/Ligation-REEAD OB (SEQ ID NO: 15) and 500 mM NaCI was added to the squares of the slide and incubated for 60 min at 37°C. Slides were washed for 1 min at room temperature in wash buffer 1 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.3% SDS) followed by 1 min at room temperature in wash buffer 2 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.05% Tween-20). Finally, the slides were dehydrated in 99.9% ethanol for 1 min and air-dried.

The circularization reactions were completed by the addition of 10 unit/pl T4 DNA ligase in a buffer containing 50 mM Tris-HCI pH 7.5, 10 mM MgCI₂, ImM ATP for 60 min at 25°C. The slides were washed in wash buffers 1 and 2, and dehydrated.

Rolling circle DNA amplification (RCA) was performed for 60 min at 37 °C in lx Phi29 buffer (50 mM Tris-HCI, 10 mM MgCI₂, 10 mM (NH₄)₂S04, 4 mM DTT pH 7.5) supplemented with 0.2 pg/pl BSA, 250 pM dNTP, and 1 unit/pl Phi29 DNA polymerase. The reactions were stopped by washing in wash buffers 1 and 2 and 99.9% ethanol and air-dried.

The Rolling Circle Products RCPs were detected by hybridization to 0.17 pM of FAM-Topo-probe (SEQ ID NO: 16) in a buffer containing 20% formamide, 2xSSC (300mM NaCI, 30 mM Sodium citrate) and 5% glycerol for 30 min at 37 °C. The slides were washed in wash buffers 1 and 2, dehydrated, mounted with with Vectashield (Vector laboratories), and visualized in the Olympus 1X73 fluorescent microscope. 15 pictures for every square of the slide were taken using a 63x objective and the hTOPl activity was quantified counting the fluorescent dots using ImageJ software.

Detection of hTOPl activity bv Liaation-REEAD

The reactions were carried out onto primer-coupled High Density Glass slides (#DHDl-0023 Surmodics). 25 mm² squared hydrophobic areas were drawn on the glass surface using a mini pap pen (#008877 Thermo Fisher). The 5-amine-anti- ID33 primer (SEQ ID NO: 13) was coupled to the squares of the slide according to the Surmodics manufacturer descriptions.

Purified hTOPl [15] was incubated with 0.1 mM Cleavage/Ligation-REEAD OA substrate (SEQ ID NO: 14) in a standard hTOPl reaction buffer containing 10 mM Tris-HCI, pH 7.5, 5 mM CaCL·, 5 mM MgCL·, lOOmM NaCI for 30 min at 37 °C. Subsequently, a buffer containing 1 mM Cleavage/Ligation-REEAD OB (SEQ ID NO: 15) and 500 mM NaCI (or increasing concentration of NaCI 150-300-350-400-500 mM, see Figure 2) was added to the squares of the slide and in presence of 0.1 % DMSO, or 50 pM CPT or 50 pM 6d [16] and incubated for 15 min at 37°C. Slides were washed for 1 min at room temperature in wash buffer 1 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.3% SDS) followed by 1 min at room temperature in wash buffer 2 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.05% Tween-20). Finally, the slides were dehydrated in 99.9% ethanol for 1 min and air-dried.

The circularization reactions were completed by the addition of 10 unit/pl T4 DNA ligase in a buffer containing 50 mM Tris-HCI pH 7.5, 10 mM MgCh, ImM ATP) for 60 min at 25°C. The slides were washed in wash buffers 1 and 2, and dehydrated. Rolling circle DNA amplification (RCA) was performed for 60 min at 37 °C in lx Phi29 buffer (50 mM Tris-HCI, 10 mM MgCL·, 10 mM (NH₄)2S04, 4 mM DTT pH 7.5) supplemented with 0.2 pg/pl BSA, 250 pM dNTP, and 1 unit/pl Phi29 DNA polymerase. The reactions were stopped by washing in wash buffers 1 and 2 and 99.9% ethanol and air-dried. The Rolling Circle Products (RCPs) were detected by hybridization to 0.17 mM of FAM-Topo-probe (SEQ ID NO: 16) in a buffer containing 20% formamide, 2xSSC (300mM NaCI, 30 mM Sodium citrate) and 5% glycerol for 30 min at 37 °C. The slides were washed in wash buffers 1 and 2, dehydrated, mounted with Vectashield (Vector laboratories), and visualized in the Olympus 1X73 fluorescent microscope. 15 pictures for every square of the slide were taken using a 63x objective and the hTOPl activity was quantified counting the fluorescent dots using ImageJ software.

