5 '-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure

Oligonucleotide conjugates bearing two pyrene residues attached to 5 ' -pho sphate through a phosphoramide bond were synthesised. Fluorescence spectra of the conjugates show a peak typical of monomer emission (lambda (max) 382 nm) and a broad emission peak with lambda (max) 476 nm, which indicates th e excimer formation between the two pyrene residues. Conjugation of these t wo pyrene residues to the 5 ' -phosphate of oligonucleotides does not affec t the stabilities of heteroduplexes formed by conjugates with the correspon ding linear strands. A monomer fluorescence of the conjugates is considerab ly affected by the heteroduplex formation allowing the conjugates to be use d as fluorescent hybridisation probes. The 5 ' -bis-pyrenylated oligonucleo tides have been successfully used for investigation of affinity and kinetic s of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occ urring during hybridisation depended on the structure of the binding sites: hybridisation to heavily structured parts of RNA resulted in quenching of the excimer fluorescence, while binding to RNA regions with a loose seconda ry structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.