Ethidium bromide solution

Genomic DNA was extracted using GenElute Mammalian Genomic DNA (Merck KGaA, Darmstadt, Germany). The bisulfite treatment of the genomic DNA was performed using an EZ DNA Methylation Kit (Zymo Research Corporation, Tustin, CA, USA). A PCR was performed using an EpiScope^® MSP Kit (Takara Bio Inc., Shiga, Japan). We obtained the DNA sequences of the CpG island from the UCSC Genome Browser (https://genome-asia.ucsc.edu/index.html) accessed on 25 March 2024 and designed the primers for the MSP using Meth-Primer (https://www.urogene.org/cgi-bin/methprimer/methprimer.cgi) accessed on 25 March 2024. The sequences of primers for the MSP are listed in Table S1. PCR products were subjected to 3% gel agarose electrophoresis. The gels were stained with Ethidium Bromide Solution (1:20,000, NACALAI TESQUE, Inc., Kyoto, Japan), and the images were captured using Amersham ImageQuant 800 (Global Life Sciences Technologies Japan K.K., Tokyo, Japan).

The intercalation of bio-psoralen into underwound DNA was examined using an in vitro crosslinking assay^{26 (link)}. DNA was crosslinked by bio-psoralen under UV irradiation, linearized, and denatured by heat. As bio-psoralen crosslinking prevents DNA heat denaturation, the intercalation of bio-psoralen can be evaluated based on the amount of non-denatured DNA after heat treatment. Relaxed and underwound DNA were prepared by incubating plasmid pBR322 (Takara Bio) with topoisomerase I (TopI, Takara Bio) and gyrase (TopoGEN), respectively, at 37 °C for 1 h, and purified using NucleoSpin^® Gel and a PCR Clean-up kit (Macherey-Nagel). Bio-psoralen (200 µM; EZ-Link^® Psoralen-PEG₃-Biotin, Thermo Fisher Scientific) was added to DNA and exposed to 3.6 kJ/m² of long-wave (365 nm) UV (LUV-4; AS ONE) on ice for 30 min. For linearization, DNA was cleaved with EcoRI (Toyobo) at 37 °C for 1 h. For heat denaturation, one half of each sample was boiled for 5 min and immediately chilled in ice-cold water. Agarose gel electrophoresis was run at 50 V for 1 h, and the gel was stained with 0.1 µg/mL ethidium bromide solution (Nacalai Tesque) for 30 min. Images were acquired using ImageQuant^TM LAS500 (Cytiva) and DNA bands were quantified using Fiji^{59 (link)}. The fraction of bio-psoralen-crosslinked DNA was calculated by dividing the amount of non-denatured DNA by the total amount of DNA.