DNA Nicking assay

HTOPl activity was assayed using a DNA nicking assay by incubating 330 ng/mI of hTOPl with 0.5 mg of negatively supercoiled pUC18 in 20 mI of reaction buffer (20 mM Tris-HCI, 0.1 mM EDTA, 10 mM MgCI₂, 10 mM CaCI₂ and 150 mM NaCI, pH 7.5) and in presence of 0.1 % DMSO or 50 mM CPT or 50 mM 6d. The reactions were performed at 37 °C, stopped by the addition of 0.5% SDS after indicated time intervals. The samples were protease digested, electrophoresed in a horizontal 1% agarose gel in lxTBE (50 mM Tris, 45 mM boric acid, 1 mM EDTA) containing 0.5 mg/ml EtBr at 26V during 20 h. The picture was taken using a gel doc imager.

Example 3 - Salt sensitivity of the Cleavage-REEAD or Ligation REEAD assay compared with REEAD

Aim of study and theory

To differentiate between the cleavage and the ligation step of the TOP1B catalytic cycle using the model shown in Figure 1, a salt titration comparing this novel assay with the REEAD as performed.

The enzymatic activity of TOP1B is affected by the ionic strength in the reaction solution. In the REEAD assay, TOP1B can perform a cleavage-ligation equilibrium because the substrate used is one DNA oligonucleotide. TOP1B can complete an entire catalytic cycle on the DNA molecule and can dissociate from the DNA to start another cycle on the same molecule or another one. However, under physiological condition (150 mM NaCI), the ligation reaction is favoured over the cleavage reaction. As a product of the cleavage-ligation reaction, more closed DNA molecules are obtained than the nicked ones, which will in turn be amplified by RCA. If the ionic strength in the reaction is increased, TOP1B cannot cleave and ligate the DNA substrate because the initial step of the catalytic cycle, the DNA binding, is prevented. When a potential TOP1B inhibitor is added to the reaction it is possible to measure the effect on the cleavage-ligation equilibrium in a quantitative manner.

In the case of the Cleavage-REEAD or Ligation-REEAD, the effect of a potential inhibitor can be measured on the two separated reactions. First, TOP1B binds and cleaves OA remaining covalently bound to OA (Figure 1, II). In a second moment, OB is added to the reaction and TOP1B can ligate OA and OB together (Figure 1, III). If, in this step of the reaction, the ionic strength is increased, the reaction is shifted towards the ligation, obtaining the maximum amount of OA and OB ligated together. Indeed, after OA and OB have been ligated together, TOP1B dissociates from the DNA but the high ionic strength does not allow for a new catalytic cycle to happen.

Materials and methods See also example 2.

Results and conclusion

For the Cleavage-REEAD and Ligation-REEAD to be used as a drug-screening tool, this "ionic strength" hypothesis has been validated using a salt titration comparing REEAD and Ligation-REEAD. As control, the reactions were also performed in presence of human TOP1B (hTOPl) but without T4 DNA ligase or without hTOPl but in presence of T4 DNA ligase. As expected, the number of signals obtained with REEAD decreased as a function of the increased NaCI concentration (see Figure 2). In contrast, the Ligation-REEAD proved to be effective at up to 500 mM NaCI and the two negative controls had a very low number of signals, demonstrating the specificity of the reaction.

Example 4 - Small molecule drug effect on the Cleavage-REEAD or the Ligation-REEAD

Aim of study

To demonstrate the ability of the Cleavage-REEAD and the Ligation-REEAD to measure and determine the type of inhibitor tests with Camptothecin (CPT) were performed. CPT a well-known selective inhibitor of the TOP1B Ligation [11] and the 6d molecule, published as hTOPl inhibitor on the basis of the inhibition of the relaxation assay [16] .

Materials and methods

See also example 2 for further information.

Cleavage-REEAD:

The reactions were carried out onto glass slide coated with 5-amine-anti-ID33 primer (SEQ ID NO: 13).

