3.3. FRET Melting Analysis

Fluorescence resonance energy transfer (FRET) melting assay was performed with an excitation wavelength of 470–505 nm and a detection wavelength of 523–543 nm using the DNA Engine Option 2 Real-Time Cycler PCR detection system (Biorad, Hercules, CA, USA). The dual fluorescently labeled oligonucleotides were used in this protocol (Table S1). The donor fluorophore was 6-carboxyfluorescein (FAM) and the acceptor fluorophore was 6-carboxytetramethylrhodamine (TAMRA). All purified nucleotides (Sigma Genosys, Tokyo, Japan) were dissolved as stock solutions (100 μM) in MilliQ water to be used without further purification. Further dilutions of the oligonucleotides were performed with a 60 mM potassium cacodylate buffer (pH 7.4), and FRET experiments were carried out with a 0.4 μM oligonucleotide solution. Dual-labeled DNA was annealed by heating at 99 °C for 5 min, and then slowly cooled to room temperature. Ligands were prepared as DMSO stock solutions (10 mM) and diluted to 1 mM using DMSO, and then diluted to 100 μM using a 60 mM potassium cacodylate buffer (pH 7.4). Next, the annealed DNA (20 μL, 0.4 μM) and the compound solution (20 μL, 2 μM) were distributed across 96-well plates (Takara), with a total volume of 40 μL, with the labeled oligonucleotide (0.2 μM) and the compound (1.0 μM). The plates were incubated at 25 °C for 12 h. Subsequent experiments used the following temperature procedure in RT-PCR, finishing as follows: 25 °C for 20 min, and then a stepwise increase of 1 °C every minute from 25 °C until 99 °C. During the procedures, we measured the FAM fluorescence after each step. The change in the melting temperature at 1.0 μM compound concentration (ΔT_m (1.0 μM)) was calculated from at least three experiments by subtraction of the blank from the averaged melting temperature of each compound.