FLUORESCENCE ENERGY-TRANSFER BETWEEN 2 TRIPLE HELIX-FORMING OLIGONUCLEOTIDES BOUND TO DUPLEX DNA

An 11-mer oligopyrimidine was covalently linked via its 5'-phosphate t o an acridine derivative (acridine-ll-mer), and a 13-mer was covalentl y linked via its 3'-phosphate to an ethidium derivative (13-mer-ethidi um). Each of them formed a triple helix with a 31-bp DNA fragment cont aining two oligopurine-oligopyrimidine sequences, 11 and 13 bp in leng th, separated by a variable number of base pairs. When both oligonucle otides were bound to the 31-bp DNA fragment, fluorescence energy trans fer (FET) from acridine to ethidium was observed, as revealed by a que nching of acridine fluorescence and a sensitized ethidium emission. FE T vc;as temperature-dependent and occurred only when both oligonucleot ides were simultaneously bound to the DNA matrix. A single base-pair c hange in one of the target sequences strongly reduced the energy-trans fer efficiency. This method was used to discriminate between a fully c omplementary and a mismatched target sequence.