The Cleavage-REEAD was performed by incubation of hTOPl with OA (SEQ ID NO: 14) in the presence of 50 mM CPT or 50 pM of 6d and 100 mM NaCI, to allow hTOPl to bind and cleave OA. As a control, DMSO was used, being the solvent of the two small molecule drugs. After 30 minutes of incubation, a mild wash was performed to remove the drugs. Subsequently, the OB (SEQ ID NO: 15) was added and the salt concentration was increased to 500mM to allow hTOPl to ligate all the cleaved OA with OB. Finally, T4 ligase was added, and the RCA performed. The RCPs were quantified using a fluorescent microscope and the results normalized to DMSO.

Ligation-REEAD:

In this case, the OA (SEQ ID NO: 14) was incubated with hTOPl and 100 mM NaCI for 30 minutes. After the cleavage reaction, DMSO, CPT or 6d were added together with the OB (SEQ ID NO: 15) and the salt concentration was increased to 500 mM. In this way, it was possible to measure the effect of the drugs on the ligation step of hTOPl.

Results

The results of the Cleavage-REEAD are plotted in Figure 3. As expected, CPT does not decrease hTOPl cleavage activity, being a well-known inhibitor of the ligation step only. The drug 6d showed instead a 2.5-fold drop of the hTOPl cleavage activity. The results of the Ligation-REEAD are plotted in Figure 4. As shown in Figure 4, CPT decreased the ligation activity of hTOPl, as expected and 6d showed the same extent of inhibition of CPT, showing to be a ligation inhibitor.

Conclusion

The method of the invention is indeed able to differentiate how a drug influences the cleavage and ligation step of Topoisomerase 1.

Example 5 - Agarose-gel based nicking assay

Aim of study

To confirm this result and validate the presented Cleavage-REEAD and Ligation- REEAD, we performed a state-of-the-art agarose-gel based nicking assay.

Materials and methods See also example 2.

In short, in this assay, TOP1B is incubated with a supercoiled plasmid in presence or absence of a small molecule inhibitor. After incubation, the DNA is run in an agarose gel, in presence of a DNA intercalator, such as Ethidium bromide. If a compound increases the TOP1B cleavage activity or decreases the TOPI ligation activity, the amount of plasmid DNA that is nicked in one of the two strands is increased. This can be visualized by the different mobility of the DNA in the agarose.

Results

As shown in Figure 5, CPT increases the amount of nicked DNA (compare lane 4-5 with lanes 2-3). This is consistent with CPT slowing down the ligation activity without inhibiting the cleavage activity, thus causing an increase of the amount of nicked DNA molecules, with TOP1B covalently bound to them. 6d drugs on the other hand, does not shown any increases of the nicked DNA (compare lanes 6-7 with lanes 2-3). This is consistent with 6d inhibiting the cleavage reaction thus preventing the generation of TOP1B-DNA cleavage complexes and the lack of nicked DNA molecules.

Conclusion With the present work we have, for the first time, designed two new tools, named Cleavage-REEAD and Ligation-REEAD, to measure the cleavage and the ligation step of the TOP1B catalytic cycle, respectively. The assays are fast, easy, and can be adapted to fluorescent or colorimetric readout, providing a reliable and easy accessible tools to researchers e.g. for use within the field of drug screening. The experimental conditions to be used in these new designed assays were verified by comparison with the state-of-the-art cleavage-ligation equilibrium REEAD.

By using two small molecules drugs, we have been able to measure the hTOPl cleavage or ligation activity separately. The use of CPT, a well-known TOP1B poison, enabled to validate the assays. Moreover, we have determined the mechanism of action of the small molecule 6d, which was previously published as an hTOPl inhibitor [16] and we found it to be both a cleavage and a ligation inhibitor, e.g. a catalytic inhibitor. This mechanism of action was confirmed by comparison with the nicking assay.

In conclusion, the Cleavage-REEAD and Ligation-REEAD are assays that can determine and measure the mechanism of action of the class IB of Topoisomerases enzymes. These tools allows easy, fast, effective, and quantitative analysis of TOP1B drugs for use in research or in the clinic as anticancer, anti-bacterial, or anti-parasite drugs.

