Thiazole orange (TO) with different tethers were synthesized to be attached to oligonucleotides. The FRET of TO in solution with double-stranded DNA (dsDNA) was investigated with BlackHole (BHQ1) or ((4-dimethylamino)phenyl)azo)benzoic acid (DABCYL) quenchers which decreased the fluorescence 2.9 +/- 7% and 2.5 +/- 10% times, respectively. A quenching mechanism could therefore be designed to transduce hybridization.The FRET of N,N,N,N-tetramethylcarboxyrhodamine (TAMRA) and IowaBlackRQ RTM (IABLK) linked to complementary oligonucleotides immobilized on glass substrates was investigated; IABLK quenched TAMRA fluorescence. However, surface bound dsDNA caused some self-quenching of TAMRA.Solution FRET using TAMRA/IABLK at 24.5°C and 60°C with complementary and mismatched DNA was measured to investigate potential for mismatch detection. The probe sequence was based on the determinate for Spinal Muscular Atrophy (SMA). Signal intensity differed between complementary and the mismatch samples at 60°C, indicating mismatch detection potential. Results suggest the possibility of designing tethered fluorophore-quencher pairs for transduction of hybridization for development of optical nucleic acid biosensors for SMA screening.