Fluorescence energy transfer as a probe for tetraplex formation: The i-motif

The secondary structure of cytosine-rich oligodeoxynucleotides has been inv estigated with fluorescent probes. Intramolecular folding of an oligonucleo tide into an i-DNA motif led to fluorescence excitation energy transfer bet ween a donor (fluorescein) and an acceptor (tetramethylrhodamine) covalentl y attached to the 5' and 3' ends of the DNA, respectively, provided that a suitable linker was chosen. The conjugation of the dyes to the oligonucleot ide had an influence on the thermodynamics of i-motif formation as well as on the kinetics of folding. Intramolecular folding was demonstrated from th e concentration independence of FRET over a wide concentration range. Foldi ng of the oligonucleotide was confirmed by UV absorption melting experiment s. The folding of the i-motif could be followed at concentrations as low as 50 pM. Fluorescence energy transfer can thus be used to reveal the formati on of multistranded DNA structures.