Example 6 - Easy and fast readout for rolling-circle-based assays

Aim of study and theory

To develop easy and fast readout for the rolling-circle-based assays that does not require the use of a special equipment such as a fluorescent microscope or a fluorescent scanner. This allows for the detection of all the enzymatic activities, where a DNA substrate is converted into a closed circle, to be performed also in non-specialized laboratories.

Materials

The reactions were carried out onto primer-coupled High Density Glass slides (#DHDl-0023 Surmodics). A custom silicone isolator grid (called Wellmaker in the following) was designed and purchased from Grace-biolabs (#RD501118) and attached to the slide to create well- defined and separated squared areas (wells). The 5-amine-antitopo primer (SEQ ID NO: 1) was coupled to the wells according to the Surmodics manufacturer descriptions. Serial dilution of purified hTOPl was incubated with 0.1 mM REEAD hTOPl substrate (SEQ ID NO: 11) in a standard hTOPl reaction buffer containing 10 mM Tris-HCI, pH 7.5, 5 mM CaCh, 5 mM MgCh, and 50mM NaCI for 60 min at 37 °C in a humidifier chamber.

Circularization reactions were terminated by heat-inactivation for 5 min at 95 °C. Subsequently, the circularized substrates were hybridized to the primer-coupled slide for 60 min at 37 °C in a humidifier chamber. The slides were washed for 1 min at 25°C in wash buffer 1 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.3% SDS) followed by 1 min at room temperature in wash buffer 2 (0.1 M Tris-HCI, pH 7.5, 150 mM NaCI, and 0.05% Tween-20). Finally, the slides were dehydrated in 70% ethanol for 1 min and air-dried.

Rolling circle DNA amplification (RCA) was performed for 60 min at 37°C in a humidifier chamber in lx Phi29 buffer (50 mM Tris-HCI, 10 mM MgCh, 10 mM (NH4)2S04, 4 mM DTT pH 7.5) supplemented with 0.2 pg/pl BSA, 100 pM dATP, 100 pM dTTP, 100 pM dGTP, 90 pM dCTP, 10 pM biotin-dCTP (Jena #NU-809- BI016) and 1 unit/pl Phi29 DNA polymerase. The reactions were stopped by washing in wash buffers 1, 2 and 70% ethanol and air-dried.

The wells-coupled biotinylated Rolling Circle Products (RCPs) were incubated with 1:300 HRP-anti-biotin antibody (Merck, # A4541) in a lx TBST buffer (20 mM Tris-HCL, 150 mM NaCI, 5% non-fat dry milk, 5% BSA, 0.05% tween20, pH 9) for 1 hour at 25°C in a humidifier chamber. The wells were washed 3 times for 3 minutes in lxTBST buffer. The detection was performed either by incubation with 2pl of the ECL mixture 1: 1 (Cytiva #RPN2236) and visualized in a CCD camera or upon incubation with 4pl of TMB substrate (1-step Ultra TMB-Elisa #34028 Thermofisher) and imaged with a smartphone camera.

Methods

In this setup, the DNA circles are generated upon incubation of a DNA substrate with a specific DNA enzyme (Topoisomerases, restriction enzymes, other endonucleases, DNA repair enzymes) (Figure 6, 1). Upon hybridization to a solid support (glass slide, bead, nylon membrane), the circles are amplified by RCA in the presence of biotinylated nucleotides (Figure 6, II-III). This allows for subsequent coupling of Horse-Radish-Peroxidase (HRP) conjugated anti-biotin- antibody (Figure 6, IV). In the presence of luminol and hydrogen peroxide, the HRP enzyme then catalyses a chemical reaction for generating a recordable and quantitative signal in the form of light (Enhanced Chemiluminescence, Figure 6,

V a). The emitted light can be captured with X-ray film or by using a CCD camera imaging device that detect chemiluminescence. Alternatively, HRP catalyses the conversion of a chromogenic substrate 3,3',5,5'-Tetramethylbenzidine (TMB) into a soluble blue colour for a colorimetric visualization of the signals (Figure 6, V b).

