Fluorescence Energy Transfer between Two Triple Helix-Forming Oligonucleotides Bound to Duplex DNA

An 11-mer oligopyrimidine was covalently linked via its 5′-phosphate to an acridine derivative (acridine-11-mer), and a 13-mer was covalently linked via its 3′-phosphate to an ethidium derivative (13-mer-ethidium). Each of them formed a triple helix with a 31-bp DNA fragment containing two oligopurine-oligopyrimidine sequences, 11 and 13 bp in length, separated by a variable number of base pairs. When both oligonucleotides were bound to the 31-bp DNA fragment, fluorescence energy transfer (FET) from acridine to ethidium was observed, as revealed by a quenching of acridine fluorescence and a sensitized ethidium emission. FET was temperature-dependent and occurred only when both oligonucleotides were simultaneously bound to the DNA matrix. A single base-pair change in one of the target sequences strongly reduced the energy-transfer efficiency. This method was used to discriminate between a fully complementary and a mismatched target sequence.