Fluorescence Intercalator Displacement (FID) Method

Bacterial genomic DNA (ATCC 43300, DSM 13661; ATCC 27853, DSM1117; ATCC 700603, DSM 26371; ATCC 25922, DSM 1103; ATCC 19606, DSM 30007; ATCC 51299, DSM 12956; Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH) or salmon genomic DNA (deoxyribonucleic acid sodium salt from salmon testes, D1626, Merck) dissolved in 1 mM phosphate buffer pH 7.4 (containing 0.27 mM potassium chloride, 13.7 mM sodium chloride) to a concentration of 100 μg/mL in SybrSafe (SYBR Safe DNA Gel Stain, ×10,000 in DMSO, S33102 Invitrogen) was used as supplied by the manufacturer in DMSO, and MGB-BP-3 was prepared as 10 mM stock in DMSO. These stock solutions were diluted appropriately with each other and 1 mM phosphate buffer to give a test solution comprised of 20 μM S-MGB, 12,500-fold dilution of SybrSafe and 3.92 μg/mL DNA. Control solutions of gDNA and SybrSafe, gDNA, and SybrSafe at these concentrations were also prepared. Test and control solutions were heated to 30 °C and the fluorescence of each solution was measured using the SYBER filter setting of a StepOnePlus using melt analysis mode (StepOne Software v2.3). The reduction of fluorescence due to the binding of MGB-BP-3 to the gDNA was calculated as a normalized percentage based on the fluorescence measured due to the control with SybrSafe and gDNA as maximum and the control with only SybrSafe as minimum. Low normalized percentage indicates a greater ability to displace SybrSafe from the gDNA and suggests strong binding to gDNA. Three independent experiments were carried out and the results were presented as average values ± standard error of the mean.