Results and conclusions

Rolling circle amplification-based assays provide powerful, isothermal and easy tools for the measurement of DNA modifying enzymes (DNA Topoisomerases, endonucleases, DNA repair enzymes) either as purified form or in crude extracts from biological samples. The DNA circles, converted by enzymes, represent the substrate for the isothermal amplification that can be carried out in several ways, depending on the readout. By using the fluorescent microscopic readout is possible to obtain single-molecule resolution with the potential of detecting enzyme activities even in a single cell [9]. However, this require access to a fluorescence microscope, which is not possible for all laboratory setups. Therefore, we here investigated an alternative readout based on a simple ECL- or colorimetric-based method that does not require the use of special equipment. In this setup, biotinylated nucleotides are incorporated during RCA. Like the fluorescence microscope dependent readout, the biotinylated nucleotides-based readout provides a directly quantitative measure as demonstrated by the TOPI titration experiment shown in Figure 6, V a. In this experiment, decreasing concentrations of TOPI were assayed. The results were visualized using the ECL readout and show a linear relationship with the increasing amount of TOPI.

Similar results, but semiquantitative, can be obtained using a TMB-colorimetric based readout as showed in Figure 6, V b. The new presented readout provides and easy method to measure DNA binding enzymes activities as an alternative the state-of-the art assay, which are more tedious and requires specialized equipment and training.

References

[1] J.J. Champoux, DNA TOPOISOMERASES : Structure , Function , and Mechanism, Annu. Rev. Biochem. 70 (2001) 369-413. doi: 10.1146/a nnu rev. biochem.70.1.369.

[2] Y. Pommier Editor, DNA Topoisomerases and Cancer, 2011. https://www.springer.com/us/book/9781461403227 (accessed January 7, 2019).

[3] M. Stougaard, J.S. Lohmann, A. Mancino, S. Celik, F.F. Andersen, J. Koch, B.R. Knudsen, Single-molecule detection of human topoisomerase I cleavage-ligation activity., ACS Nano. 3 (2009) 223-33. doi: 10.1021/nn800509b.

[4] C. Tesauro, S. Juul, B. Arno, C.J.F. Nielsen, P. Fiorani, R.F. Frohlich, F.F. Andersen, A. Desideri, M. Stougaard, E. Petersen, B.R. Knudsen, Specific detection of topoisomerase i from the malaria causing P. falciparum parasite using isothermal Rolling Circle Amplification, Proc. Annu. Int. Conf. IEEE Eng. Med. Biol. Soc. EMBS. (2012) 2416-2419. doi: 10.1109/EMBC.2012.6346451.

[5] C. Tesauro, A.K. Simonsen, M.B. Andersen, K.W. Petersen, E.L. Kristoffersen, L. Algreen, N.Y. Hansen, A.B. Andersen, A.K. Jakobsen, M. Stougaard, P. Gromov,

B.R. Knudsen, I. Gromova, Topoisomerase I activity and sensitivity to camptothecin in breast cancer-derived cells : a comparative study, BMC Cancer. (2019) 1-15.

[6] A.K. Jakobsen, K.L. Lauridsen, E.B. Samuel, J. Proszek, B.R. Knudsen, H. Hager, M. Stougaard, Correlation between topoisomerase I and tyrosyl-DNA phosphodiesterase 1 activities in non-small cell lung cancer tissue, Exp. Mol. Pathol. 99 (2015) 56-64. doi : 10.1016/j.yexmp.2015.05.006.

[7] S. Juul, C.J.F. Nielsen, R. Labouriau, A. Roy, C. Tesauro, P.W. Jensen, C. Harmsen, E.L. Kristoffersen, Y.L. Chiu, R. Frohlich, P. Fiorani, J. Cox-Singh, D. Tordrup, J. Koch, A.L. Bienvenu, A. Desideri, S. Picot, E. Petersen, K.W. Leong,

Y.P. Ho, M. Stougaard, B.R. Knudsen, Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing plasmodium parasites based on enzyme activity measurement, ACS Nano. 6 (2012) 10676-10683. doi : 10.1021/nn3038594. [8] M.S. Hede, P.N. Okorie, S.K. Fruekilde, S. Fjelstrup, J. Thomsen, O. Franch, C.

