FIGURE 1.

SPA used for HTS of DNMT inhibitors. A, schematic of assay used to screen for DNMT3A inhibitors. Two custom oligos were utilized as the substrate. One was a 58-mer oligo, biotinylated (Biotin) on the 5′ end and methylated at 12 CpG sites (black circles). The other oligo was an antisense 58-mer without biotinylation or methylation. The two oligos were annealed to produce a biotinylated, hemi-methylated double-stranded oligo template. Assay components were mixed and the reaction initiated by the addition of tritiated S-adenosylmethionine ([³H]AdoMet). If the compound inhibited the reaction, the double-stranded oligo should remain hemimethylated. If the compound did not inhibit the reaction, the double-stranded oligo would be methylated and AdoMet incorporated. Addition of streptavidin-coated PVT (dark gray SPA beads) allows for light production when bound to double-stranded oligos with [³H]AdoMet incorporated. B, screening strategy for DNMT inhibitors. See text describing the HTS assay under “Experimental Procedures” and “Results” for more details. Norm, normalized.