A nonfluorescent, broad-range quencher dye for Förster resonance energy transfer assays

We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Förster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 substrates were prepared by conjugating the quencher, IRDye QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum approximately 540 nm), Cy3 (approximately 570 nm), Cy5 (approximately 670 nm), IRDye 680 (approximately 700 nm), IRDye 700DX (approximately 690 nm), or IRDye 800CW (approximately 790 nm). The Förster distance R(0) values are calculated as 41 to 65A for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change on complete cleavage by recombinant caspase-3 and ranged from 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes.