Sybrg 2

The 94nt MALAT1 RNA (also termed M1^TH) was transcribed by T7 RNA polymerase using a MALAT1 HDV ribozyme plasmid (a generous gift from Prof. Jessica Brown, University of Notre Dame). For this assay, a fixed concentration of 94nt MALAT1 (500 nM) and SYBRG II (Invitrogen) (4×) was used. A total of 5 μL of compound (in DMSO) and 95 μL of RNA/dye complex in assay buffer (5 mM sodium cacodylate at pH 6.5, 50 mM KCl, 1 mM MgCl₂, 0.1 mM EDTA, 0.01% Triton-x100) were added to assay plate (black nunc 96-well plate, Fisher Scientific) and incubated at room temperature for 30 min, and fluorescence intensity values were measured (485 ± 5 nm excitation, 525 ± 5 nm emission) using a Tecan plate reader. IC₅₀ values were determined by normalizing fluorescence intensity of each well to an average value for the fluorescence intensity of RNA/dye complex and using a nonlinear regression fitting of the competition curves (GraphPad Prism 7.0 software).

A fixed concentration of HBV $\text{ε}$ RNA (500 nM) and SYBRG II (Invitrogen) (4x) was used. 5 μL of compound (in DMSO) and 95 μL of RNA/dye complex in assay buffer D (5 mM sodium cacodylate pH 6.5, 50 mM KCl, 1 mM MgCl₂, 0.1 mM EDTA, 0.01% Triton-X100) were added to black Nunc 96-well plates (Fisher Scientific), incubated at room temperature for 30 min and fluorescence intensity values were measured (485 ± 5 nm excitation, 525 ± 5 nm emission) using a Tecan plate reader. K_d values were determined by normalizing fluorescence intensity of each well to an average value for the fluorescence intensity of RNA/dye complex (GraphPad Prism 7.0 software). Errors in K_d values are reported as the standard error of triplicate experiments.