Tesauro, M.T. Bugge, M. Christiansen, S. Picot, F. Ltsch, G. Mombo-Ngoma, J. Mischlinger, A. A. Adegnika, F.S. Pedersen, Y.P. Ho, E. Petersen, M. Stougaard, M. Ramharter, B.R. Knudsen, Refined method for droplet microfluidics-enabled detection of Plasmodium falciparum encoded topoisomerase i in blood from malaria patients, Micromachines. 6 (2015) 1505-1513. doi : 10.3390/mi6101432.

[9] J.G. Keller, C. Tesauro, A. Coletta, A.D. Graversen, Y.-P. Ho, P. Kristensen, M. Stougaard, B.R. Knudsen, On-slide detection of enzymatic activities in selected single cells, Nanoscale. 9 (2017). doi: 10.1039/c7nr05125e.

[10] N.M. Baker, R. Rajan, A. Mondragn, Structural studies of type I topoisomerases, Nucleic Acids Res. 37 (2009) 693-701. doi: 10.1093/nar/gknl009. [11] M.A. Cinelli, Topoisomerase IB poisons: Over a half-century of drug leads, clinical candidates, and serendipitous discoveries, Med. Res. Rev. 39 (2019) 1294-1337. doi : 10.1002/med.21546.

[12] S. Castelli, A. Coletta, I. D'Annessa, P. Fiorani, C. Tesauro, A. Desideri, Interaction between natural compounds and human topoisomerase I., Biol. Chem. 393 (2012) 1327-40. doi : 10.1515/hsz-2012-0240.

[13] F.F. Andersen, M. Stougaard, H.L. Jorgensen, S. Bendsen, S. Juul, K. Hald, A.H. Andersen, J. Koch, B.R. Knudsen, Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level., ACS Nano. 3 (2009) 4043-54. doi : 10.1021/nn9012912. [14] M.B. Andersen, C. Tesauro, M. Gonzalez, E.L. Kristoffersen, C. Alonso, G.

Rubiales, A. Coletta, R. Frohlich, M. Stougaard, Y.-P. Ho, F. Palacios, B.R. Knudsen, Advantages of an Optical Nanosensor System for Mechanistic Analysis of a Novel Topoisomerase I Targeting Drug: A case story, Nanoscale. (2016) 1886-1895. doi : 10.1039/C6NR06848K. [15] M. Lisby, J.R. Olesen, C. Skouboe, B.O. Krogh, T. Straub, F. Boege, S.

Velmurugan, P.M. Martensen, A.H. Andersen, M. Jayaram, O. Westergaard, B.R. Knudsen, Residues Within the N-terminal Domain of Human Topoisomerase I Play a Direct Role in Relaxation, J. Biol. Chem. 276 (2001) 20220-20227. doi : 10.1074/jbc.M010991200. [16] C. Alonso, M. Fuertes, E. Martin-Encinas, A. Selas, G. Rubiales, C. Tesauro, B.K. Knudssen, F. Palacios, Novel topoisomerase I inhibitors. Syntheses and biological evaluation of phosphorus substituted quinoline derivates with antiproliferative activity, Eur. J. Med. Chem. 149 (2018) 225-237. doi: 10.1016/j.ejmech.2018.02.058.

Claims

1. A method for determining the activity of a type IB topoisomerase (1) in a sample, the method comprising: a) providing a first oligonucleotide (2) being a substrate for the type IB topoisomerase (1); said first oligonucleotide (2) comprising a first hairpin structure, comprising: i. a first double stranded stem region (3) comprising a binding and cleavage site for the type IB topoisomerase (1); ii. a first single stranded loop region (4); and iii. a first 5'-single-stranded overhang (5); b) providing a sample comprising the type IB topoisomerase (1); c) incubating the first oligonucleotide (2) with the sample comprising the type IB topoisomerase (1); d) providing a second oligonucleotide (6) comprising a second hairpin structure comprising i. a second double stranded stem region; ii. a second single stranded loop region (7); and iii. a second 5'-single stranded overhang (8), complementary to the first 5'-single stranded overhang (5) of the first oligonucleotide (2) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1); e) incubating the first oligonucleotide (2) from step c) with the second oligonucleotide (6) from step d); thereby allowing the type IB topoisomerase (1), covalently coupled to the 3'-end of the first oligonucleotide (2), to ligate the 5'-end of the second oligonucleotide substrate (6) to the new 3'-end of the first oligonucleotide substrate (2); f) ligating the 3'-end of the second oligonucleotide (6) to the 5'-end of first oligonucleotide substrate (2), preferably using a ligase, thereby forming a circular oligonucleotide of the first oligonucleotide (2) and the second oligonucleotides (6); and g) determining the presence of the circular oligonucleotide, preferably using RCA.

