Viaflo pipet

The NIH Chemical Collection (NIHCC) was purchased from Evotec and contains a total of nine 96-well plates (700 compounds at 10 mM concentration at 10 μL volumes). For screening, compound was diluted to 250 μM in a separate working drug dilution plate. A volume of 2 μL of compound was transferred to 50 μL final assay volume via an Integra Viaflo pipet, for a final compound concentration of 10 μM.

Fluorescence polarization assay was performed in 1/2 area black plates (Corning 3686). Reaction mixtures were a total volume of 50 μL and contained final concentrations of 125 nM MSUT2 RNABP and 10 nM FAM-RNA in PBS, transferred using an Integra Viaflo pipet with a 96/50 μL head. Then 2 μL of stock compound was transferred yielding a 10 μM final concentration. Plates were then incubated at room temperature, without shaking, for 20 min in a BioSpa8 automated incubator and transferred by robotic arm to a Biotek Cytation 5 with a preconfigured green polarization filter cube (8040561) at excitation 485/20 and emission 528/20, a dichroic mirror at 510 nm, and a read height of 10 mm. Fluorescence polarization was calculated by first subtracting background from a buffer-only control well and then using the equation $P=\frac{F_{\parallel }-F_{\perp }}{F_{\parallel }+F_{\perp }}$ to determine the polarization (P).