**1. Introduction**

Colorectal cancer is the third most common cancer worldwide with more than 1.9 million new cases in 2020 [1]. Camptothecin (CPT) is the mother compound of a class of molecules that specifically targets the Topoisomerase 1 enzyme (TOP1) [2–4]. Currently, derivatives of the camptothecin (CPTs) family such as irinotecan are in clinical use for treatment of advanced stage of colorectal malignancies [5–7]. However, despite promising results [5], only a subset of the patients respond well to CPTs-based treatment and the development of chemoresistance remains a major issue [4, 8–10]. Tumor cells are characterized by a high degree of heterogeneity not only in morphology, but also in the functionality of the cells, including the activity of intracellular enzymes [11, 12]. Hence, investigation of TOP1 activity is a good biomarker for determining the response to CPTs-based anticancer treatment.

TOP1 maintains the genomic DNA integrity by regulating the DNA topology during replication and transcription. This is achieved by introducing a transient nick in the double-stranded DNA and the formation of a DNA-TOP1 cleavage complex (TOP1cc). The TOP1 enzyme becomes covalently attached to the 3′end of the DNA, and this is followed by a rapid religation of the scissile strand. These cleavage and ligation reactions allow for the relaxation of the supercoiling state of the DNA [13]. Although the cleavage-ligation reactions are fast, CPTs are able to reversibly bind to the interface of TOP1cc and selectively inhibit the religation step of the TOP1 catalysis, thereby prolonging the half-life of TOP1cc [14]. Upon collision with the replication- or transcription machinery, TOP1cc is converted to permanent double-stranded breaks resulting in genome fragmentation, which potentially can cause cell death [15, 16]. Hence, CPTs convert the activity of TOP1 into a cell poison, explaining the direct correlation between TOP1 activity and the TOP1 susceptibility to CPTs [17–20]. Consistent with the cytotoxic effect of CPTs, high level of TOP1 activity is associated with high CPTs' sensitivity, and these drugs are indeed particularly effective on fast dividing cells, such as cancer cells, where TOP1 is generally upregulated to manage the increased number of S-phase cycles [15, 21, 22]. Therefore, common mechanisms behind chemoresistance toward CPTs include downregulation of TOP1 level [17, 19, 20, 23–28] or mutations in the TOP1 gene leaving the enzyme insensitive toward CPTs [8, 29–32]. Cancer cells are frequently observed to have an upregulated activity of TOP1 [33–35], and enzyme activity can be regulated posttranslationally and not necessarily correlate to the TOP1 protein levels in the cells [36, 37]. Hence, a central aspect in investigating biomarkers for drug resistance is the measurement of enzymatic activity rather than RNA level or protein amount alone.

Over the years, a number of assays have been developed to investigate the activity of the TOP1 enzyme [38] to allow for the enzyme mechanism to be dissected [39], to investigate the inhibition of potential new small-molecule compounds [40], or to validate TOP1 as a cancer biomarker in cell lines [41, 42]. Among the most used assays, we have the gold standard relaxation assay [43], the DNA suicide cleavage-ligation assay [44, 45], the electrophoretic mobility shift assay [46], and the *in vivo* complex of enzymes (ICE) assay [47]. These assays have been extensively used to dissect the steps of the TOP1 catalytic cycle, but they have a lot of limitations. They require either gel electrophoresis, which involves DNA intercalating agents, or highly specialized expertise and training, and they all usually perform optimally when using a large amount of purified TOP1 enzyme or cell extract. For all these reasons, these assays have been used only in research settings, making the potential of investigating TOP1 as a predictive marker for anticancer response very limited.

We have previously developed a rolling circle enhanced enzyme activity detection (REEAD) assay [48] that enables the specific detection of TOP1 activity at the single catalytic event level [49]. In the REEAD assay, the cleavage and ligation reaction of TOP1 converts a specifically designed DNA substrate to a closed circle. This reaction can either be performed in solution, where the generated DNA circles are hybridized to a glass-slide-anchored primer or directly onto the glass slide upon hybridization of the DNA substrate to the primer-coupled slide (On-slide REEAD) [50, 51]. In either

### *Simple and Fast DNA-Based Tool to Investigate Topoisomerase 1 Activity, a Biomarker for Drug… DOI: http://dx.doi.org/10.5772/intechopen.105758*

case, each closed circle acts as a template for isothermal rolling circle amplification (RCA) generating ~103 tandem repeat rolling circle products (RCPs). These RCPs can then be detected in a fluorescent microscope at the single molecule level by hybridization to a fluorescently labeled DNA probe or by the incorporation of fluorescently labeled nucleotides during the RCA step. Using this setup, the assay proved to be highly sensitive, as each TOP1-mediated cleavage-ligation generates one closed DNA circle that results in one detectable product in the microscope, and thereby the assay is directly quantitative. For these reasons, REEAD is a powerful tool that allows the investigation of TOP1 activity in crude extract from small biological samples. Indeed, using the described REEAD setup, we have been able to measure the activity of TOP1 in biopsies from cancer patients [52], and in single cells [50, 51] and to predict the CPT cytotoxicity in cancer cell lines [42]. Moreover, REEAD allowed to measure the activity and CPT sensitivity of rare subpopulation of colon cancer cell lines, showing a high degree of chemoresistance [42, 50, 51]. Finally, we have recently developed a new REEAD-based assay, called REEAD C/L that allows for the cleavage and ligation steps of the catalytic cycle to be investigated separately [53]. This enables the identification of new small-molecule compounds as potential TOP1 poisons, which specifically inhibit the relegation step or as TOP1 catalytic inhibitors, which inhibit the DNA binding/cleavage of the enzyme catalysis.

However, both the basic REEAD setup and the REEAD C/L have some limitations. Using a fluorescently labeled probe or fluorescently labeled nucleotides requires a fluorescent microscope for the detection of the RCPs, and to use such a microscope requires specialized training. Moreover, this setup is not well adaptable to non-specialized laboratories or clinical settings. Therefore, the development of other readout methods is highly relevant.

In this chapter, we present two newly developed simple and fast readout methods for the REEAD assay that do not require the use of a fluorescent microscope. In these setups, TOP1 converts a specific DNA substrate to a closed circle, and the RCA is performed in the presence of biotinylated nucleotides. This allows for the detection *via* the two readouts, by enhanced chemiluminescence (ECL) or by color development directly onto the slide. In this way the assay becomes easy adaptable to all laboratory settings, including clinics, where a screening for the patient response to treatment can be performed with results in only few hours.
