Example 4
Bacterial Phage Lambda DNA (Sigma-Aldrich), CpG DNA (Enzo Life, Plymouth Meeting, Pa.), Torula yeast RNA (Sigma-Aldrich) were completely dissolved in Tris-acetate-EDTA (TAE) buffer (pH=8) by gentle inversion overnight at 4° C. Lambda DNA, CpG DNA, yeast RNA stock solutions were characterized by spectrophotometry to determine the concentration and purity. Total mouse heart RNA was extracted from C57Bl6 mouse hearts using TRI Reagent (Sigma-Aldrich) and characterized with a NanoDrop spectrophotometer (Thermo Fisher) as described in Feng Y, Chen H, et al., J Biol Chem. 2015; 290: 26688-98.
To measure the fluorescence response in the presence of nucleic acid (NA), serial dilutions of the NA solutions with ratio of 1:3 were prepared in a Corning Costar 96-Well black clear-bottom plate (Thermo Fisher). 50 nM of fluorochrome equivalents, based on the common benzothiazole ring, were added to the NA-containing wells and incubated for 2 hours. Fluorescence was measured with the GloMax-Multi Detection System and snap-in fluorescent optical kits (Promega, Madison, Wis.). EC50 was determined using the dose-response curve fitting function implemented in Prism (GraphPad, La Jolla, Calif.).