title: Perylene attached to 2'-amino-LNA: synthesis, incorporation into oligonucleotides, and remarkable fluorescence properties in vitro and in cell culture.
abstract: 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.
authors.affiliation: Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark.
authors.forename: Irina V
authors.initials: IV
authors.lastname: Astakhova
authors.forename: Vladimir A
authors.initials: VA
authors.lastname: Korshun
authors.forename: Kasper
authors.initials: K
authors.lastname: Jahn
authors.forename: Jørgen
authors.initials: J
authors.lastname: Kjems
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
mesh_terms: D001483:Base Sequence; D018929:Cell Culture Techniques; D002460:Cell Line; D005453:Fluorescence; D006801:Humans; D009691:Nucleic Acid Denaturation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010569:Perylene / Q000031:analogs & derivatives*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013194:Staining and Labeling; D044366:Transition Temperature
chemical_list: D009841:Oligonucleotides; C477371:locked nucleic acid; D010569:Perylene