Bioconjugate Chemistry · 2008 · 52 citations · 83 references
PharmacyChemical BiologyMolecular BiologyBiophysical ChemistryNucleic Acid BiochemistryNucleic Acid ChemistryDna ChemistryFluorescence SpectroscopyMolecular Probe DevelopmentNucleic Acid LabelingOligonucleotide ChemistryPhotophysics Of Fluorescent ProbesFluorescence Quantum YieldFluorescent Nucleic Acid ProbesFluorescent ProbesLna SynthesisNucleic Acid Base PairingNucleic Acid FluorescenceOrganic FluorophoresThermal Dna Denaturation
During recent years, fluorescently labeled oligonucleotides have been extensively investigated within diagnostic approaches. Among a large variety of available fluorochromes, the polyaromatic hydrocarbon perylene is an object of increasing interest due to its high fluorescence quantum yield, long-wave emission compared to widely used pyrene, and photostability. These properties make perylene an attractive label for fluorescence-based detection in vitro and in vivo. Herein, the synthesis of 2'- N-(perylen-3-yl)carbonyl-2'-amino-LNA monomer X and its incorporation into oligonucleotides is described. Modification X induces high thermal stability of DNA:DNA and DNA:RNA duplexes, high Watson-Crick mismatch selectivity, red-shifted fluorescence emission compared to pyrene, and high fluorescence quantum yields. The thermal denaturation temperatures of duplexes involving two modified strands are remarkably higher than those for double-stranded DNAs containing modification X in only one strand, suggesting interstrand communication between perylene moieties in the studied 'zipper' motifs. Fluorescence of single-stranded oligonucleotides having three monomers X is quenched compared to modified monomer (quantum yields Phi F = 0.03-0.04 and 0.67, respectively). However, hybridization to DNA/RNA complements leads to Phi F increase of up to 0.20-0.25. We explain it by orientation of the fluorochrome attached to the 2'-position of 2'-amino-LNA in the minor groove of the nucleic acid duplexes, thus protecting perylene fluorescence from quenching with nucleobases or from the environment. At the same time, the presence of a single mismatch in DNA or RNA targets results in up to 8-fold decreased fluorescence intensity of the duplex. Thus, distortion of the duplex geometry caused by even one mismatched nucleotide induces remarkable quenching of fluorescence. Additionally, a perylene-LNA probe is successfully applied for detection of mRNA in vivo providing excitation wavelength, which completely eliminates cell autofluorescence.
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