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.
Mergny
J L
JL
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM U201, CNRS UA481, Paris, France.
Garestier
T
T
Rougée
M
M
Lebedev
A V
AV
Chassignol
M
M
Thuong
N T
NT
Hélène
C
C
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004735:Energy Transfer; D004996:Ethidium / Q000737:chemistry; D006860:Hydrogen Bonding; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013696:Temperature
D009838:Oligodeoxyribonucleotides; D004247:DNA; D004996:Ethidium