**4. Material and methods**

### **4.1 Materials**

### *4.1.1 Oligonucleotides*

All oligonucleotides for construction of the TOP1-specific REEAD substate and the REEAD primer were synthesized by Merck Life Science A/S, Søborg, Denmark. The sequences of the oligonucleotides were as follows:

5′-amine REEAD primer: 5′-/5AmMC6/CCAACCAACCAACCAAGGAGCCAAA CATGTGCATTGAGG

TOP1 dumbbell substrate:

5′-AGAAAAATTTTTAAAAAAACTGTGAAGATCGCTTATTTTTTTAAAAATTTTTCT AAGTCTTTTAGATCCCTCAATGCACATGTTTGGCTCCGATCTAAAAGACTTAGA

### *4.1.2 Reagents*

CodeLink HD Activated slides (#DHD1-0023) and BioFX TMB enhanced one compound HRP (ESPM-0100-01) were from SurModics and the custom silicon isolator grids were from Grace-Biolabs. Vectashield without DAPI (#H-1000) was from Vector Laboratories. ATTO-488 dUTP (#95387) and biotin-16-dCTP (NU-809- BIO16L) were from Jena Bioscience. Anti-Biotin HRP conjugated antibody (#A4541) was from Merck, and ECL mixture (#RPN2236) was from Cytiva. The synthetic gene of the phi29 polymerase was from GenScript, and the GST Gravitrap columns (#28952360) were from GE Healthcare.

### **4.2 Methods**

## *4.2.1 Cell culture*

Caco2 cells were cultured in MEM supplemented with 20% FBS, 1% non-essential amino acids, 1% penicillin-streptomycin. Cells were incubated in a humidified incubator (5% CO2/95% air atmosphere) at 37°C and harvested by trypsin treatment. Fresh cell pellets were used for all analyses.

### *4.2.2 Phi29 purification*

The synthetic gene of the phi29 polymerase was purchased from GenScript and cloned into the pGEX vector resulting in a recombinant N-term GST-tagged phi29 Polymerase expression plasmid. *E. coli* competent cells BL21 (Promega) were transformed with the plasmid and grown in 2xTY media supplemented with 100 μg/ ml of ampicillin. Expression of the fusion protein was induced in log phase cells at OD600 = 0.8, by addition of 1 mM isopropyl b-D-1-thiogalactopyranoside at 37°C for 2 h. Cells were harvested after induction and resuspended in sonication buffer (50 mM Tris-HCL pH 7.5, 2.5 M NaCl, 1 mM EDTA, 1 mM DTT, 10 mg/ml of Lysozyme). Following 1 h of incubation on ice, the cells were then lysed by freezing and thawing in liquid N2 followed by sonication. After centrifugation, the lysate was mixed with 4% Streptomycin Sulfate for 1 h at 4°C. The insoluble particles were removed by centrifugation and the lysate was filtered by using a 0.45 μm filter. The lysate was loaded onto a pre-equilibrated GST Gravitrap column (GE Healthcare)

following manufacturer's instructions. The column was washed in 10-time volumes of sonication buffer. Protein was eluted in 10-time column volumes of elution buffer (10 mM Tris-HCl pH 8, 5 mM Glutathione, 500 mM NaCl) and collected in fractions. The fractions were analyzed on a protein gel. The fractions were then adjusted to 50% glycerol, 0.5% Tween20, 1 mM DTT, and 0.5% NP40 and stored at −20°C.

### *4.2.3 Preparation of slides*

A custom-designed silicone isolator grid, the Wellmaker (Grace-bio lab, USA), was attached to the CodeLink HD slides (Surmodics, USA). The 5′-amine REEAD primer was coupled to the slides in print buffer (300 mM Na3PO4, pH 8) and incubated overnight in a humidity chamber with saturated NaCl. The slides were blocked in 50 mM Tris, 50 mM Tris-HCl, 50 mM Ethanolamine, pH 9 for 30 min at 50°C, and subsequently washed in 4xSSC, 0.1% SDS for 30 min at 50°C.

### *4.2.4 Circularization and rolling circle amplification*

The circularization of the TOP1-specific dumbbell substrate was carried out by incubating a serial dilution of cell extract from Caco2 cells with 0.1 μM substrate in 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 50 mM NaCl for 1 h at 37°C in a humidifier chamber. The circularization reaction was terminated by heat inactivation for 5 min at 95°C. Subsequently, the circles were hybridized to the primer-coupled slides for 1 h at 37°C in a humidifier chamber. The slides were washed for 1 min at room temperature in wash buffer 1 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% SDS) followed by 1 min wash at room temperature in wash buffer 2 (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20). Finally, the slides were dehydrated for 1 min in 70% ethanol and air-dried.

Rolling circle amplification was carried out for 2 h at 37°C in a humidifier chamber in 1× Phi29 buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT) supplemented with 0.2 μg/μL BSA, 100 μM dATP, 100 μM dTTP, 100 μM dGTP, 90 μM dCTP, 10 μM biotin-dCTP, and 1 unit/μL Phi29 polymerase for colorimetric readout. Alternatively, the rolling circle amplification was carried out in 1× Phi29 buffer supplemented with 0.2 μg/μL BSA, 100 μM dATP, 100 μM dCTP, 100 μM dGTP, 90 μM dTTP, 10 μM ATTO-488-dUTP, and 1 unit/μL Phi29 polymerase for fluorescent readout. The reaction was stopped by washing the slide in wash buffer 1 and 2 for 5 min, dehydrated in 70% ethanol, and air-dried.

### *4.2.5 Detection of rolling circle products*

For the fluorescent readout, the slide was mounted with Vectashield without DAPI and visualized using a 60x objective in a fluorescent microscope (Olympus IX73). The signals detected in an average of 12 images were counted in ImageJ and plotted as mean.

Alternatively, the slide was blocked in 1xTBST (20 mM Tris-HCl pH 9, 150 mM NaCl, 0.05% Tween-20) supplemented with 5% nonfat dry milk and 5% BSA for 30 min at room temperature followed by a 2-min wash in 1xTBST. This was repeated before an incubation with 1:300 HRP conjugated anti-Biotin antibody in a 1xTBST supplemented with 5% nonfat dry milk and 5% BSA buffer for 50 min at room temperature in a humidifier chamber. The slide was washed three times for 3 min in 1xTBST buffer. The chemiluminescent detection was performed by adding 2 μL

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

1:1 ECL mixture and visualized in a CCD camera. The colorimetric detection was performed by incubation with 2 μL TMB for 30 min followed by 1 min wash in 70% EtOH. The slide was air-dried, and a picture of the color development was taken using the camera of a smartphone.

### *4.2.6 Statistical analysis*

Data were analyzed using GraphPad Prism software and expressed as mean ± standard deviation.

Statistical significance between two groups was assessed with a two-tailed unpaired Student's t-test, applying Welch correction.

### **Acknowledgements**

The authors would like to thank laboratory technician Noriko Y. Hansen (Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus, Denmark) for technical assistance in relation to Phi29 enzyme purifications. We like to acknowledge the support from Food & Bio Cluster Denmark.
