5'-bis-pyrenylated oligonucleotides displaying excimer fluorescence provide sensitive probes of RNA sequence and structure.
Oligonucleotide conjugates bearing two pyrene residues attached to 5'-phosphate 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 the excimer formation between the two pyrene residues. Conjugation of these two pyrene residues to the 5'-phosphate of oligonucleotides does not affect the stabilities of heteroduplexes formed by conjugates with the corresponding linear strands. A monomer fluorescence of the conjugates is considerably affected by the heteroduplex formation allowing the conjugates to be used as fluorescent hybridisation probes. The 5'-bis-pyrenylated oligonucleotides have been successfully used for investigation of affinity and kinetics of antisense oligonucleotides binding to the multidrug resistance gene 1 (PGY1/MDR1) mRNA. The changes of excimer fluorescence of the conjugates occurring 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 secondary structure was accompanied by an enhancement of the excimer fluorescence. Potentially, these conjugates may be considered as fluorescent probes for RNA structure investigation.
Kostenko
E
E
Novosibirsk Institute of Bioorganic Chemistry, Lavrentiev Avenue 8, Novosibirsk 630090, Russia.
Dobrikov
M
M
Pyshnyi
D
D
Petyuk
V
V
Komarova
N
N
Vlassov
V
V
Zenkova
M
M
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics; D001483:Base Sequence; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D005453:Fluorescence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics*; D011721:Pyrenes / Q000737:chemistry*; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013696:Temperature
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D015342:DNA Probes; D009841:Oligonucleotides; D011721:Pyrenes; D012333:RNA, Messenger; D012313:RNA; C030984:pyrene