2. The method according to claim 1, wherein the first oligonucleotide (2) comprises a 5' phosphorylation.

3. The method according to claim 1 or 2, wherein the first oligonucleotide (2) comprises a 3' blocking moiety, such as an amino group, a biotin group, a 2- MeORNA group, or a digoxigenin group, preferably an amino group or a 2- MeORNA group.

4. The method according to any of the preceding claims, wherein the first oligonucleotide (2) is hybridized to a third oligonucleotide (9) in the loop region.

5. The method according to any of the preceding claims, wherein the third oligonucleotide (9) is coupled to a solid support, preferably at the 5'-end.

6. The method according to any of the preceding claims, wherein the sample in step b) comprises purified and/or isolated type IB topoisomerase (1) or the sample is a biological sample such as a selected from the group consisting of tissue samples, saliva, blood, food samples, surface swipes, such as from catheters, environmental sample, such as liquid, water, soil, air, and plant samples, such as seeds.

7. The method according to any of the preceding claims, wherein the type IB topoisomerase (1) forms a covalent enzyme-DNA intermediate before self-re- ligating the DNA again, via a 3'-DNA-enzyme covalent intermediate such as via a tyrosine residue.

8. The method according to any of the preceding claims, wherein in step c) binding of the type IB topoisomerase (1) to the first oligonucleotide (2) results in covalent coupling of the type IB topoisomerase (1) to the 3' end of the cleavage site and removal of the 3' fragment (10) of the first oligonucleotide (2).

9. The method according to any of the preceding claims, wherein in step c) a drug to be tested is present, such as wherein the drug is under investigation of affecting the DNA cleavage activity of the type IB topoisomerase (1), such as inhibiting or stimulating the DNA cleavage activity of the type IB topoisomerase (1), preferably for inhibiting the DNA cleavage activity of the type IB topoisomerase (1).

10. The method according to any of the preceding claims, wherein in step e) a drug to be tested is present, such as wherein the drug is under investigation of affecting the DNA ligation activity of the type IB topoisomerase (1), such as inhibiting or stimulating the DNA ligation activity of the type IB topoisomerase (1), preferably for inhibiting the DNA ligation activity of the type IB topoisomerase (1).

11. The method according to any of the preceding claims, wherein in step c) or step e) a drug to be tested is present.

12. The method according to any of the preceding claims, wherein said determination step g) is performed by a method selected from the group consisting of Rolling Circle Amplification (RCA), PCR, real-time-PCR, Southern blotting, quantitative PCR (qPCR), restriction fragment length dimorphism-PCR (RFLD-PCR), primer extension, DNA array technology, LAMP and isothermal amplification.

13. The method according to any of the preceding claims, wherein said method is for in vitro use for drug screenings.

14. A kit comprising:

- a first container comprising a first oligonucleotide (2) being a substrate for a type IB topoisomerase (1); said first oligonucleotide (2) comprising a first hairpin structure, comprising: i. a first double stranded stem region comprising a binding and cleavage site (3) for the type IB topoisomerase (1); ii. a first single stranded loop region (4); and iii. a first 5'-single-stranded overhang (5); - a second container comprising a second oligonucleotide (6) comprising a second hairpin structure comprising i. a second double stranded stem region; ii. a second single stranded loop region (7); and iii. a second 5'-single stranded overhang (8), complementary to the first 5'-single stranded overhang (5) of the first oligonucleotide (2) after enzymatic cleavage of the first oligonucleotide (2) by the type IB topoisomerase (1);

- optionally, a third container comprising a type IB topoisomerase (1); - optionally, a fourth container comprising a ligase;

- optionally, a fifth container comprising a polymerase;

15. Use of a kit according to any of claim 14 for screening of drugs, such as screening the drugs for modulating topoisomerase IB enzyme activity.