**2. Results and discussion**

### **2.1 Detection of TOP1 activity using REEAD with easy-to-perform readout formats**

Here, we present alternative readout methods for the quantitative and sensitive detection of TOP1 activity using the previously described REEAD assay [48]. In the new assay setup, the fluorescent detection of TOP1 generated products has been substituted with either chemiluminescent or a colorimetric readout. The original and the modified REEAD assays are schematically depicted in **Figure 1**. The setup uses a specially designed dumbbell-shaped DNA substrate that contains a doublestranded stem and two single-stranded loops. The stem contains a TOP1 preferred cleavage site three bases upstream from the 3′ end (**Figure 1**, **I**). Cleavage of the substrate results in temporary covalent binding of TOP1 to the 3′-end and diffusion of the three-base fragment, allowing the 5′ hydroxyl overhang to anneal to the

#### **Figure 1.**

*Schematic representation of the REEAD assay. (I) The dumbbell-shaped substrate contains a preferred TOP1 cleavage site in the double-stranded stem, as well as a primer binding (PB) sequence and an identifier (ID) element in the two single-stranded loops. The substrate acts as a specific template for TOP1 and is upon the TOP1 cleavage and ligation reaction converted into a closed circle (II). (III) The anchored circles are amplified by rolling circle amplification (RCA) initiated by Phi29 polymerase. The RCA can be performed either by incorporation of fluorescent nucleotides (IV) or biotinylated nucleotides (VI). The fluorescent rolling circle products are visualized using a fluorescent microscope (V). The biotinylated rolling circle products are incubated with an anti-biotin antibody conjugated with horse radish peroxidase (HRP) (VII), which binds specifically to the incorporated biotin molecules. The signal development is mediated by the HRP enzyme bound to the rolling circle products. The signals are then visualized by enhanced chemiluminescence (ECL) (VIII, left) and detected in a CCD camera or using Kodak films. Alternatively, HRP catalyzes the conversion of a chromogenic substrate TMB into a blue color for a colorimetric visualization of the signals (VIII, right).*

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

substrate, thus positioning itself for TOP1 mediated ligation. The religation reaction results in the conversion of the dumbbell substrate from an open conformation to a closed DNA molecule, named circle in the following (**Figure 1**, **II**). The closed DNA circle is hybridized to a surface-anchored oligonucleotide, which is complementary to the region in one of the single-stranded loops of the TOP1 substrate (loop PB, **Figure 1**, **I**). RCA is initiated from this surface-anchored oligonucleotide using the phi29 polymerase, which is able to perform RCA with a high degree of strand displacement (**Figure 1**, **III**). RCA generates a long tandem repeat product complementary to the initial DNA circle. In the original REEAD assay, after RCA is performed, a fluorescent probe, complementary to the other loop of the dumbbell substrate (loop ID, **Figure 1**, **I**), is hybridized to the RCPs, thus allowing for the visualization in the fluorescence microscope. Alternatively, the RCA can be carried out in the presence of fluorescently labeled nucleotides (**Figure 1**, **IV**) generating bright fluorescent dots as a product that can be visualized in the microscope. In both cases, the RCPs will appear as fluorescent dots, and given the sensitivity of the assay, each dot will correspond to one cleavage-religation reaction (**Figure 1**, **V**). Upon taking pictures of the fluorescent RCPs coupled to the slide, these dots can be counted using a software and plotted as a direct measure of the number of the TOP1-mediated cleavage-religation reactions.

To enable the visualization of the RCPs to be performed without the use of a big, expensive, and not easy to use instrument, and without the time-consuming image analysis, the RCA can be performed in the presence of biotinylated nucleotides (**Figure 1**, **VI**). This generates long tandem repeat products with several incorporated biotins. The detection of the products can then be achieved by incubation with horse radish peroxidase (HRP)-conjugated anti-biotin antibodies that will bind the biotin molecule on the RCPs (**Figure 1**, **VII**). This enables visualization in two ways by adding specific substrates for HRP. The substrate can be the components of an ECL kit resulting in a chemiluminescence readout. Alternatively, the substrate can be 3,3′,5,5′-Tetramethylbenidine (TMB) that is oxidized by HRP and converted from colorless to blue giving a colorimetric readout (**Figure 1**, **VIII**). Both detection methods enable a fast, simple, and quantitative detection of TOP1 activity.
