title: Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays.
abstract: Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.
authors.affiliation: Nucleic Acid Center, Department of Chemistry, University of Southern Denmark, DK-5230 Odense M, Denmark.
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Hrdlicka
authors.forename: B Ravindra
authors.initials: BR
authors.lastname: Babu
authors.forename: Mads D
authors.initials: MD
authors.lastname: Sørensen
authors.forename: Niels
authors.initials: N
authors.lastname: Harrit
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
mesh_terms: D004247:DNA / Q000032:analysis; D008856:Microscopy, Fluorescence / Q000379:methods; D015394:Molecular Structure; D009696:Nucleic Acids / Q000032:analysis*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000138:chemical synthesis / Q000737:chemistry*; D011721:Pyrenes / Q000737:chemistry*; D012313:RNA / Q000032:analysis; D013696:Temperature
chemical_list: D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D011721:Pyrenes; C477371:locked nucleic acid; D012313:RNA; D004247:DNA