PicoGreen RT Assay.

The RT assay was performed with the EnzCheck Reverse Transcriptase Assay kit (Molecular Probes, Invitrogen), as described by the manufacturer. The assay is based on the dsDNA quantitation reagent PicoGreen. This reagent shows a pronounced increase in fluorescence signal upon binding to dsDNA or RNA/DNA heteroduplexes. Single-stranded nucleic acids generate only minor fluorescence signal enhancement when a sufficiently high dye to base pair ratio is applied (49). This condition is met in the assay. Five µL of 1 mg/mL poly(rA) template of ∼350 bases long was annealed with 5 µL of a 50 µg/mL oligo(dT)16 primer at a molar ratio of 1:1.2 (60 min at room temperature). The annealed template/primer was then diluted 180-fold in polymerization buffer (60 mM Tris⋅HCl, pH 8.1, 60 mM KCl, 8 mM MgCl₂, 13 mM DTT, and 100 µM dTTP) and 17 µL of this RNA/DNA was brought into each well of a 96-well flat-black plate. To test the RT inhibition, 3 µL of compound in dimethyl sulfoxide (DMSO) was added to each well before the addition of RT enzyme solution. Control wells without compound contained the same amount of DMSO. Five microliters of RT enzyme solution, diluted to a suitable concentration in the enzyme dilution buffer (50 mM Tris⋅HCl, pH 7.5, 20% glycerol, and 1 mM DTT) was added. The reactions were incubated at 25 °C for 40 min and then stopped by adding 2 µL of 200 mM EDTA. Heteroduplexes were then detected by addition of 173 µl 0.73 µM PicoGreen. Signals were read using an excitation wavelength of 490 nm and emission detection at 523 nm in a spectrofluorometer (Safire 2, Tecan). The results (summarized in Table 1 and detailed in SI Appendix, Table 2) are expressed as relative fluorescence, i.e., the fluorescence signal of the reaction mix with compound divided by the signal of the same reaction mix without compound.